52 publications mentioning mmu-mir-193a

Open access articles that are associated with the species Mus musculus and mention the gene name mir-193a. Click the [+] symbols to view sentences that include the gene name, or the word cloud on the right for a summary.

1

Having shown that miR-193a negatively regulates WT1 expression and inhibits metastasis, we directly address the role of WT1 downregulation in the anti-metastasis activity.

MiR-193a was reported to be a suppressive miRNA and overexpression of miR-193a suppressed proliferation and promoted apoptosis via targeting several oncogenes, such as c-KIT [23] and MCL-1 [24].

was performed using Student’s t-test To determine potential target genes by miR-193a, several miRNAs predicting software including miRwalk, TargetScan, and PicTar were used to predict possible target genes.

The further decrease of miR-193a by TGF-β loses the ability of suppressing WT1 and finally enhances the expression of WT1, which leads to the decreased expression of E-cadherin (Additional file 6: Figure S4B).

Because miR-193a expression was more decreased in lung cancer cells with greater migration and invasion abilities, we speculated that TGF-β1 inhibited the expression of miR-193a.

Hypermethylation of miR-193a leads to the low expression of miR-193a, which loses the ability to inhibit WT1 expression.

In addition to WT1, miR-193a targeted several important oncogene, such as Yin Yang1 (YY1) [33], phosphoinositide-3-kinase regulatory subunit (3PIK3R3), and the mammalian target of rapamycin (mTOR) [34].

Overexpression of miR-193a significantly decreased the protein levels of WT1, but increased the expression of the epithelial marker E-cadherin (Fig.   3d), which was reported to be negatively regulated by WT1 in NSCLC [15].

Ectopic overexpression of WT1 partially reverses miR-193a -induced inhibition of metastasis.

Overexpression of miR-193a inhibited migration and invasion via modulating WT1-E-cadherin axis.

As expected, TGF-β1 reduced the expression of miR-193a by 30 %–50 % (Fig.   6f) and subsequently increased the expression of WT1 in A549 and H1299 cells (Fig.   6g).

To confirm the low expression of miR-193a in lung cancer cells, the relative expression of miR-193a was detected in lung cancer tissues and adjacent non-tumor tissues.

To select the cells stably expressing miR-193a, 2 μg/ml puromycin (Medchemexpress, Princeton, USA) was added into the supernatant for 1 week.

Further, overexpression of WT1 partially prevented miR-193a -induced growth inhibition (Fig.   5d and e).

MiR-193a inhibited the metastasis of lung cancer cells by deregulating the expression of tumor-related proteins [26].

Further, overexpression of miR-193a decreased the colony formation (Fig.   2e) and suppressed cell proliferation (Fig.   2f).

Thus, restoring the expression of miR-193a or decreasing the expression of WT1 might provide alternative therapeutic strategy for NSCLC patients.

Ectopic expression of miR-193a inhibited cell proliferation, colony formation, migration, and invasion in A549 and H1299 cells.

Because WT1 is implicated in the metastasis of NSCLC through inhibiting the expression of E-cadherin [15], we hypothesized that one mechanism of anti-metastasis activity of miR-193a might perform by modulating WT1-E-cadherin axis.

Consistently, ectopic overexpression of miR-193a inhibits metastasis and the EMT-like conversion in NSCLC.

Ectopic expression of miR-193a inhibits metastasis in lung cancer cells.

Similarly, besides miR-193a, miR-125a was identified to suppress WT1 expression via binding 3′UTR of WT1 in myeloid leukemia cells [51].

Consistent with the results from cell lines, the protein levels of WT1 were significantly decreased but E-cadherin expressions were increased in two tumors obtained from miR-193a -overexpressed mice in comparison with control mice (Fig.   7e).

Thus, miR-193a might have multiple different mRNA targets other than WT1, and these additional targets may also play important roles in carcinogenesis.

Furthermore, overexpression of WT1 partially prevents miR-193a -induced inhibition of migration and invasion, suggesting that WT1 plays an important role in the anti-metastasis by miR-193a.

For example, miR-193a inhibited invasion by negatively regulating ERBB4/PIK3R3/mTOR/S6K2 signaling pathway in NSCLC [25].

Decreased expression of miR-193a regulated TGF-β1 -induced EMT progress.

Consistently, miR-193a-3p, the another product of pre-miR-193a, was also downregulated in lung cancer [25, 34].

Downregulation of miR-193a in lung cancer tissues.

Indeed, many studies have indicated that miR-193a was down-regulated in various types of cancers [32– 34].

Overexpression of miR-193a decreased the luciferase activities by approximately 40 %, which were almost abolished by the mutation in putative miR-193a -binding sites (Fig.   3b).

However, whether miR-193a can regulate E-cadherin expression is not determined.

Importantly, knockdown of WT1 resembled the anti-cancer activity by miR-193a and overexpression of WT1 partially reversed miR-193a -induced anti-cancer activity, indicating that WT1 plays an important role in miR-193a -induced anti-cancer activity.

f and g Cell proliferation was detected by CCK-8 assay in A549 (f) and H1299 (g) cells transfected with sh-NC or sh-WT1 for 24, 48, and 72 h. * P < 0.01 versus sh-NC Because miR-193a inhibited metastasis via targeting WT1 protein, which acts as an oncogene in multiple types of cancers including lung cancer, we are interested in examining whether WT1 counteracts miR-193a -induced anti-metastasis.

MiR-193a directly targets WT1 and indirectly modulates E-cadherin.

Mechanistically, miR-193a reduced the expression of WT1, which negatively regulated the protein level of E-cadherin, suggesting that miR-193a might prevent EMT via modulating WT1-E-cadherin axis.

Collectively, our results have revealed an important role of miR-193a-WT1-E-cadherin axis in metastasis, demonstrated an important molecular cue for EMT, and suggested a therapeutic strategy of restoring miR-193a expression in NSCLC.

Our finding that miR-193a inhibits TGF-β -induced EMT through WT1-E-Cadherin axis augments the role of miRNAs in EMT of NSCLC.

Finally, overexpression of miR-193a decreased the growth of tumor xenografts in mice.

Our results indicate that decreased expression of miR-193a contributes to proliferation, metastasis, and TGF-β -induced EMT through WT1-E-cadherin pathway.

Human WT1 CDS including the predicted miR-193a target sites was amplified by PCR and cloned into pMIR-REPORT vector named as pMIR-WT1CDS (Ambion, Dallas, USA).

Correspondingly, overexpression of WT1 partially reversed the anti-migration (Fig.   5b) and anti-invasion (Fig.   5c) induced by miR-193a.

Therefore, our results suggest that miR-193a exerts tumor-suppressive effects through WT1-E-cadherin pathway.

A statistically significant correlation between miR-193a versus WT1 expression was observed by Pearson’s method.

TGF-β1 -induced EMT is attenuated by the overexpression of miR-193a.

d WT1 and E-cadherin expressions were performed in A549 and H1299 cells transfected with lentivirus LVX-miR-193a or LVX-NC.

n = 62. g The plotting of miR-193a versus WT1 expression showed an inverse correlation between them.

Finally, IHC staining indicated that the expression of WT1 was decreased while E-cadherin was increased in tumor xenograft from mice inoculated with A549-miR-193a than that inoculated with A549-miR-NC (Fig.   7f).

MiR-193a was downregulated in about 81 % of tumors (P < 0.01, 50 of 62 patients), with about 40 % reduction relative to their adjacent non-tumor tissues by qRT-PCR (Fig.   1b).

Recently, miR-193a has been reported to be a suppressive miRNA in multiple types of cancers, but its underlying anti-oncogenic activity in non-small cell lung cancers (NSCLC) is not fully elucidated.

TGF-β1 -induced EMT was partially prevented by the overexpression of miR-193a (Fig.   6d and e).

Here, we report that miR-193a -mediated pathway may take part in TGF-β -induced inhibition of E-cadherin (Additional file 6: Figure S4A).

Restoration of miR-193a inhibits migration, invasion, and TGF-β1 -induced EMT through modulating WT1-E-cadherin axis.

As shown in Additional file 4: Figure S2A-C, overexpression of WT1 CDS (Mut) did not prevent the anti-migration and anti-invasion induced by miR-193a.

We further analyzed the relationship between WT1 and miR-193a expressions.

Importantly, a significant inverse correlation between miR-193a and WT1 mRNA expressions was found in lung cancer tissues (Fig.   3g).

The expression of miR-193a in lung cancer tissues was decreased comparing to adjacent non-tumor tissues due to DNA hypermethylation in lung cancer tissues.

However, miR-193a minics failed to decrease WT1 expression in cells transfected with pMIR-WT1CDS (Fig.   3c, Right).

The mutation on miR-193a -binding sites in human WT1 CDS was generated by the site-directed mutagenesis kit (Agilent Technologies, Palo Alto, USA).

Relationship between the expression of miR-193a and clinicopathologic parameters.

f and g The expressions of miR-193a (f) and WT1 (g) were measured in A549 and H1299 cells treated with TGF-β1 for 3 days by qRT-PCR and western blot, respectively Finally, we determined whether overexpression of miR-193a could reduce the tumorigenicity in a xenograft mo del.

l The expression of miR-193a was detected in A549 and H1299 cells treated with 5 μM AZA for 3 and 5 days.

Therefore, targeting miR-193a-WT1-E-cadherin axis might provide a novel strategy to improve survival in lung cancer patients.

No statistically significant correlations were observed between the miR-193a expressions and age, gender, tumor size, or degree of differentiation, respectively.

Intriguingly, miR-193a expression is decreased in TGF-β -induced EMT, suggesting that miR-193a-WT1-E-cadherin pathway was mediated in TGF-β -induced EMT.

n = 62. c The expression of miR-193a was detected in lung cancer tissues with metastasis (n = 24) or without metastasis (n = 38).

Meanwhile, overexpression of miR-193a significantly decreased the wound healing activity in A549 (Fig.   2c) and H1299 cells (Fig.   2d).

In our study, the levels of miR-193a were lower in lung cancer tissues than those in normal noncancerous tissues, suggesting that miR-193a might be involved in the pathogenesis of lung cancer as a tumor suppressor.

The expressions of miR-193a (miR-193a-5p) in human lung cancer tissues and cell lines were detected by real-time PCR.

Fig. 6Overexpression of miR-193a partly reverses TGF-β1 -induced EMT.

Moreover, overexpression of miR-193a partially reversed tumor growth factor-β1 (TGF-β1) -induced epithelial-to-mesenchymal transition (EMT) in NSCLC cells.

Furthermore, miR-193a minics decreased the protein expression of WT1 in cells transfected with pMIR-WT1CDS (Mut) (Fig.   3c, Left).

Further, lower expression of miR-193a was related with greater migration and invasion.

These observations are compatible with the work by Heller et al. that miR-193a was silenced by DNA hypermethylation through genome-wide miRNA expression profiling [35].

AZA increased miR-193a expression in a time -dependent manner (Fig.   1l).

a The expression of miR-193a was detected in several lung cancer cell lines and normal lung epithelial cell line BEAS-2B by qRT-PCR.

Ectopic overexpression of WT1 CDS (Mut) fails to prevent miR-193a -induced anti-metastasis activity.

Dual-luciferase reporter assay was used to identify the direct target of miR-193a.

Also, miR-193a expression was reduced in lung cancer patients with metastasis compared with those without metastasis (Fig.   1c and Additional file 1: Table S1).

CDS of WT1 including putative miR-193a -binding sites was subcloned into pMIR vector to produce wide-type vector pMIR-WT1CDS and mutation vector pMIR-WT1CDS(Mut) (Fig.   3a).

Immunofluorescent analysis also showed that the expression of WT1 was decreased but E-cadherin was increased in miR-193a -transfected cells compared with negative control (Fig.   3e and Additional file 3: Figure S1A).

MiR-193a expression is decreased due to DNA hypermethylation in NSCLC specimens.

To determine whether WT1 knockdown resembles the effects of miR-193a in our experimental mo del, lung cancer cells were transfected with specific shRNA for WT1 (sh-WT1).

Therefore, TGF-β-miR-193a-E-cadherin pathway complements the routine regulatory net in EMT.

Fig. 4Knockdown of WT1 resembles the anti-metastasis activity by miR-193a.

Thus, these results suggest that miR-193a might regulate the metastasis in NSCLC.

As shown in Fig.   5a, miR-193a -induced inhibition of WT1 was prevented by transfection of MSCV-WT1 compared with negative control.

In addition, miR-193a regulated metastasis in solid cancers including NSCLC.

However, the expression of miR-193a was significantly decreased in lung cancer with advanced stages (TNM III and IV) compared with early stages (Stage I + II, Additional file 1: Table S1).

The relative expressions of miR-193a were elevated by 93- and 102-fold in LVX-miR-193a -transfected cells compared with normal control in A549 and H1299 cells (data not shown), respectively.

To construct the plasmid that expresses miR-193a in mammalian cells, the primary sequence of has-pre-miR-193a and its flanking regions were amplified by specific primer pairs and then were cloned into lentivirus vector pLVX-puro (Clontech, Palo Alto, USA).

Lung cancer cells transfected with pLVX-miR-193a or pLVX-NC were seeded into 96-well plates (6.0 × 10 [3] cells per well).

e The methylation status of miR-193a was analyzed by MSP in several lung cancer cell lines and BEAS-2B.

Our data affirm these results and indicate a new TGF-β-miR-193a-E-cadherin pathway to promote TGF-β -induced EMT in lung cancer.

d and e A549 and H1299 cells were transfected with LVX-NC or LVX-miR-193a, followed with treatment with TGF-β1 for 3 days.

Finally, migration and invasion were measured in miR-193a -overexpressed A549 cells transfected with MSCV-NC or MSCV-WT1 CDS (Mut).

Our studies also indicate that hypermethylation of miR-193a is frequently occurred in lung cancer cell lines and lung cancer tissues but not in adjacent non-tumor tissues.

To further explore the role of miR-193a in lung cancer cells, A549 and H1299 cells were transfected with lentivirus vector LVX-miR-193a or negative control.

b A549 cells were transfected with wide-type pMIR-WT1CDS, pMIR-WT1CDS (Mut), pMIR-NC, and the pRL-SV40 containing Renilla luciferase gene for 24 h, followed by the transfection with miR-193a mimic or Scramble for another 24 h. Firefly and Renilla luciferase activities were both detected and histograms showed the Firefly luciferase activities normalized to Renilla luciferase activities.

f IHC staining of WT1 and E-cadherin was done in tumor xenografts from mice inoculated with A549-miR-193a or A549-miR-NC cells.

A total of 1 × 10 [7] viable A549-LVX-NC or A549-LVX-miR-193a cells were trypsinized and resuspended with 200 μl sterile 1× PBS and injected subcutaneously into right flank of each nude mouse.

Here, we report a miR-193a-WT1-E-cadherin axis in NSCLC.

f– k The methylation status of miR-193a was analyzed by MSP in 27 lung cancer tissues (f– h) and 27 adjacent non-tumor tissues (i– k).

Additionally, miR-193a partially prevented TGF-β1 -induced EMT, suggesting that miR-193a plays an important role in TGF-β1 -induced EMT.

Hypermethylation of miR-193a was observed in seven lung cancer cell lines (Fig.   1e).

We speculated that pMIR-WT1CDS binds miR-193a minics and consequently prevents miR-193a -induced decrease of WT1.

Fig. 7The anti-tumor effects of miR-193a in tumor xenograft.

To further determine the role of miR-193a in the TGF-β1 -induced EMT, A549 and H1299 cells transfected with LVX-miR-193a or negative control were treated with TGF-β1 for 3 days.

b The expression of miR-193a was measured in lung cancer tissues and adjacent non-tumor tissues.

Migration and invasion were measured in miR-193a -overexpressed A549 and H1299 cells, which were transfected with MSCV-NC or MSCV-WT1.

e Colony formation was performed by methylene blue staining in A549 and H1299 cells transfected with LVX-miR-193a or LVX-NC.

n = 7. a A photograph of tumors from mice inoculated with A549-miR-193a or A549-miR-NC.

a Schematic of putative binding sites for miR-193a in WT1 CDS.

a Western blot was analyzed in A549 and H1299 cells transfected with LVX-miR-193a and retrovirus MSCV-NC or MSCV-WT1.

About 1 × 10 [7] viable A549 cells transfected with pLVX-miR-193a (A549-miR-193a) or pLVX-miR-NC (A549-miR-NC) were subcutaneously injected into right flank of each nude mouse.

These vectors were co -transfected together with miR-193a mimics or its negative control into A549 cells.

After 24 h, LVX-puro-miR-193a and MSCV-puro-WT1 vectors were cotransfected with packaging and envelope vectors into HEK293T cells.

Tumors in mice inoculated with LVX-miR-193a were significantly smaller than those in control mice (Fig.   7a).

In contrast, a complete absence of miR-193a methylation was found in BEAS-2B cells (Fig.   1e).

Furthermore, tumor growth was significantly reduced in mice inoculated with LVX-miR-193a (Fig.   7b).

Scale bar = 50 μm To investigate the role of miR-193a in lung cancer cells, the expression of miR-193a was detected in several lung cancer cell lines and normal lung epithelial cell line BEAS-2B.

d and e Cell proliferation was detected by CCK-8 assay in A549 (d) and H1299 (e) cells, which were transfected with LVX-miR-193a and retrovirus MSCV-NC or MSCV-WT1 for 24, 48, and 72 h. * P < 0.01 compared with MSCV-NC The observation that overexpression of miR-193a inhibited metastasis through WT1-E-cadherin axis urged us to investigate whether miR-193a is mediated in the EMT of lung cancer cells.

To further explore whether DNA hypermethylation mediates the silencing of miR-193a, A5459 and H1299 cells were treated with DNA methyltransferase inhibitor 5′-azacytidine (AZA) and the levels of miR-193a was measured.

To further determine whether the CpG islands of pre-miR-193a were hypermethylated in a tumor-specific manner, the methylation status of miR-193a was analyzed by methylation-specific PCR (MSP) in several lung cancer cell lines and BEAS-2B.

Fig. 2The anti-metastasis activity of miR-193a in lung cancer cells.

After growth for 24 h, each well was transiently cotransfected with 100 ng pMIR-REPORT plasmid containing 10 ng internal control vector pRL-SV40 (Promega, Madison, USA), wild-type or mutant pMIR-WT1CDS, and 60 pmol scramble or miR-193a mimics using Hiperfect transfection reagent (Qiagen).

e The protein levels of WT1 and E-cadherin were detected in two tumor lysates from mice inoculated with A549-miR-193a or A549-miR-NC.

In summary, we explore the role of miR-193a-WT1-E-cadherin axis in the metastasis and EMT in NSCLC.

However, further studies are needed to determine the underlying mechanism by which miR-193a is decreased by TGF-β.

The relationship between miR-193a expression levels and clinicopathological characteristics of the NSCLC patients were indicated in Additional file 1: Table S1.

To determine whether miR-193a impacts tumorigenesis, 14 four-week-old mice were randomly divided into two equal groups.

In addition to miR-193a, several other miRNAs are mediated in the EMT of NSCLC.

Using MethPrimer software [27], we identified typical CpG islands around the region encoding pre-miR-193a, suggesting that miR-193a was likely regulated by DNA methylation (Fig.   1d).

To investigate the role of miR-193a in lung cancer cells, the expression of miR-193a was detected in several lung cancer cell lines and normal lung epithelial cell line BEAS-2B.

The reduced expression of miR-193a prompted us to investigate the biological role of miR-193a in lung cancer.

2

Further, ectopic expression of miR-193a in SW1990 cells transfected with miR-193a angomir reduced the protein expression of TGF-β2, TGF-βRIII and E2F6, while inhibition of miR-193a in AsPC-1 cells transfected with miR-193a antagomir could increase the expression of these proteins compared to the controls (Fig. 3c).

In our study, besides miR-193a could regulate TGF-β2 expression, miR-193a was also found to directly target TGF-βRIII.

Furthermore, restoration of TGF-β2 in SW1990-EgmiR-193a cells could abolish the inhibition of p-SMAD2, p-SMAD3, E2F6 and the increase of c-Myc expression caused by ectopic expression of miR-193a.

Meanwhile, miR-193a expression was also upregulated (Fig. 1a, Right).

Rather, neutralizing TGF-β2 in PANC-1-hU6miR-193a-IN cells could abrogate the increased expression of p-SMAD2, p-SMAD3, E2F6 and the decreased expression of c-Myc caused by knockdown of miR-193a (Fig. 3e).

Fig. 6Targeting miR-193a suppresses cancer repopulation and metastasis in PDX mo del.

We found that miR-193a expression was upregulated in the irradiated SW1990 and PANC-1 cells compared with the untreated ones (Fig. 1b).

It was found that ectopic expression of miR-193a could significantly decrease E-cadherin and N-cadherin expression, and vice versa (Fig.   4a).

Further, restoration of TGF-β2 in SW1990-EgmiR-193a cells could abolish the repressed expression of ARHGEF15 and ABL2 caused by ectopic overexpression of miR-193a.

These data demonstrated that knockdown of miR-193a or restoration of TGF-β2/TGF-βRIII signalings in pancreatic cancer cells could inhibit cancer repopulation and metastasis after radiation, and prolong the survival.

However, knockdown of miR-193a in PANC-1 feeder cells inhibited the repopulation after radiation.

The luciferase/GFP-labeled pancreatic cancer cells (with or without miR-193a overexpression or knockdown) were subcutaneously injected into nude mice.

We further developed stable SW1990 cells with overexpression of miR-193a (SW1990-EgmiR-193a) (> 200-fold changes, Additional file  6: Figure S5A, Left), and PANC-1 cells with knockdown of miR-193a (PANC-1-hU6miR-193a-IN) (< 0.1-fold changes, Additional file 6: Figure S5A, Right) for analysis of their biological effects.

Rather, neutralizing TGF-β2 in PANC-1-hU6miR-193a-IN cells could abrogate the increased expression of ARHGEF15 and ABL2 caused by knockdown of miR-193a (Fig. 4g).

miR-193a accelerated pancreatic cancer cell cycle and stimulated cell proliferation and repopulation through inhibiting TGF-β2/TGF-βRIII/SMADs/E2F6/c-Myc signaling, and even destroyed normal intercellular junctions and promoted metastasis via repressing TGF-β2/TGF-βRIII/ARHGEF15/ABL2 pathway.

Schematic physical maps of the constructs concerning miR-193a targets.

These results indicated that miR-193a could destroy intercellular junctions of pancreatic cancer cells, and promote metastasis through inhibiting TGF-β2/TGF-βRIII/ARHGEF15/ABL2 signaling.

These results indicated that TGF-β2 could counteract the effects of pancreatic cancer cell proliferation and repopulation induced by miR-193a overexpression.

Furthermore, higher miR-193a expression was observed in the stimulated SW1990 and PANC-1 reporter cells co-cultured with corresponding irradiated feeder cells than in those with untreated ones (Fig. 1c).

ARHGEF15 and ABL2 were the target genes of miR-193a.

As shown, miR-193a was found to be highly expressed in the irradiated pancreatic cancer cells, and be transferred into the surviving pancreatic cancer cells.

The sequence of miR-193a inhibitor (miR-193a-IN) was synthetized by Sangon Biotech (Shanghai, China) and was cloned into the vector pLV-hU6shRNA/CvG2L (10,704 bp).

The putative counterparts (WTs and MTs) of miR-193a-target sequence in the 3’-UTR of TGF-β2 (GenBank NM_001135599), TGF-βRIII (GenBank, NM_003243), ABL2 (GenBank, NM_005759), E2F6 (GenBank, NM_001278278) and ARHGEF15 (GenBank, NM_025014) were synthetized by Sangon Biotech (Shanghai, China) and also inserted into the same reporter vector, and named as pmirGLO-TGB2U-WT, pmirGLO-TGB2U-MT, pmirGLO-TGBRIIIU-WT, pmirGLO-TGBRIIIU-MT, pmirGLO-ABL2U-WT, pmirGLO-ABL2U-MT, pmirGLO-E2F6U-WT, pmirGLO-E2F6U-MT, pmirGLO-AGEF15U-WT and pmirGLO-AGEF15U-MT, respectively.

Our findings demonstrated that miR-193a stimulated pancreatic cancer cell repopulation and metastasis through modulating TGF-β2/TGF-βRIII signalings, and miR-193a might be a potential therapeutic target for pancreatic cancer repopulation and metastasis.

The PDX mice with miR-193a antagonist treatment effectively inhibited tumor repopulation and metastasis, and exhibited an improved prognosis after radiotherapy.

Notably, E2F6 was also similarly predicted as the target of miR-193a (Fig. 3a, Lower).

miR-193a was highly expressed in the irradiated pancreatic cancer dying cells, accordingly elevated the level of miR-193a in surviving cells, and further promoted pancreatic cancer repopulation and metastasis in vitro and in vivo.

b & c RT-qPCR for detecting miR-193a expression changes in pancreatic cells (±X-ray), and in the stimulated SW1990 and PANC-1 reporter cells co-cultured with their corresponding feeder cells (±X-ray).

The basal miR-193a expression in multiple pancreatic cells was tested (Additional file  4: Figure S3A).

Therefore, the expression level of miR-193a in pancreatic cancer tissues might contribute to screen the beneficial patients for radiotherapy.

The amplified pri-miR-193a was confirmed by sequencing (Sangon Biotech, China), and further cloned into the expression plasmid of pLEX-EgmiR-CvG2L vector [24] by double-digestion with BamHI/NheI (NEB).

In PDX mo dels, the treatment in combination with miR-193a antagonist and radiation was found to dramatically inhibit pancreatic cancer cell repopulation and metastasis, and further improved the survival after radiation.

The amplified pri-miR-193a was cloned into the expression plasmid of pLEX-EgmiR-CvG2L, and resulted in pLEX-EgmiR-193a/CvG2L.

We further explored whether miR-193a expression changed in vivo.

Intriguingly, miR-193a -binding sites were found in the 3’-UTR of TGF-β2 and its indispensable receptor TGF-βRIII according to the prediction of TargetScanHuman software (http://www.

The treatment of radiation in combination with miR-193a antagonist could effectively inhibit pancreatic cancer cell repopulation and metastasis.

Obvious increase of miR-193a expression was also observed in the co-culture system with the irradiated SW1990-EgmiR-193a feeder cells (> 10-fold changes).

Accordingly, miR-193a expression was detected in the tissues of the irradiated and the untreated tumor, and found to increase in the irradiated tumors (> 1.5-fold changes, Fig. 1e).

Then on the one hand, the elevated miR-193a in the surviving cells accelerated cell cycle through inhibiting TGF-β2/TGF-βRIII/SMADs/E2F6/c-Myc signaling, and led to cell proliferation and repopulation.

n = 5. (E) RT-qPCR for assessing miR-193a expression changes in mice (±X-ray) with pancreatic tumors.

These results suggested that TGF-β2, TGF-βRIII and E2F6 were the targeted genes of miR-193a.

In other words, miR-193a could serve as a potential therapeutic target to resensitize cancer cell to radiation, and abrogate repopulation after radiation in pancreatic cancer.

Similarly, ARHGEF15 and ABL2 were also confirmed to be the targets of miR-193a (Additional file  11: Figure S6).

a RT-qPCR for detecting miR-193a expression changes in the stimulated SW1990 and PANC-1 reporter cells co-cultured with their corresponding feeder cells (±X-ray).

Together, miR-193a was highly expressed in irradiated pancreatic cancer tissues, and might stimulate pancreatic cancer repopulation and promote metastasis.

Moreover, higher miR-193a expression was detected in the stimulated PANC-1-reporter cells co-cultured with the irradiated PANC-1-hU6shR-NC feeder cells than those with untreated PANC-1-hU6shR-NC ones (Fig. 2a, Lower).

The synthetized miR-193a inhibitor (miR-193a-IN) was cloned into the vector pLV-hU6shRNA/CvG2L, and resulted in pLV-hU6miR-193a-IN/CvG2L.

miR-193a antagonist suppresses cancer repopulation and progression in PDX mo del.

miR-193a might be a potential therapeutic target for pancreatic cancer repopulation and metastasis after chemoradiation treatment.

However, no significant alterations of miR-193a expression was observed in the stimulated PANC-1-reporter cells co-cultured with PANC-1-hU6miR-193a-IN feeder cells before and after radiation.

Notably, we speculated that TGF-β2 might be the target of miR-193a according to the prediction of several software on miRNAs.

d Knockdown of miR-193a blocked pancreatic cancer repopulation in vivo (±X-ray).

The results indicated that knockdown of miR-193a blocked pancreatic cancer repopulation in vivo.

Fig. 5Knockdown of miR-193a or restoration of TGF-β2/TGF-βRIII signaling blocks cancer repopulation and metastasis.

Moreover, knockdown of miR-193a in PANC-1 cells obviously slowed down cell growth, and resulted in a significant decrease in tumor volume (Fig. 2f).

Knockdown of miR-193a or restoration of TGF-β2/TGF-βRIII signalings blocks cancer repopulation and metastasis after radiation.

However, the irradiated PANC-1 feeder cells could only stimulate the growth of the co-cultured PANC-1-hU6shR-NC reporter cells, yet knockdown of miR-193a in PANC-1 reporter cells blocked its stimulation from the irradiated PANC-1 feeder cells.

Ulteriorly, in gain- and loss-of-function assay, higher miR-193a in the feeder cells either induced by ectopic expression or radiation could promote the growth of SW1990-reporter cells, leading to the living pancreatic cancer cell repopulation.

Knockdown of miR-193a or restoration of TGF-β2/TGF-βRIII signaling in pancreatic cancer cells was found to block pancreatic cancer repopulation and metastasis after radiation.

The EdU assay showed that overexpression of miR-193 promoted pancreatic cancer cell proliferation, and vice versa (Fig. 2c).

These data indicated that the elevated miR-193a from the irradiated dying pancreatic cancer cells caused miR-193a increased in the living pancreatic cancer cells and promoted tumor repopulation in vitro.

miR-193a accelerates cancer cell cycle, and promotes cell proliferation and repopulation.

In fact, microRNA-193a (miR-193a) was found to be induced by radiation in glioma and cervical carcinoma [19], and contributed to chemoradiation resistance in oesophageal carcinoma [20].

We herein identified that the elevated miR-193a from the irradiated dying pancreatic cancer cells could promote the surviving cancer cell repopulation and metastasis through modulating TGF-β2/TGF-βRIII signaling.

We further explored whether miR-193a promoted pancreatic cancer repopulation through TGF-β2/TGF-βRIII signalings.

In this study, we further revealed that miR-193a promoted pancreatic cancer repopulation and metastasis through modulating TGF-β2/TGF-βRIII signaling (Fig.   7).

The elevated miR-193a from irradiated dying cells promotes accelerated repopulation and metastasis of surviving cancer cells.

The 3’-UTR of predicted miR-193a binding site in TGF-β2, TGF-βRIII, ABL2, E2F6 and ARHGEF15, and their putative counterparts (WTs and MTs) were inserted into the reporter plasmid pmirGLO.

Moreover, the irradiated PDX group with miR-193a antagonist treatment lived much longer than the only irradiated or the untreated one (Fig. 6d).

a Schematic diagrams of the predicted miR-193a -binding sites in the 3’-UTR of TGF-β2, TGF-βRIII and E2F6.

In addition, we found that pancreatic cancer cells or the loaded-tumors of mice with low-level miR-193a failed to undergo repopulation after radiation.

Taken together, our findings indicated that miR-193a antagonist could effectively block pancreatic cancer repopulation, reduce spontaneous liver metastasis and prolong the survival through modulating TGF-β2/TGF-βRIII signalings.

Whilst, miR-193a intervention might help to improve pancreatic cancer radiotherapy.

Notably, the irradiated PDX group with miR-193a antagonist treatment shrank slowly after radiation, and failed to recover (Fig. 6a Lower & 6b).

On the other hand, miR-193a destroyed normal intercellular junctions through repressing TGF-β2/TGF-βRIII/ARHGEF15/ABL2 signaling, and caused the repopulated cancer cells to deviate from the primary sites and migrate for metastasis.

Fig. 7 miR-193a from the irradiated dying cells stimulates pancreatic cancer cell proliferation, repopulation and metastasis In terms of mechanisms, a complex network of cellular responses was evoked to the ionizing radiation damage [39].

d- g for detecting TGF-β2/TGF-βRIII/SMADs/E2F6/c-Myc signaling respectively in SW1990-EgmiR-193a and PANC-1-hU6miR-193a-IN cells (d), in SW1990-EgmiR-193a (±TGF-β2 cytokine, 10 ng/ml) and PANC-1-hU6miR-193a-IN cells (±TGF-β2 neutralization antibody, 2.5 μg/ml) (e), in stimulated SW1990 and PANC-1 reporter cells co-cultured with the corresponding feeder cells (±X-ray) (f), and in tumor tissues from burdened-mice (±X-ray) with SW1990-G2L cells (g)Accordingly, the 3’-UTR of TGF-β2, TGF-βRIII and E2F6, as well as their corresponding miR-193a -binding sequences (WT) and mutant counterparts (MT) were cloned into the reporter plasmids, and the relative luciferase activities were further assessed.

d PDX mice survival curve on X-ray radiation with miR-193a antagonist treatment, n = 11 In addition, there appeared a higher rate of spontaneous liver metastasis in PDX with radiation than the untreated group, and even reached 100% of liver metastasis.

The miR-193a -mediated loss-of-function for TGF-βRIII made TGF-β2 unable to combine with TGF-βRII, further inactivating TGF-β2 signaling.

d PDX mice survival curve on X-ray radiation with miR-193a antagonist treatment, n = 11In addition, there appeared a higher rate of spontaneous liver metastasis in PDX with radiation than the untreated group, and even reached 100% of liver metastasis.

microRNA-193a TGF-β2 TGF-βRIII Cancer repopulation Cancer metastasis Pancreatic cancer Pancreatic cancer is one of the most known malignancies with a five-year survival rate of ~ 5% [1].

We further explored the underlying mechanisms of miR-193a on pancreatic cancer cell proliferation and repopulation.

miR-193a promotes cancer repopulation through modulating TGF-β2/TGF-βRIII/SMADs/E2F6/c-Myc signaling.

These results showed that miR-193a antagonist treatment could block pancreatic cancer repopulation in PDX mo del.

Meanwhile, another group of the irradiated PDX mice were treated with miR-193a antagonist.

As described above, we found that miR-193a promoted pancreatic cancer repopulation after radiation.

We herein found that miR-193a, which increased in the irradiated cells, could accelerate cell cycle and destroy intercellular junctions to promote metastasis by modulating TGF-β2/TGF-βRIII signalings.

When tumor volume reached ~500mm [3], miR-193a antagonist (RiboBio, China) was used for intra-tumor injection of the irradiated mice.

b PDX tumor growth curve on X-ray radiation with miR-193a antagonist treatment.

Collectively, miR-193a was indispensable for pancreatic cancer repopulation in vitro.

Of note, the numbers of the metastatic nodules in the irradiated PDX group with miR-193a antagonist treatment (0 at average) were much less than that of the only irradiated one (> 100 at average, 36~ 254 in range), and even less than that of the untreated one (< 5 at average) (Fig. 6c, Upper right).

We found that higher miR-193a occurred in the stimulated SW1990-reporter cells co-cultured with the irradiated feeder cells than that of the controls (Fig.   2a, Upper).

We thought miR-193a might promote metastasis through repressing TGF-β2/TGF-βRIII/ARHGEF15/ABL2 signaling.

d- g for detecting TGF-β2/TGF-βRIII/SMADs/E2F6/c-Myc signaling respectively in SW1990-EgmiR-193a and PANC-1-hU6miR-193a-IN cells (d), in SW1990-EgmiR-193a (±TGF-β2 cytokine, 10 ng/ml) and PANC-1-hU6miR-193a-IN cells (±TGF-β2 neutralization antibody, 2.5 μg/ml) (e), in stimulated SW1990 and PANC-1 reporter cells co-cultured with the corresponding feeder cells (±X-ray) (f), and in tumor tissues from burdened-mice (±X-ray) with SW1990-G2L cells (g) Accordingly, the 3’-UTR of TGF-β2, TGF-βRIII and E2F6, as well as their corresponding miR-193a -binding sequences (WT) and mutant counterparts (MT) were cloned into the reporter plasmids, and the relative luciferase activities were further assessed.

Every mouse was injected 10 nmol miR-193a antagonist in 100 μl volume at twice one week for 4 weeks.

We wondered whether miR-193a could affect the intercellular junctions and further promote metastasis.

This coincidence might portend that miR-193a would mediate repopulation after radiation through modulating of TGF-β2/TGF-βRIII signaling.

To further explore the necessity of miR-193a in pancreatic cancer repopulation and metastasis, the feeder cells, PANC-1-hU6shR-NC and PANC-1-hU6miR-193a-IN, were treated with or without X-ray, and then co-cultured with their corresponding reporter cells.

At the last observation time-point, the tumor volume of the irradiated PDX with miR-193a antagonist treatment (190mm [3] at average) was far less than those of the untreated (1869mm [3] at average) and the irradiated (2161mm [3] at average) group, and even smaller than its volume before radiation (493mm [3] at average) (Fig. 6a & 6b).

293T cells were cultured in 96-well plates, and co -transfected with the reporter plasmids and miR-193a angomirs (RiboBio, China) using Lipofectamine 2000 (Invitrogen, USA).

We found that the dying cells from chemoradiation would release highly elevated miR-193a, which further blockaded TGF-β2/TGF-βRIII signalings and stimulated the surviving tumor cell proliferation, and eventually caused pancreatic cancer repopulation and promoted tumor metastasis.

Moreover, restoration of TGF-β2 signaling with cytokine could block miR-193a -induced repopulation in pancreatic cancer.

Together, we believed that miR-193 promoted cell proliferation and accelerated repopulation through TGF-β2/TGF-βRIII/SMADs/E2F6/c-Myc signaling.

These results indicated that miR-193a could accelerate pancreatic cancer cell cycle, and promote cell proliferation and repopulation in vitro and in vivo.

These data indicated that miR-193a antagonist in pancreatic cancer could repress metastasis after radiation, and improve the survival.

miR-193a destroys intercellular junctions and promotes metastasis through repressing TGF-β2/TGF-βRIII/ARHGEF15/ABL2 signaling.

We further explored the effects of miR-193a on pancreatic cancer cell proliferation in vivo.

We further explored whether miR-193a was necessary in pancreatic cancer repopulation and metastasis in vivo.

However, no tumor metastasis appeared in the irradiated PDX group with miR-193a antagonist treatment (Fig. 6c, Upper left & Lower).

3

The CT26 cells overexpressing miR-193a (Fig. 4c, left panel) have significantly downregulated Caprin1 mRNA expression (Fig. 4c, right panel) as well as Caprin1 protein expression (Fig. 4d).

To further determine whether our finding as described above can be translated into a clinical application, three upregulated (miR-193a, miR-126 and miR-148a) and one downregulated miRNA (miR-196b) found in the exosomes isolated from metastatic liver in a mouse colon cancer were analysed in samples from colon cancer patients by qPCR.

Oncosuppressor miR-193a directly targets Caprin1We further hypothesize that exporting tumour-suppressive miRNA such as miR-193a from exosome donor cells into exosomes is a benefit for colon cancer metastasis to the liver.

Our findings on the mechanism of sorting tumour suppressor miR-193a into exosomes have implications for other known tumour suppressor miRNAs, such as those listed in Table 2. Our findings also support future studies to determine whether tumour suppressor miRNAs have unique signal(s) recognized by the cellular complex.

The tumour suppression role of miR-193a was further supported by the fact that overexpression of miR-193a inhibited CT26 cell proliferation and significantly prolonged survival of colon cancer-bearing mice (Fig. 4g).

Inhibition of miR-193a expression reversed the effects of MVP KO on repressive expression of Caprin1, CCND2 and c-MYC (Fig. 5d), eventually leading to induction of cell growth (Fig. 5e).

In addition to miR-193a targeting of caprin-1, other potential molecules could be targeted by miR-193a, such as GTPase Rab27b, which regulates exosome biogenesis 49.

Knock down of miR-193a expression by miR-193a inhibitor enhanced tumour metastasis in liver and decreased survival of colon cancer-bearing mice (Fig. 5g, right panel).

Oncosuppressor miR-193a directly targets Caprin1.

The impact of miR-193a overexpression on the inhibition of cell proliferation was further confirmed by the Caprin1 siRNA knockdown in CT26 colon cancer cells (Fig. 4e).

miR-193a suppresses the progression of CT26 colon cancer by directly targeting Caprin1.

As summarized in Table 2, we noticed that most of the miRNAs (miR-10a-5p, miR-193a-3p, miR-200b-5p, miR-222-3p) that are actively sorted into exosomes have tumour suppressive effects involving cell growth suppression, whereas miRNAs (miR-196a/b, miR-181d-5p, miR-155-5p) that have oncogenic effects are retained in the tumour cells even though the levels of the oncogenic miRNAs are higher in their donor cells than in the exosomes.

Three public miRNA databases (TargetScan, Pictar and MicroRNA) all predicted that cell cycle -associated protein Caprin1 might be a target for miR-193a (Fig. 4a), and the 3′-UTR of Caprin1 contains a highly conserved binding site from position 2288 to 2309 for miR-193a (Fig. 4a,b).

Moreover, overexpression of anti-sense miR-193a (miRNA inhibitor) caused induction of luciferase; however, there was no inductive effect of the anti-sense miR-193a on the activity of the reporter with a mutant 3′UTR of Caprin1 (Fig. 4f).

The data presented in this study indicate that miR-193a targets the 3′UTR of Caprin-1 mRNA, leading to inhibition of production of caprin-1 protein.

Accumulation of miR-193a in tumour cells may also inhibit the release of exosomes via miR-193a-3p -mediated targeting of GTPase Rab27b, which has been reported by other groups 50.

However, we demonstrated that knockout of MVP in CT26 cells leads to miR-193a accumulation in the cells without affecting the levels of miR-126a or miR-148a, suggesting that MVP selectively targets miR-193a.

Therefore, it is speculated that accumulation of miR-193a in tumour cells by knockdown of MVP may prevent exosome release, thus contributing to inhibition of tumour progression as well.

This repression on tumour growth was partially reversed by knocking down the expression of miR-193a (Fig. 5h,i).

The downregulation of miR-193a in tumour tissue was stage -dependent (Fig. 6c, second panel from left).

To determine the ability of miR-193a to target the 3′ UTR- Caprin1 activity, a luciferase reporter containing 3,243 bp of the Caprin1 3′ UTR in the pEZX-MT01 vector was purchased from GeneCopoeia (Cat [#]: MmiT072744-MT06, Rockville, MD, USA).

The results generated from MVP gene overexpression in CT-26 cells suggest that the higher the level of MVP, the lower the level of miR-193a (Supplementary Fig. 7b,c).

The impact of miR-193a -mediated interruption of the caprin-1/G3BP-1/c-MYC/Cyclin D2 complex could be a potential target for anti-cancer therapeutic applications.

qPCR-quantification of mature miR-193a, MVP, Caprin1, CyclinD and c-MYC expressed in CT26 cells (left panel) and CT26 exosomes (right panel) after the cells were treated as indicated.

We found that higher levels of miR-193a in tumour cells cause cell cycle G1 arrest and cell proliferation repression through reduction of caprin-1 expression.

The normalized luciferase activity of transfected control mimic miRNA was set as a relative luciferase activity of 1. (g) Survival of BALB/c mice after intrasplenic injection of CT26 cells with miR-193a overexpression (n=6 mice per group).

Higher levels of exosomal miR-193a lead to more aggressive disease.

We first searched miRNA databases for potential miR-193a targets that may contribute or promote tumour progression.

To determine whether miR-193a could target Caprin1 in colon cancer cells, we transfected the mature mouse miR-193a mimic into CT26 cells.

qPCR analysis of miR-193a, miR-126a and miR-148a expression in colon cancer tissue and adjacent non-tumour tissue from the same patients (right panel).

Since higher levels of exosomal miR-193a in the peripheral blood of colon cancer patients is correlated with more aggressive disease, the level of circulating miR-193a in A33 [+] exosomes can be quantitatively analysed with qPCR.

Our study demonstrates that accumulation of miR-193a in the tumour cell leads to inhibition of tumour growth.

As summarized in Fig. 7, MVP binds to tumour suppressor miR-193a, forming an MVP protein-miR-193a complex.

Expression of miR-193a (b), miR-18a (c) and miR-21 (d) in the exosomes and exosome donor tissues, including primary colon cancer and liver metastasis of colon cancer, were assessed by qPCR.

These results demonstrate that Caprin1 is a target of miR-193a in colon cancer cells.

MVP KO in CT26 colon tumour cells resulted in the inhibition of liver metastasis of colon cancer (Fig. 5f, middle and right panels), and this result correlates with an increase in miR-193a in CT26 cells and a decrease in miR-193a in exosomes (Fig. 5g, left panel).

We further hypothesize that exporting tumour-suppressive miRNA such as miR-193a from exosome donor cells into exosomes is a benefit for colon cancer metastasis to the liver.

This finding was further demonstrated by reverse transcription-quantitative PCR (RT-qPCR) analysis of tumour-suppressive miR-193a (Fig. 2b), miR-18a (Fig. 2c) and oncogenic miR-21 (Fig. 2d), as an example.

As proof of concept, we used tumour suppressor miR-193a to demonstrate this phenomenon.

Overexpression of miR-193a decreased the luciferase activity of the reporter with the 3′UTR of Caprin1 by approximately 56% in CT26 cells (Fig. 4f).

Collectively, these data suggest that MVP regulates miR-193a sorting into exosomes and that the accumulation of miR-193a in the exosomal donor cells as a result of KO MVP is detrimental to tumour cells.

We found that CCND2 and c-MYC, which are regulated by Caprin1, are also decreased as a result of miR-193a treatment (Fig. 4c,d).

MVP regulates the loading of miR-193a to exosomes.

However, the mutation that disrupted the binding site for miR-193a entirely restored luciferase activity.

The fact that MVP knockout causes miR-193a accumulation in cells instead of exosomes further supports this finding.

To ascertain the direct effect of miR-193a on Caprin1, a mutant construct that would disrupt the predicted miR-193a binding site was generated from pEZX-MT01-Caprin1 containing a full length 3′UTR of Caprin1 mRNA (Gene Accession: NM_001111289).

CT26 cells transfected with MVP knock out (KO) MVP sgRNA CRISPR Lentivirus (>1 × 10 [7] IU ml [−1]) have low levels of MVP detected (Fig. 5c,d) but the level of miR-193a is increased in the cells (Fig. 5c, left panel).

Different concentrations of MVP were washed with buffer (PBS, 50 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 0.1 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM sodium fluoride, 10 mg ml [−1] phenylmethylsulfonyl fluoride, 2 μM aprotinin, 100 mM sodium orthovanadate) and were incubated with 2 μg of mature miR-193a in immunoprecipitation buffer (150 mM NaCl, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA (pH 8.0), 0.2 mM sodium ortho-vanadate, 0.2 mM PMSF, 1% Triton X-100, 0.5% NP-40) for 2 h at 4 °C.

Our results indicate that the level of miR-18a and miR-193a in the exosomes from either primary colon tumour tissue or metastatic liver of colon tumour is higher than in their donor tumour tissues.

Next, to determine whether MVP interacts with miR-193a, immunoprecipitation of MVP was carried out.

A typical staining pattern of the Bio-miR-193a complex obtained from CT26 exosomal extracts on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is shown in Fig. 5a, left panel.

The level of miR-193a in the tumour was lower than in adjacent tissue in all three stages of colon cancer.

The results generated from qPCR show that miR-126a, miR-148a and miR-193a are significantly higher in the exosomes released from metastatic CT26 cells and circulating in the peripheral blood of metastatic colon cancer in the liver, but not from primary colon cancer or subcutaneous xenografts.

Caprin1-pEZX-MT01 (100 ng) or mutant luciferase reporter (100 ng) were transfected using FuGENE HD Transfection Reagent (Roche Applied Science, Indianapolis, IN, USA) with 10 pmol of mimic mmu-miR-193a and Opti-MEM Reduced Serum Medium (Invitrogen, Carlsbad, CA, USA).

Proposed mo del for the mechanism of colon cancer metastasis to the liver involves exporting miR-193a via exosomes sorted by MVP.

The qPCR results indicate that MVP interacts with miR-193a in an MVP immunocomplex dose -dependent manner (Supplementary Fig. 7a).

To further confirm the interaction of MVP and miR-193a, MVP was pulled down from CT26 cell lysates using anti-MVP antibody (Protein Tech) and protein G beads (ThermoFisher Scientific).

To test our hypothesis, biotin -labelled miR-193a complex was isolated from exosomal lysates using streptavidin beads.

The miR-193a seed matches in the Caprin1 3′UTR are mutated at the positions as indicated.

Cell cycle assessment suggested that miR-193a causes a G1 phase arrest in the cell cycle (Fig. 4h,i).

Subsequently, this complex is packed into exosomes leading to the reduction of cytoplasmic miR-193a.

Sorting of miR-193a from cell to exosomes through major vault protein (MVP).

This prospective study indicated that colon cancer patients with high levels of miR-193a in exosomes from peripheral blood have a higher risk of metastasis (Fig. 6b).

Collectively, these data suggest that miR-193a sorting into exosomes is MVP -dependent.

In this study, we demonstrated that MVP transports miR-193a from tumour cells to exosomes.

How to cite this article: Teng, Y. et al. MVP -mediated exosomal sorting of miR-193a promotes colon cancer progression.

The miR-193a/MVP complex was pulled-down, and the miR-193a remaining in the supernatant was collected for qPCR analysis.

In-gel digestion-MALDI-TOF mass spectrometry (MS) analysis was carried out for identification of proteins that are specifically present in the Bio-miR-193a complex sample but not in the control bio-miRNA complex.

Analysis of miR-193a levels in tumours by qPCR indicated that MVP KO caused the accumulation of miR-193a in cells (Fig. 5i, left panel), with a concomitant decrease of miR-193a in the exosomes (Fig. 5i, right panel).

Therefore, exosomal miR-193a may be used as a biomarker for predicting progression of colon cancer.

By contrast, reduction of miR-193a by MVP -dependent sorting into exosomes leads to tumour cell proliferation and to a faster cell cycle, eventually enhancing tumour cell growth and metastasis.

Induction of exosome miR-193a in peripheral blood increases the risk of metastatic colon cancer in the liver of patients.

The MVP -mediated promotion of tumour progression through miR-193a is also demonstrated by subcutaneous injection of human colon cancer SW620 cells into nude mice (Fig. 5h).

This finding is inversely correlated with the levels of miR-193a in the exosomes (Fig. 5c, right panel).

The accumulation of miRNA in the MVP KO CT26 cells is miR-193a-specific since no change of miR-126a was observed due to MVP KO (Supplementary Fig. 6b).

The results generated from a mouse colon cancer mo del with liver metastasis further supports that miR-193a exported via exosomes by MVP promotes tumour progression (Fig. 5f).

Major vault protein (MVP) was subsequently identified as a potential miR-193a binding protein by MS (Fig. 5a, right panel), and this interaction was verified by western blot (Fig. 5b, Supplementary Fig. 6a).

4

In the present study, we verified that MCL1 silencing suppressed GC cells proliferation, and we further demonstrated that miR-193a-3p overexpression inhibited GC tumor growth by directly targetting at MCL1.

So far, we have demonstrated that up-regulation of LINC00152 suppressed miR-193a-3p, contributing to enhancement of MCL1 expression and afterward, promoting proliferation of GC cells.

Figure 7LINC00152 enhanced MCL1 expression by down -regulating miR-193a-3p(A) The mRNA expression levels of MCL1 when LINC00152 and miR-193a-3p were differentially expressed.

Figure 5 MiR-193a-3p inhibited GC cells proliferation in vitro(A) qRT-PCR revealed that the expression of miR-193a-3p was increased in the mimics group and decreased in the inhibitor group; linc00152 could reverse the increased miR-193-3p level of mimics group in AGS and BGC-823 cells, respectively.

LINC00152 enhanced MCL1 expression by down -regulating miR-193a-3p To further investigate that whether MCL1 expression was regulated by LINC00152 through miR-193a-3p, qRT-PCR, and Western blot were conducted to analyze the mRNA and protein expression of MCL1 in linc00152 or miR-193a-3p transfected cells.

It has been shown that down-regulation of miR-193-3p inhibits proliferation, migration, and chemotherapy resistance of tumor cells in GC through regulating PTEN gene [13].

Besides, previous work has confirmed that the transfection of miR-193a-3p decreases the expression of MCL1, and MCL1 is a direct target of miR-193a-3p [6].

In this work, we worked over the relationship between LINC00152 expression and the cell multiplication of GC, and we verified both in vivo and in vitro that LINC00152 promoted gastric cells proliferation by regulating miR-193a-3p and its target gene MCL1.

Figure 4LINC00152/ miR-193a-3p or miR-193a-3p/ MCL1 had direct interaction(A) TargetScan prediction revealed that linc00152-wt could directly bind with miR-193a-3p.

The following groups were set: negative control (NC) group to be the group transfected with pcDNA3.1 empty plasmid vector, mimics group to be the group transfected with both miR-193a-3p mimics and pcDNA3.1 empty plasmid vector, inhibitor group to be the group transfected with both miR-193a-3p inhibitor and pcDNA3.1 empty plasmid vector, LINC00152 + mimics (mix) group to be the group transfected with miR-193a-3p mimics + pcDNA3.1-LINC00152.

Besides, cells in both the miR-193a-3p inhibitor group and LINC00152 group showed a higher MCL1 expression than in the control group (Figure 7A).

LINC00152 relieved cells proliferation inhibition induced by miR-193a-3p To investigate the influence of miR-193a-3p on GC cells proliferation and the negative regulation effect of LINC00152 on miR-193-3p, we transfected mimics NC, miR-193a-3p mimics, miR-193a-3p inhibitor, miR-193a-3p mimics, and pcDNA3.1-LINC00152 into AGS and BGC-823 cells, respectively.

LINC00152 enhanced MCL1 expression by down -regulating miR-193a-3p.

The colony formation assay revealed that the GC cells proliferation was suppressed after transfection with miR-193a-3p mimics, and the suppression was reversed by LINC00152 (P<0.01, Figure 5B).

It was indicated that the target binding of linc00152-mut and miR-193a-3p was reduced (P<0.01, Figure 4B), and the target binding of MCL1-mut and miR-193a-3p was also reduced (P<0.01, Figure 4D), which was evidenced by significantly higher luciferase activity in mut groups compared with wt groups.

Amongst the five groups, the expression of MCL1 was lowest in the miR-193a-3p mimics groups, while MCL1 expression was enhanced in linc00152 and miR-193a-3p co -transfected groups compared with the mimics groups (P<0.05).

To further investigate that whether MCL1 expression was regulated by LINC00152 through miR-193a-3p, qRT-PCR, and Western blot were conducted to analyze the mRNA and protein expression of MCL1 in linc00152 or miR-193a-3p transfected cells.

Collectively, our study showed LINC00152 might function as an oncogenic lncRNA to drive cancer development through LINC00152/ miR-193a-3p/ MCL1 pathway and have great potential to become a promising biomarker and therapeutic target in GC.

LINC00152 enhanced MCL1 expression by down -regulating miR-193a-3p.

Besides, the FCM analysis indicated that miR-193a-3p overexpression significantly promoted the arrest of both cell lines in G [1]/G [0] phase, with an obvious decrease in the number of S-phase cells (P<0.05).

LINC00152 relieved cells proliferation inhibition induced by miR-193a-3p.

The interaction between LINC00152/ miR-193a-3p and miR-193a-3p/ MCL1 TargetScan prediction output revealed that LINC00152 might contain a binding site for miR-193a-3p (Figure 4A), and miR-193a-3p might contain a binding site for MCL1 (Figure 4C).

Thus, the inhibition of MCL1 given by miR-193a-3p was again confirmed by comparing the mix group to the LINC00152 group (P<0.01).

Besides, miR-193a-3p was identified as an antiproliferative miRNA inhibiting endothelial colony forming cells (ECFC)-derived cell proliferation [29].

Moreover, the overexpressed LINC00152 increased MCL1 level, while the silenced LINC00152 increased miR-193-3p level.

That was to say, LINC00152 could relieve cells proliferation inhibition induced by miR-193a-3p.

Expression levels of LINC00152, miR-193a-3p, and MCL1 were determined by qRT-PCR.

Finally, we came to a conclusion that LINC00152 overexpression promoted GC cells proliferation by LINC00152/ miR-193a-3p/ MCL1 pathway.

MiR-193a-3p mimics, miR-193a-3p inhibitor, and pcDNA3.1 plasmid vectors were purchased from GenePharma (Shanghai, China).

TargetScan prediction output revealed that LINC00152 might contain a binding site for miR-193a-3p (Figure 4A), and miR-193a-3p might contain a binding site for MCL1 (Figure 4C).

Researchers have demonstrated that miR-193a-3p serves as a neoplasm suppressor in lung cancer [26, 27] and colorectal cancer [28].

The lower expression of miR-193a-3p in the mix group compared with the mimics group validated that LINC00152 negatively regulated miR-193a-3p (P<0.01, Figure 5A).

Taken together, these data suggested that LINC00152 could directly interact with miR-193a-3p, and miR-193a-3p could directly interact with MCL1 in GC cells.

To investigate the influence of miR-193a-3p on GC cells proliferation and the negative regulation effect of LINC00152 on miR-193-3p, we transfected mimics NC, miR-193a-3p mimics, miR-193a-3p inhibitor, miR-193a-3p mimics, and pcDNA3.1-LINC00152 into AGS and BGC-823 cells, respectively.

MiR-193a-3p inhibited GC cells proliferation in vitro.

LINC00152/ miR-193a-3p or miR-193a-3p/ MCL1 had direct interaction.

MCL1 promoted GC cells proliferationNow that we have validated the direct interaction between MCL1 and miR-193a-3p, we further confirmed the effect of MCL1 on GC cells by transfecting NC, MCL1, sh- MCL1 into AGS and BGC-823 cells, respectively.

Now that we have validated the direct interaction between MCL1 and miR-193a-3p, we further confirmed the effect of MCL1 on GC cells by transfecting NC, MCL1, sh- MCL1 into AGS and BGC-823 cells, respectively.

In our study, a direct relationship between LINC00152 and miR-193a-3p was demonstrated.

In summary, our study first proposed the LINC00152/ miR-193a-3p/ MCL1 pathway involved in GC tumor development, and indicated the critical role of LINC00152 in GC cell proliferation.

MiR-193-3p could also negatively regulate MCL1 level (P<0.01, Figure 4E).

By the same method, MCL1-WT and MCL1-mut sequences were inserted into AGS and BGC-823 cells by the pGL3 plasmid, co -transfected with miR-193a-3p mimics or mimics NC.

Furthermore, an interesting discovery is that LINC00152 works as a competing endogenous RNA (ceRNA) through sponging miR-193a-3p and shares the identical responsive elements of miR-193a-3p with some signaling pathway factor like erb-b2 receptor tyrosine kinase 4 (ERBB4) [14].

The interaction between LINC00152/ miR-193a-3p and miR-193a-3p/ MCL1.

Furthermore, miR-193a-3p notably reduced proliferation and viability of GC cells, which was consistent with the results given by Pekow et al. [30].

Meanwhile, Lipofectamine 3000 transfection reagent (Life Technologies, U. S. A. ) was used to co-transfect miR-193a-3p mimics or scrambles (mimics NC) into AGS or BGC-823 cells.

5

Yu T Li J Yan M Liu L Lin H Zhao F Sun L Zhang Y Cui Y Zhang F Li J He X Yao M MicroRNA-193a-3p and-5p suppress the metastasis of human non-small-cell lung cancer by downregulating the ERBB4/PIK3R3/mTOR/S6K2 signaling pathwayOncogene 2014 ᅟ ᅟ 28.

Therefore, the miR-193a-3p’s impact on the BCa chemoresistance is significantly achieved by its targeting repression of both LOXL4 and SRSF2 expression.

A full length of the human LOXL4 3’-untranslated region (1325 bp) with a wide type and mutant target sequence for miR-193a-3p were cloned into 3’ flank of luciferase coding sequence of pGL3 (Invitrogen, Carlsbad, CA, USA) to construct pGL3-luc-LOXL4 WT and pGL3-luc-LOXL4 Mut, respectively.

Chemically modified mimic oligonucleotides (agomir) were synthesized to regulate miR-193a-3p/5p expression in vivo.

To conclude that LOXL4 gene is a direct target of miR-193a-3p, we put the wild type or mutant 3′-UTR region (1325 bp) at the downstream of the firefly luciferase gene of pGL3 vector (Promega) to create pGL3-LOXL4 UTR WT and the PGL3-LOXL4 UTR Mut, respectively (Figure 3D).

Therefore, the miR-193a-3p mediated regulation at the post-transcriptional levels likely has a predominant role in the LOXL4 expression in BCa multi-chemoresistance (Figure 3A and B).

Getting all these together, LOXL4 is indeed, a direct target of miR-193a-3p and may execute the miR-193a-3p’s effect on the BCa chemoresistance.

LOXL4 mRNA is a direct target of miR-193a-3p in BCa cells.

We further demonstrated that lysyl oxidase-like 4 (LOXL4) gene [GenBank: NM_032211.6] is a direct target of miR-193a-3p and executes the former’s impact on bladder cancer chemoresistance.

In the present work, LOXL4 as another direct target of miR-193a-3p has been shown having an active role in an implementation of the miR-193a-3p’s positive impact on the BCa chemoresistance (Figures 1, 2 and 3) to great extent.

Figure 3 The LOXL4 is a direct target of miR-193a-3p in BCa cells.

We show here that miR-193a-3p promotes the BCa multi-drug resistance phenotype via its repression of the lysyl oxidase-like 4 (LOXL4) gene, a newly identified direct target of miR-193a-3p.

Importantly, we showed for the first time that miR-193a-3p can positively regulates the OS pathway, most likely achieved by its repressing effect on the LOXL4 expression (Figure 5B) A network analysis to link the LOXL4 gene to the OS pathway was performed by combining the literature mining with an analysis of the public protein-protein interaction databases: Wiki-PI [http://severus.

Besides SRSF2 [29], miR-193a-3p’s influence on the cancer chemoresistance is expected to be accomplished via repression of its other targets.

D, The sequences (the wild-type in shadow and mutant in red) in UTR region of LOXL4 gene targeted by miR-193a-3p.

For example, miR-193a-3p was shown to promote in vivo tumorigenesis of metastatic medullary thyroid carcinoma [28], and to enhance both tumor growth in nude mice and chemoresistance of HCC by targeting of the SRSF2 gene [29].

LOXL4 is one of several dozen genes that differentially expresses in a pattern opposite to the miR-193a-3p’s.

It is clear that further efforts are needed to address how the miR-193a-3p affected the OS pathway through its repression of the LOXL4 expression in the content of the BCa chemoresistance.

In this study, we demonstrated that miR-193a-3p promotes the multi-chemoresistance of BCa via repressing of the LOXL4 expression and therefore activating the OS pathway.

In addition to a new mechanistic insight, our results provide a set of the essential genes in this newly identified miR-193a-3p/LOXL4/Oxidative Stress axis as the diagnostic targets for a guided anti-bladder cancer chemotherapy.

miR-193a-3p promotes Pa chemoresistance of BCa viarepressing both SRSF2 and LOXL4 expression in BCa tumor xenografts in nude mice.

miR-193a-3p promotes Pa chemoresistance of BCa viarepressing both SRSF2 and LOXL4 expression in BCa tumor xenografts in nude miceTo minimize the inter-mouse bias, 5637 (1.5 × 10 [7] cells/site) or H-bc (0.7 × 10 [7] cells/site) cells were subcutaneously injected at two back sites of six mice each.

We also show here that the Oxidative stress (OS) pathway is the predominant pathway affected by miR-193a-3p via its repression of LOXL4 expression.

Figure 1 The miR-193a-3p expression differs in the chemoresistance distinct bladder cancer cell lines.

This study also provided a new set of genes in this newly identified miR-193a-3p/LOXL4/Oxidative Stress axis as the diagnostic targets for the guided anti-bladder cancer chemotherapy, including the level of the miR-193a-3p gene, both LOXL4 and SRSF2 gene and the key OS pathway associated genes in both cancer tissues and urine sediments.

Both constructs and pGL3 were transfected into 5637 and H-bc cells respectively, to determine whether the chemoresistance associated expression of miR-193a-3p in BCa cells is indeed functional.

The first indication that miR-193a-3p might be involved in tumor suppression was the observation that it was silenced by DNA methylation during oral carcinogenesis [12].

MiR-193a-3p also induces an accumulation of intracellular reactive oxygen species (ROS) and DNA damage in cancer cells via targeting Mcl-1 [26].

Dysregulation of miR-193a-3p was also reported in other types of cancer, such as non-small cell lung cancer (NSCLC) [13], prostate cancer [14], breast cancer [15], Head and Neck Squamous Cell Carcinomas [16], and colorectal cancer [17].

F, The level of the OS pathway related mRNA s in the miR-193a-3p antagomiR versus the NC transfected H-bc cells.

From the 4 [th] day after cell injection, all 5637 generated tumors on the left back of nude mice were intratumorally injected with 2nM miR-193a-3p agomiR/Mock, while H-bc generated tumors on the left back of nude mice were injected with 4nM miR-193a-3p/Mock antagomiR.

Figure 5 The effects of the forced reversal of both miR-193a-3p and LOXL4 levels on the activity of the signaling pathways in 5637 versus H-bc cells.

Indeed, along with a rise of miR-193a-3p level and reduction of the LOXL4 level in 5637 cells, the drug-triggered cell death was rescued (Figure 5).

Figure 4 The effects of a forced reversal of the miR-193a-3p or LOXL4 levels on the chemoresistance of 5637 and H-bc cells.

LOXL4 miR-193a-3p Chemoresistance Bladder cancer Oxidative stress pathway Bladder cancer (BCa) is the second most frequent type of cancer in male around the world [1].

An intratumor injection of miR-193a-3p agomiR in comparison with the mock into the 5637 cell derived tumor xenograft resulted in a bigger tumor mass of 5637 cell derived tumor xenografts (0.81/0.53 = 1.53).

E, The relative luciferase activity (fold) of the reporter with wild-type (WT) or mutant (Mut) LOXL4-UTR or with no UTR (Vec) were determined in the miR-193a-3p mimic (in 5637) or antagomir (in H-bc) or Mock transfected BCa cells.

The carcinogenic impact of miR-193a-3p has been attributed to its repression of c-Kit [18] and the PTEN/PI3K signaling pathway in acute myeloid leukemia [19], of KRAS and PLAU in colon cancer [20], of PLAU [21] and EGFR -driven cell-cycle network proteins [22] in breast cancer, of ARHGAP19, CCND1, ERBB4, KRAS and Mcl-1 in epithelial ovarian cancer [23], of PLAU in hepatocellular carcinoma (HCC) [24], and of Mcl-1 in NSCLC [25].

The intratumor injection of either miR-193a-3p’s agomiR (into 5637)/antagomiR (into H-bc tumor) indeed led the expected changes of both SRSF2 and LOXL4 proteins in tumor tissues (Figure 6E), and consolidate the conclusion that miR-193a-3p promotes both the growth and chemoresistance of the BCa cell derived tumor xenografts in nude mice.

Although no or marginal effect on the activities of the DNA damage, NF-κB and Myc/Max pathways was observed, both OS and Notch pathways were activated in the LOXL4 siRNA transfected to a similar extent observed in the mimic transfected 5637 cells (Figure 5B) with the OS pathway’s response most drastically, Therefore, the LOXL4’s role to relay the miR-193a-3p’s effect on the BCa chemoresistance is principally accomplished via its effect on the OS pathway.

We further determined the LOXL4 level in both miR-193a-3p mimic transfected 5637 and the antagomiR transfected H-bc cells versus the mock transfected.

In our previous study, we showed that SRSF2, as a key executor of the miR-193a-3p’s impact on the 5-FU resistance in HCC [29].

The activities of these three pathways were reduced in the miR-193a-3p mimic transfected 5637 (Figure 5B) and elevated in the antagomiR transfected H-bc cells (Figure 5C), indicating a negative association of which with the BCa chemoresistance.

Therefore, the effect on the OS pathway (Figure 5) by miR-193a-3p via repression of LOXL4 seems to be produced by other unknown mechanism(s).

The level of miR-193a-3p (A), the LOXL4 mRNA l (B) and protein (C) in the miR-193a-3p mimic (3 PM) transfected 5637 cells and the miR-193a-3p antagomiR (3PA) transfected H-bc cells versus the negative control (NC), determined by qRT-PCR or Western analyses.

From the detailed studies of a multi-chemosensitive (5637) versus a chemoresistant (H-bc) bladder cancer cell lines, we showed that miR-193a-3p [GenBank: NR_029710.1] promotes the multi-chemoresistance of bladder cancer cells.

Despite this difference, LOXL4 indeed plays an essential part in the miR-193a-3p’s effect on the multi-chemoresistance of BCa cells, except for the Ad resistance.

As expected, a miR-193a-3p antagomiR transfection raised the mRNA level of LOXL4 by over 37 folds (Figure 3B) and the protein level by 84% in H-bc cells (Figure 3C).

Coincidently, the Nrf1, Nrf2, NQO1 and SUV39H1 mRNAs were higher in H-bc than 5637 cells (Figure 5D), and were raised in the miR-193a-3p mimic and LOXL4 siRNA transfected 5637 cells (Figure 5E), while repressed in the miR-193a-3p antagomiR transfected H-bc cells (Figure 5F).

Revealed by both a sequencing based miRomic analysis (not shown) and the qRT-PCR validation, the miR-193a-3p level was over 100 folds higher in both H-bc and UM-UC-3 than in 5637 cells (Figure 1B and C).

We have shown that LOXL4 mediated the miR-193a-3p’s chemoresistance effects to four drugs, but not the Ad’s (Figure 4A).

B, The relative pathway activities in the LOXL4 siRNA or miR-193a-3p mimic (3 PM) versus the NC transfected 5637 cells.

In parallel with the changes of the miR-193a-3p level (Figure 3A), a miR-193a-3p mimic transfection brought down the LOXL4 mRNA level by nearly 70% (Figure 3B) and the protein level by 58% (Figure 3C) in 5637 cells.

Essentially as expected, a miR-193a-3p antagomiR transfection sensitized H-bc cells to the cell death triggered by four drugs, excluding Ci (Figure 4D).

However, we found no evidence for that the force reversal of both miR-193-3p and SRSF2/LOXL4 levels in 5637 and H-bc cells have a detectable effect on the level of Nrf2 and its phosphorylated form as well as its nuclear-cytoplasmic distribution (data not shown).

In contrast, several studies reported an oncogenic role for miR-193a-3p.

The miR-193a-3p level was higher in the chemoresistant (H-bc and UM-UC-3) than the chemosensitive (5637) BCa cell lines.

All these observations suggest that miR-193a-3p may have a promoting role in the BCa chemoresistance as previously reported in the 5-FU resistance of HCC [29].

With half less cells injected, the H-bc derived tumors were significantly heavier than 5637 derived (0.81 g/0.53 g = 1.53 in tumor weight) (Figure 6C and D), suggesting a miR-193a’s promoting role in the in vivo tumor growth.

F, The effects of the forced reversal of both miR-193a-3p and LOXL4 levels on the apoptosis by FACS analysis of 5637 cells in plot and in the original.

From the day 4 [th], PBS, the miR-193a-3p agomiR/antagomiR or the scramble sequence control (Mock) was intratumourly injected into a pair of 5637/H-bc tumors on right back site (NB, the other pair of tumor was injected with the reagents that has nor relevance in this study), respectively, once every two days for 5 times as indicated.

The relative miR-193a-3p level (fold) in BCa cell lines by both miR-seq and qRT-PCR analyses is shown in Table (B) and by qRT-PCR in plot (C).

An intratumor injection of miR-193a-3p agomiR/antagomiR into the 5637/H-bc derived tumors on the left back of mice was initiated on the 4 [th] day and repeated four times once in two days.

C, The relative pathway activities in the miR-193a-3p antagomiR (3PA) versus the NC transfected H-bc cells.

It is surprising to find a significant discrepancy between the mRNA level (by qRT-PCR) and protein level (by Western blotting analysis) of SRSF2 and LOXL4 altered by miR-193-3p mimic or antagomiR, the alteration of the protein level is far limited than that of mRNA.

A siRNA mediated LOXL4 repression essentially reproduced the miR-193a-3p mimic’s effect on the chemoresistant state of 5637 cells.

A, The IC [50] dosed drug-triggered cell death of 5637 cells transfected by miR-193a-3p mimic (3 PM) or the gene specific siRNAs versus the negative control (NC).

D, The drug-triggered cell death of the miR-193a-3p antagomiR (3PA) versus the NC transfected H-bc cells.

Therefore, the miR-193a-3p is capable to promote the in vivo BCa tumor growth in nude mice.

Figure 6 The miR-193a-3p’s effect on both the in vivo growth and Pa chemoresistance of 5637 and H-bc derived xenografts in nude mice.

The cell death triggered by all the five drugs was significantly reduced in the miR-193a-3p mimic transfected 5637 cells (Figure 4A).

E, The level of the OS pathway related mRNAs in the miR-193a-3p mimic or siRNA versus the NC transfected 5637 cell.

These observations suggest that LOXL4 and SRSF2 have significantly overlapped but distinguished roles in execution of the miR-193a-3p’s impact on the BCa chemoresistance in a drug-specific fashion.

The Oxidative Stress pathway activity is drastically affected by a forced reversal of miR-193a-3p or LOXL4 levels in cell and may act at the downstream of LOXL4 gene to relay the miR-193a-3p’s impact on the multi-chemoresistance in both cultured cells and the tumor xenografts in nude mice.

6

Based on the results of quantitative real-time PCR (qPCR) assays, the expression of miR-193a, which is embedded in a CpG island, was down-regulated in the IGF-1-stimulated group and negatively correlated with c-kit expression, whereas c-kit -positive CSCs infected with lentivirus carrying micro -RNA193a displayed reduced c-kit expression, migration and proliferation.

miR-193a expression was downregulated in the IGF-1-stimulated group and negatively correlated with c-kit expression.

In addition, miR-193a targeted the 3′-UTR of the c-kit mRNA to downregulate c-kit expression in c-kit -positive mouse CSCs.

IGF-1 upregulated c-kit expression in c-kit -positive CSCs resulting in enhanced CSC proliferation and migration by activating the PI3K/AKT/DNMT signaling pathway to epigenetically silence miR-193a, which negatively modifies the c-kit expression level.

The expression of miR-193a was downregulated in the IGF-1-stimulated group and negatively correlated with c-kit expression.

IGF-1 upregulated c-kit expression in c-kit -positive CSCs, resulting in enhanced CSC proliferation and migration, by activating the PI3K/AKT/DNMT signaling pathway to epigenetically silence miR-193a, which negatively modifies the c-kit expression level.

Although the role of miR-193a in c-kit -positive CSCs was unclear, we hypothesized that IGF-1/PI3K/AKT -induced DNMT activation would epigenetically silence miR-193a expression, eventually leading to c-kit upregulation in CSCs.

In addition, IGF-1 -mediated CSC migration and proliferation were also inhibited by miR-193a overexpression (Fig.   5f, g; P < 0.05).

In our study, the expression of miR-193a and c-kit was decreased by infection with a miR-193 -expressing lentivirus or 5-AZA treatment.

Primary c-kit -positive CSCs were infected with a lentivirus expressing pLVmicro -RNA193a or pLV-miR-Ctrl, and the infection efficiency was determined by measuring the expression of green fluorescent protein (GFP) using a fluorescence microscope (Additional file 2: Figure S1) and a qPCR analysis of miR-193a expression in infected cells (Additional file 2: Figure S2).

Moreover, miR-193a expression was altered following the inhibition of the PI3K/AKT pathway.

Based on the qPCR results, the IGF-1 treatment downregulated the expression of miR-193a compared with the control group (Fig.   5a; P < 0.01).

e CSCs were infected with a control lentivirus or a lentivirus expressing miR-193a and stimulated with PBS or 100 ng/ml IGF-1 for 72 h. Western blots were performed to analyze the expression of the c-kit and GAPDH proteins.

However, the IGF-1 -induced increase in c-kit expression was significantly blunted by miR-193a overexpression (Fig.   5e; P < 0.05).

We revealed the involvement of DNA methylation and the role of miR-193a in regulating gene expression, and the results verify that IGF-1 improved the proliferation of c-kit -positive CSCs as a potential treatment for cardiac regeneration.

Isolated CSCs were treated with IGF-1, IGF-1 + 5-AZA, or 5-AZA alone to examine whether DNMTs regulated miR-193a expression.

According to the results of the qPCR assay, miR-193a expression was upregulated in the IGF-1 + 5-AZA group compared with the IGF-1 group (Fig.   5d; P < 0.05).

MicroRNA-193a represses c-kit expression and functions as a methylation-silenced tumor suppressor in acute myeloid leukemia.

MiR-193a has been shown to function as a methylation-silenced tumor suppressor in acute myeloid leukemia that represses c-kit expression [14].

a qPCR was performed to analyze the expression of miR-193a.

Our study is the first to show that miR-193a negatively changed c-kit expression is modulated by DNMT activity.

qPCR analysis showing the upregulation of miR193a in infected cells (3.14-fold) compared with control group.

Activated DNMTs then repressed miR-193a expression through promoter hypermethylation.

qPCR was performed to analyze miR-193a expression.

The lentiviral pGIPZ plasmid expressing miR-193a and a negative control miRNA were designed and synthesized by Genechem (Shanghai, China) and named pLV-micro -RNA193a and pLV-miR-Ctrl, respectively.

However, the mechanism underlying this regulatory action of IGF-1 on c-kit -positive resident CSCs through miR-193a remains unknown.

Thus, we aimed to clarify the underlying mechanism by which IGF-1 regulates c-kit -positive CSCs, and especially how miR-193a engaged in this process.

For the 3′-UTR-Renilla luciferase reporter assay, 293 T cells were seeded in 24-well plates at a density of 5.0 × 10 [4] cells/well and co -transfected with a reporter construct (psiCHECK2-ache-3′-UTR-wt or psiCHECK2-ache-3′-UTR-mut) and a miR-193a expression plasmid using the FuGENE HD transfection reagent (Roche Applied Science, Indianapolis, IN, USA) [14].

Hence, we measured miR-193a expression in cells with or without IGF-1 treatment.

Subsequently, CSCs were infected with a control lentivirus or a lentivirus carrying miR-193a with or without IGF-1 stimulation.

We analyzed the methylation status of CpGs in the has-miR-193a promoter c-kit -positive CSC control group or the group treated with IGF-1 using bisulfite genome sequencing.

According to several studies, miR-193a is embedded in a CpG island, and hypermethylation of the miR-193a promoter results in its epigenetic silencing in AML [14, 20], non-small cell lung cancer [32], and ovarian cancer [22].

d Bisulfite pyrosequencing analysis of the DNA methylation status of the miR-193a promoter region in c-kit positive CSCs.

The relative gene expression levels were calculated by normalization to the GAPDH mRNA level, whereas the miR-193a expression level was calculated by normalization to the U6 small nuclear RNA levels.

In previous studies, miR-193a has been shown to be embedded in a CpG island and epigenetically repressed by promoter hypermethylation in leukemia cell lines, primary acute myeloid leukemia (AML) blasts, and ovarian cancer stem cells [14, 22].

Infection with miR-193a lentivirus.

Furthermore, the miR-193a level was reported to be negatively correlated with c-kit levels in these cells [17].

Subsequently, luciferase activity was measured in 293 T cells co -transfected with a reporter construct (psiCHECK2-ache-3′-UTR-wt or psiCHECK2-ache-3′-UTR-mut) and a miR-193a expression plasmid.

7

Intriguingly, we found that upregulation of miR-193a two weeks after disease induction was followed by a reduction in Wt1 expression in week 4 (Fig 9).

In kidneys of Nphs2 [R140Q/-] and control animals one, two and four weeks after induction, miRNA21 expression persistently increased during the first four weeks of disease whereas miRNA-193a expression was increased by 100% within one week after induction and reached a peak after two weeks (Fig 8).

miR-193a was found highly upregulated in glomeruli isolated from FSGS patients and it has been shown that its overexpression in an inducible mouse mo del led to focal and later global glomerulosclerosis with broad capsular adhesion [15, 16].

To investigate possible post-transcriptional regulation of gene expression in our mo del, we searched for differences in the expression of miRNA-21 and miRNA-193a, previously described to be involved in chronic kidney failure [15– 21].

We observed an early and transient upregulation of miR-193a in our mo del, supporting the notion that this miRNA is an important player in the sequence of molecular events leading to FSGS in hereditary podocytopathies caused by functional deficiency of a single podocyte protein.

The major target of miR-193a is WT1, which plays an essential role in the development and maintenance of podocytes [16, 46, 47].

Repression of WT1 upon upregulation of miR-193a initiates a cascade of events resulting in structural destabilization and disrupted function of podocytes, leading to progressive FSGS [16].

In order to elucidate key molecular responses to the podocin mutated state, we chose to study the expression of miR-193a and miR-21 based on a review of the recent literature.

The analysis of miRNA-21 (5'- UAGCUUAUCAGACUGAUGUUGA-3') and miRNA-193a (5'- AACUGGCCUACAAAGUCCCAGU-3') was performed by qPCR by CBC (Comprehensive Biomarker Center GmbH, Hei delberg, Germany).

8

All three miRNAs have previously been found to be differentially regulated in MS lesions: miR-146a and miR-193a were upregulated in active MS lesions, whereas miR-181b was found to be down regulated in inactive MS lesions (8).

Based on a microarray analysis followed by verification with qPCR, we identified three miRNAs, miR-146a, miR-181b, and miR-193a that were differentially expressed in response to CPZ exposure.

By contrast, the expression level of miR-193a and miR-181b decreased in response to CPZ -induced demyelination and had returned to baseline in the full remyelination phase (p < 0.001 and p < 0.01, respectively, one-way ANOVA, LSD post hoc test) (Figures 1B,C).

Only one recent study examined miRNA expression during CPZ -induced demyelination by microarray, but miRNAs were only evaluated in sorted CNPase-EGFP [+] cells with an OPC phenotype; only part of the differentially expressed miRNAs were presented, and those did not show miR-181b, miR-146a, or miR-193a (46).

In summary, here we used a comprehensive and unbiased approach to identify three miRNAs, miR-146a, miR-181b, and miR-193a, which were differentially regulated in the corpus callosum in response to CPZ exposure.

We identified three miRNAs, miR-146a, miR-181b, and miR-193a, which were differentially expressed compared to controls confirmed by qPCR (Figure 1).

Using microarray and validation by quantitative PCR (qPCR), three miRNAs, miR-146a (A), miR-193a (B) and miR-181b (C) were differentially regulated in response to CPZ exposure in the corpus callosum.

9

The high expression of miR-122 and miR-705 combined with the downregulation of miR-193 and miR-27a might decrease the levels of fatty acid synthesis and lipid metabolism, which might be important in inhibiting the growth and development of S. japonicum in the host environment.

Some differentially expressed miRNAs in liver had important functions, such as involvement in nutrient metabolism, including a cholesterol metabolism regulator (miR-122), a lipid metabolism regulator (miR-705), an adipocyte differentiation and regulation factor (miR-27a and miR-193), and erythrocyte differentiation (miR-223 and miR-451).

miR-193 is a key regulator in adipogenesis [35], and miR-27a is known as an important negative regulator of adipocyte differentiation [36].

Among them, miR-122, miR-705, miR-193 and miR-27a were related to nutrient metabolism.

10

Some of the results are in accordance with previous studies, such as the up-regulation of mmu-miR-221 and mmu-miR222 cluster and the down-regulation of the mmu-miR-200 family, as well as of mmu-miR-204, mmu-miR-30a*, mmu-miR-193, mmu-miR-378 and mmu-miR-30e*.

On the contrary, the following miRNAs were down-regulated in WAT after HFD feeding: mmu-miR-141, mmu-miR-200a, mmu-miR-200b, mmu-miR-200c, mmu-miR-122, mmu-miR-204, mmu-miR-133b, mmu-miR-1, mmu-miR-30a*, mmu-miR-130a, mmu-miR-192, mmu-miR-193a-3p, mmu-miR-203, mmu-miR-378 and mmu-miR-30e*.

The down-regulation of mmu-miR-30a*, mmu-miR-30e*, mmu-miR-193 and mmu-miR-378 during HFD -induced obesity is consistent with previous studies [19], [37], [50].

The following 22 murine microRNAs were selected for qPCR validation of their expression: mmu-miR-1, mmu-miR-21, mmu-miR-30a*, mmu-miR-30e*, mmu-miR-122, mmu-miR-130a, mmu-miR-133b, mmu-miR-141, mmu-miR-142-3p, mmu-miR-142-5p, mmu-miR-146a, mmu-miR-146b, mmu-miR-192, mmu-miR-193a-3p, mmu-miR-200b, mmu-miR-200c, mmu-miR-203, mmu-miR-204, mmu-miR-222, mmu-miR-342-3p, mmu-miR-378 and mmu-miR-379.

11

Stable ectopic expression of RUNX1-MTG8, CBFB-MYH11, or miR-17 in U937 cells (a representative U937 clone is shown for each construct) leads to downregulation of miR-193a, a RUNX1-regulated miRNA targeting KIT (left), and miR-27a, a RUNX1-regulated miRNA involved in myeloid differentiation (right).

For instance, as shown in Additional file 1: Figure S2, left, miR-193a is significantly downregulated in U937 [miR-17], U937 [RUNX1-MTG8], and U937 [CBFB-MYH11] clones.

12

According to the literatures [26, 27], two truncated fragments of ING5 (aa 1-184 and aa 107-226) can induce cellular senescence and S arrest by down -regulating Cyclin E and Cdk2 expression, while microRNA-193 was found to have pro-proliferation effects for bone mesenchymal stem cells after low-level laser irradiation treatment by targeting ING5 to regulate Cdk2 activity.

Recently, miR-331-3p and miR-193a-3p might suppress ING5 expression to increase cell proliferation and decrease multi- chemoresistance respectively [12, 16].

13

While the cluster miR-193–365 is up-regulated by Prdm16, partially through Pparγ [14], they are not required for brown fat development and function [24].

Inhibition of miR-193a/b and/or miR-365 in mouse primary brown pre-adipocytes impairs brown adipogenesis in vitro [14].

Homeobox C9 (HOXC9) is predicted to be targeted by miR-193, 150 and -26b.

14

miRNA expression of miR-193a (A) and miR-15b (B) on lung fibroblasts from cell lines of healthy controls (HC) and patients with SSc-ILD stimulated for 18 hours with media, IL-13, TGF-beta, and IFN-alpha.

There was mild expression of miR-193a and miR-15b in lung fibroblasts in both groups after all three stimuli (Additional file 7: Figure S2A and B, respectively).

Only a few miRNA from the selected group had validated target genes: miR-155, miR-21, miR-125a, miR-31, miR-205, miR-20a, let-7f, miR-182, let-7e, miR-199b, miR-199a, miR-663, and miR-193a.

15

Indeed, by cloning the identified miR-18a binding region in Malat1 into a luciferase reporter vector, we found that transfection of precursor oligos of miR-181a (pre-181a) but not a negative control or an irrelevant miR-193a downregulated the reporter activity (Figure 6b); moreover, mutating the predicted binding site (Figure 6a, Mutant) abolished this targeting effect (Figure 6b), suggesting that indeed miR-181a directly targeted Malat1 through the above-identified site.

16

Moreover, transgenic expression of miR-193a in mice induces focal segmental glomerulosclerosis (FSGS) with downregulation of WT1, and miR-193a is overexpressed in the podocytes of patients with FSGS [17].

17

Reimplantation in the presence or absence of a miR-193a-5p antagomir of disaggregated cells from primary mouse SCCs generated by a chemical carcinogenesis protocol revealed that knockdown of this miR resulted in reduced tumor formation and enhanced chemosensitivity [23], indicating that a strategy targeting both ΔNp63 α and miR-193a-5p might be more effective in this scenario.

TAp73 and ΔNp63 α are engaged in a feedback loop involving miR-193a-5p, which is repressed by ΔNp63 α and activated by TAp73 and targets the p73 UTR.

Cisplatin treatment results in ΔNp63 α degradation and TAp73 -mediated activation of miR-193a-5p, limiting TAp73's pro-apoptotic effects and chemosensitivity [23].

18

For example, miR-146b-5p and miR-378a-3p were highly expressed in mouse but they were present at a low level of expression in bovine; inversely, miR-199b-5p, miR-423-5p and miR-193a-5p were weakly expressed in mouse but highly in bovine (Table S2).

19

miR-522, miR-139-3p, miR-520c-5p, miR-518d-5p, miR-146b-5p, miR-34a, miR-526a, miR-193a-3p, miR-221, miR-4674 were significantly upregulated and miR-760 was downregulated in ECSCs (Figure 2A).

20

Among these miRNAs, 3 were downregulated (miR-193, miR-30b and miR-29c) and 2 were upregulated (miR-199a-3p and miR-199a-5p) (Figure 9B).

21

miR-193, -30b, -30c, -26a, and -26b are highly expressed during early development, gestation and late involution; miR-141, -200a, -148a, and -146b are highly expressed during gestation, lactation, and early and late involution.

22

In contrast, only one miRNA, hsa-miR-193, was down-regulated in DMD.

miRNA Fold Change P-value mmu-miR-337-5p −5.2 0.0149 mmu-miR-3544-3p −5.1 0.0147 mmu-miR-540-5p −4.9 0.0200mmu-miR-337-3p [a] −3.0 0.0324mmu-miR-3544-5p [a] −3.0 0.0308 mmu-miR-434-3p −2.1 0.0001 mmu-miR-3071-5p −2.0 0.0004mmu-miR-136-3p [a] −2.0 0.0004mmu-miR-3071-3p [a] −1.6 0.0000 mmu-miR-136-5p −1.6 0.0000 mmu-miR-143-5p −1.2 0.0004 mmu-miR-190a-5p −1.0 0.0139 mmu-miR-872-3p −0.9 0.0152 mmu-miR-193a-3p −0.9 0.0164 mmu-miR-144-3p −0.8 0.0298 mmu-miR-127-3p −0.7 0. 0002mmu-miR-434-5p [a] −0.6 0.0082 mmu-miR-148a-3p −0.6 0.0130 mmu-miR-411-5p −0.6 0.0091 a miRNA* (passenger) strand processed from opposite arm of the mature miRNA.

23

For instance, among the 66 uniformly expressed miRNAs for which IPA assigned functions, we identified 12 candidates that have been implicated in androgen regulation, including: let-7a-5p, miR-15a-5p, miR-17-5p, miR-19b-3p, miR-23a-3p, miR-24-3p, miR-27b-3p, miR-30a-5p, miR-34a-5p, miR-140-5p, miR-193a-3p, miR-205-5p (S1 Fig).

24

Gebeshuber CA Focal segmental glomerulosclerosis is induced by microRNA-193a and its downregulation of WT1Nat.

25

Furthermore, this group reported consistent downregulation of miR-193a-3p and changes in miRPlus-E1245 levels that were specific to XMRV infection across four different cell types (DU145, LNCaP, peripheral blood lymphocytes, and monocyte-derived macrophages).

26

In particular, we identified significant indirect negative correlations between the glutamate receptor signalling and neurogenesis modules with expression indices of miR-193, miR-200, and the miR-34 families (Pearson’s correlation) (Supplementary Fig.   5 and Supplementary Table  3).

27

Focal segmental glomerulosclerosis is induced by microRNA-193a and its downregulation of WT1.

28

Finally, we profiled microRNA gene expression along the same process, leading to the identification of known general adipogenic microRNAs (e. g. miR-378), brown lineage-specific microRNAs (miR-193), as well as several microRNAs not implicated in adipogenesis so far (S5 Fig and S2 Table).

For example, the members of the bone morphogenetic protein (BMP) family, the PPARγ co-factor PGC1α, the transcription factors (TFs) PRDM16, EBF2, KLF11, the protein deacetylase SIRT1, the secreted factors IRISIN and FGF21 as well as lncRNAs Blnc1, lncBATE1, and microRNAs miR193/365 have been shown to be essential for thermogenic fat cell recruitment [6, 10– 13].

29

Lv L Deng H Li Y Zhang C Liu X Liu Q Zhang D Wang L Pu Y Zhang H He Y Wang Y Yu Y Yu T Zhu J The DNA methylation-regulated miR-193a-3p dictates the multi-chemoresistance of bladder cancer via repression of SRSF2/PLAU/HIC2 expressionCell Death Dis.

30

miRanda algorithm showed that, activin receptor 1 (ACVR1) is predicted target gene for mmu-mir-193, mmu-mir-294, mmu-mir-295 and mmu-mir132.

31

miR-431, miR-714, miR-744, miR-877, miR-130b, miR-21, miR-323-3p, miR-325, miR-409-3p, miR-154*, and miR-681 were significantly increased 4 days post-sciatic nerve crush in pre-conditioned DRGs, while miR-190, miR-1, miR-33, miR-32, miR-153, miR-335-5p, miR-193, and miR-488 showed significantly decreased expression.

32

Interestingly, in the “early” and “late” response phases, Ets1 and Nfat5, are targeted by only a single miRNA, i. e., miR-193 and miR-494, respectively.

33

Interestingly, hsa-let-7e, miR-146a, and miR-193a were over-expressed in both LPS and MEF lysate exposed PBMCs, indicating that they might represent the common miRs in DAMP- and PAMP -mediated inflammation.

34

MicroRNAs targeting MDM2: miR-192, miR-194, miR-215, miR-221, miR-605, miR-17-3p, miR-193a, miR-25, miR-32, miR-143, miR-145, miR-18b, miR-661 [reviewed in Ref.

35

However, the expression of the remaining 9 miRNAs (miR-411, miR-434-3p, miR-299*, miR-193, miR-146b, miR-379, miR-193b, miR-22, and miR-223) has not been verified.

36

Several miRNAs, such as miR-33a, miR-26a, miR-193, miR-221/222 and let-7, are regulated by metformin in breast, pancreatic, and lung cancer cells [25– 29].

37

Next, we validated all seven miRNAs in three independent experiments, which confirmed protection against APAP by all seven miRNA mimics, except miR-193a-3p (Supplementary Fig. 1A).

We found seven miRNAs fulfilling these two criteria: miR-194-5p, miR-125b-5p, miR-21-5p, let-7a-5p, miR-122-5p, miR-30c-5p and miR-193a-3p (Fig. 1c).

38

adj ssc-miR-21 -1.1788 1.45E-02 1.68E-02 -2.4642 2.07E-04 3.85E-04 ssc-miR-143-3p -1.1940 1.40E-02 1.67E-02 -2.7004 2.27E-05 5.34E-05 ssc-miR-145-3p -1.2289 2.47E-02 2.68E-02 -2.6837 6.34E-04 1.10E-03 ssc-miR-505 -1.3657 2.68E-02 2.82E-02 -2.1577 4.16E-02 4.16E-02 ssc-miR-98 -1.5185 3.46E-03 5.15E-03 -2.8061 7.55E-05 1.55E-04 ssc-miR-139-3p -1.6685 2. 54E-02 2.71E-02 -2.5158 1.69E-02 1.93E-02 ssc-miR-23b -1.7157 3.70E-03 5.42E-03 -2.3687 8.39E-03 1.10E-02 ssc-miR-224 -1.8515 1.41E-02 1.67E-02 -2.5778 1.95E-02 2.19E-02 ssc-miR-23a -1.8753 3.40E-03 5.15E-03 -2.4676 1.00E-02 1.24E-02 ssc-miR-143-5p -1.9243 1.15E-04 2.60E-04 -3.9943 1.25E-09 5.88E-09 ssc-miR-139-5p -2.1198 2.01E-02 2.24E-02 -3. 2644 1.01E-02 1.24E-02 ssc-miR-222 -2.2666 2.58E-07 1.02E-06 -2.6019 2.34E-05 5.35E-05 ssc-miR-671-5p -2.3068 1.15E-02 1.47E-02 -2.7986 3.86E-02 3.92E-02 ssc-miR-9843-3p -2.3507 9.68E-04 1.87E-03 -4.7281 5.90E-05 1.31E-04 ssc-miR-145-5p -2.7059 2.08E-03 3.50E-03 -4.3459 7.18E-05 1.51E-04 ssc-miR-221-5p -2.7136 3.21E-07 1.21E-06 -1.9513 3.02E-02 3. 22E-02 ssc-miR-221-3p -2.9643 8.31E-11 5.47E-10 -2.1967 1.74E-03 2.90E-03 ssc-miR-708-5p -4.0615 2.31E-06 7.60E-06 -2.8238 6.43E-03 8.72E-03 ssc-miR-193a-3p -4.1933 2.39E-07 1.02E-06 -4.3848 2.87E-07 9.18E-07 ssc-miR-193a-5p -4.1933 2.39E-07 1.02E-06 -7.1423 2.32E-12 1.33E-11 ssc-miR-452 -4.3025 5.55E-11 3.99E-10 -2.2057 1.53E-02 1.77E-02 ssc-miR-206 -5.3001 6. 39E-09 3.37E-08 -6.2200 3.10E-09 1.38E-08 10.1371/journal.

adj ssc-miR-21 -1.1788 1.45E-02 1.68E-02 -2.4642 2.07E-04 3.85E-04 ssc-miR-143-3p -1.1940 1.40E-02 1.67E-02 -2.7004 2.27E-05 5.34E-05 ssc-miR-145-3p -1.2289 2.47E-02 2.68E-02 -2.6837 6.34E-04 1.10E-03 ssc-miR-505 -1.3657 2.68E-02 2.82E-02 -2.1577 4.16E-02 4.16E-02 ssc-miR-98 -1.5185 3.46E-03 5.15E-03 -2.8061 7.55E-05 1.55E-04 ssc-miR-139-3p -1.6685 2. 54E-02 2.71E-02 -2.5158 1.69E-02 1.93E-02 ssc-miR-23b -1.7157 3.70E-03 5.42E-03 -2.3687 8.39E-03 1.10E-02 ssc-miR-224 -1.8515 1.41E-02 1.67E-02 -2.5778 1.95E-02 2.19E-02 ssc-miR-23a -1.8753 3.40E-03 5.15E-03 -2.4676 1.00E-02 1.24E-02 ssc-miR-143-5p -1.9243 1.15E-04 2.60E-04 -3.9943 1.25E-09 5.88E-09 ssc-miR-139-5p -2.1198 2.01E-02 2.24E-02 -3. 2644 1.01E-02 1.24E-02 ssc-miR-222 -2.2666 2.58E-07 1.02E-06 -2.6019 2.34E-05 5.35E-05 ssc-miR-671-5p -2.3068 1.15E-02 1.47E-02 -2.7986 3.86E-02 3.92E-02 ssc-miR-9843-3p -2.3507 9.68E-04 1.87E-03 -4.7281 5.90E-05 1.31E-04 ssc-miR-145-5p -2.7059 2.08E-03 3.50E-03 -4.3459 7.18E-05 1.51E-04 ssc-miR-221-5p -2.7136 3.21E-07 1.21E-06 -1.9513 3.02E-02 3. 22E-02 ssc-miR-221-3p -2.9643 8.31E-11 5.47E-10 -2.1967 1.74E-03 2.90E-03 ssc-miR-708-5p -4.0615 2.31E-06 7.60E-06 -2.8238 6.43E-03 8.72E-03 ssc-miR-193a-3p -4.1933 2.39E-07 1.02E-06 -4.3848 2.87E-07 9.18E-07 ssc-miR-193a-5p -4.1933 2.39E-07 1.02E-06 -7.1423 2.32E-12 1.33E-11 ssc-miR-452 -4.3025 5.55E-11 3.99E-10 -2.2057 1.53E-02 1.77E-02 ssc-miR-206 -5.3001 6. 39E-09 3.37E-08 -6.2200 3.10E-09 1.38E-08 10.1371/journal.

39

Roy S. Benz F. Vargas Cardenas D. Vucur M. Gautheron J. Schneider A. Hellerbrand C. Pottier N. Alder J. Tacke F. miR-30c and miR-193 are a part of the TGF-β -dependent regulatory network controlling extracellular matrix genes in liver fibrosis J. Dig.

40

miR-30c and miR-193 are a part of the TGF-β -dependent regulatory network controlling extracellular matrix genes in liver fibrosis.

41

In addition to miR-122-5p, the miRNA species miR-22, miR-29b, miR-29c, miR-130a and miR-193 were increased in both mice and humans.

In rats, in addition to miR-122-5p, the increase of miR-22, miR-193 and miR-194 was in accordance with our human data 23.

42

Similar observations were made for miR-124, miR-137, miR-193, miR-210, miR-2, miR-79 and miR87 across species, with miRNAs following the loop-counting rule having lower arm abundances of 5′-isomiRs.

Besides shifted seeds, many well-conserved 5′-isomiRs with the same or nearly identical seed regions had different arm abundances among the four species, exemplified by miR-124, miR-193, miR-210, miR-2 and miR-87 (Table 3).

43

The impact of non-coding RNA, especially miRNA, should not be ignored; e. g., miR-137 or miR-193a, as predicted by miRWalk (http://www.

44

031hsa-miR-6362.70.0052hsa-miR-301a2.80.032hsa-miR-172.00.0057hsa-miR-2173.90.033hsa-miR-449b3.20.0069hsa-miR-6522.00.039hsa-miR-130b2.20.0082hsa-miR-1861.50.039hsa-miR-1922.50.0095hsa-miR-19a1.80.040hsa-miR-4483.10.010hsa-miR-8721.50.041hsa-miR-4253.00.011hsa-miR-148b1.80.043hsa-miR-193a-3p2.60.011hsa-miR-4512.40.

45

Although it has been previously demonstrated that the microinjection of a single microRNA can both alter the molecular makeup of the embryo and induce phenotypic outcomes in adult offspring (eg microRNA-221/222 [38]; microRNA-1 [37]; microRNA-124 [39]; microRNA-193-5p (partial phenotype) [41]).

46

This analysis also identified three mouse miRNA homologues: miR-193, miR-10 and miR-200, within the top five most abundant secreted miRNAs.

47

2 mmu-miR-204* mmu-let-7b mmu-miR-30c mmu-miR-500* mmu-miR-219-3p mmu-miR-362-3p mmu-miR-455* mmu-miR-3098-5p mmu-miR-193 DEmiRNAs in bold were confirmed using.

48

Functions of the other three miRNAs, miR149, miR193 and miR466a-3p, have not been previously examined in the intestinal epithelium in vitro or in vivo.

49

99E-0249mmu-miR-185-5pmir-1850.198.912.60E-033.38E-0241mmu-miR-191-5pmir-1910.199.711.25E-031.94E-0228mmu-miR-193-3pmir-1930.256.553.70E-048.12E-0329mmu-miR-1952mir-19520.219.183.70E-048.12E-0372mmu-miR-1961mir-19610.196.581.03E-029.11E-0234mmu-miR-204-5pmir-2040.317.746.87E-041.29E-0226mmu-miR-222-3pmir-2210.2310.222.37E-045.81E-0335mmu-miR-221-3pmir-2210.259.098.47E-041.

50

Plasma miR-22, miR-101b, miR-122, miR -133a, miR135a*, miR-192, miR193 and miR486 have been shown to be affected by liver damage [39].

51

These are mir-26b, mir-29a, mir-34b, mir-92-1, mir-93, mir-133a-1, mir-133a-2, mir-193, mir-221, mir-223, mir-301, mir-323 and mir-346.

52

The expression of mature miRNAs was assayed using TaqMan MicroRNA Assays (Applied Biosystems, Foster City, CA) specific for hsa-miR-486 (ID 001278), hsa/mmu-miR-21a (ID 000397), hsa-miR-455 (ID 001280), hsa-miR-151-3p (ID 002254), mmu-miR-1a (ID 002222), mmu-miR-133b (ID 002247), mmu-miR-5128 (ID 462199_mat), mmu-miR-223 (ID 002295), mmu-miR-146b (ID 001097), mmu-miR-133a (ID 001637), mmu-miR-449a (ID 001030), mmu-miR-122 (ID 002245), mmu-miR-351-3p (ID 464446_mat), mmu-miR-193a-5p (ID 002577), mmu-miR-151-3p (ID 001190), mmu-miR-574-3p (ID 002349), mmu-miR-3107/486 (ID 001278).