12 publications mentioning gga-mir-7b

Open access articles that are associated with the species Gallus gallus and mention the gene name mir-7b. Click the [+] symbols to view sentences that include the gene name, or the word cloud on the right for a summary.

1

Overlay of the miR-7 predicted target genes with down-regulated genes with a functional annotation in neovascularization led to selection of OGT as putative novel target of miR-7. OGT is an enzyme that is involved in the hexosamine biosynthetic pathway which adds an O-GlcNAc moiety to the free hydroxyl group of select serine and threonine residues on a diverse population of nuclear and cytosolic proteins [35, 36].

Both siRNA against OGT and Alloxan did not inhibit EC migration or EC tube formation in vitro, suggesting that OGT is a good marker gene of miR-7, but not the prime miR-7 target through which EC cell migration and tube formation are inhibited (Supplementary Fig. S3 and S4).

The majority, 1317, of the miR-7 modulated genes were down-regulated while 1183 were upregulated.

These data suggest that inhibition of angiogenesis is the prime mechanism for the N2A tumor growth suppression upon intratumoral delivery of miR-7. The lack of efficacy on tumor cell proliferation in vivo is corroborated by the observation that miR-7 did not inhibit cell viability of N2A cells in vitro (Supplementary Fig. S5).

Furthermore, not only tumor vessel density was reduced, expression of the nuclear proliferation marker Ki-67 and expression of the newly identified miR-7 target gene OGT were also affected by miR-7 treatment in vivo.

Differentially expressed genes that are regulated by miR-7 and are a predicted target genes of miR-7 are plotted.

Similar to EC, downregulation of OGT in U-87 MG cells by RNAi interference did not inhibit cell viability (Supplementary Fig. S9), indicating that also in tumor cells OGT is a good marker for miR-7 delivery but not for miR-7 efficacy.

First, the set of regulated genes was overlaid with the 1051 predicted miR-7 targets, based on the TargetScan prediction tool in IPA.

It is noted that other miR-7 targets, such as EGFR and PI3K were not down-regulated in miR-7 treated EC based on the RNA-seq analysis (Supplementary Table S7), suggesting that the anti-proliferative activity of miR-7 occurs through other pathways in EC than in tumor cells.

In total 282 predicted miR-7 targets were up- or downregulated within the set of 2500 genes (Fig. 2b).

Here we show for the first time that OGT is a target of miR-7, suggesting that the anti-angiogenic effect of miR-7 in EC can, at least partly, be mediated by downregulation of OGT.

Anti-angiogenic activity of miR-7 mimic in vivo– systemic tumor neovasculature targetingClinical application of miRNA -based therapeutics is dependent on systemic administration and intracellular delivery of the miRNA (mimic) to the target site.

Investigation of gene expression levels of known miR-7 target shows that EGFR and PI3K and most of the other known miR-7 targets are not affected in HUVEC after miR-7 transfection (Supplementary Fig S10).

Eight out of 11 genes that are associated to novel anti-angiogenic drugs are down regulated Genes highly involved in tumor angiogenesis Expression ratio after miR-7 treatment versus control (log2(ratio)) Example of drug in clinical trial VEGF-B 1.443 Aflibercept (Regeneron) VEGF-C −0.778 VGX-100 (Circadian) Angiopoeitin-2 −0.805 PF-04856884 (Pfizer) PDGF-D 0.899 CR-002 (CuraGen) Jagged-1 −0.739 RO4929097 (Roche) ADAM-10 −0.870 INCB3619 (InCyte) FGF-2 −1.732 Gal-F2 (Galaxy/Roche) CXCR4 1.169 BMS-936564 (Bristoll Myers Squibb) S1PR1 −1.108 Fingolimod (Novartis) S1PR3 −1.035 Fingolimod (Novartis) NRP1 −0.801 MNRP1685A (Genentech) antibodyIPA was also used to identify biological processes that are regulated by miR-7 (Supplementary Table S3).

Differential expression analysis showed that 2500 genes were significantly up- or downregulated after transfection of HUVEC with miR-7 mimic (p-value < 0.05, compared to miR-Scr, Fig. 2a).

However, inhibition of OGT in EC does not inhibit EC tube formation or migration, which indicates that OGT is a suitable biomarker for miR-7 activity, but does not explain the anti-angiogenic activity of miR-7 in EC.

The dual targeting of the cRGD-coated nanoparticles to both endothelial as well as cancer cells provides potential for high therapeutic efficacy of miR-7. Together with the well-known role of miR-7 as regulator of invasion and migration in cancer, the novel anti-angiogenic property of miR-7 and its regulation of diverse genes involved in angiogenesis strengthen its potential value as therapeutic agent for the treatment of cancer Human Umbilical Vein Endothelial Cells (HUVEC) (Lonza) were cultured in EBM-2 medium (Lonza) supplemented with bullet kit (EGM-2, Lonza) containing several growth factors and 10% Fetal Calf Serum (FCS) (Sigma).

Eight out of 11 genes that are associated to novel anti-angiogenic drugs are down regulated Genes highly involved in tumor angiogenesis Expression ratio after miR-7 treatment versus control (log2(ratio)) Example of drug in clinical trial VEGF-B 1.443 Aflibercept (Regeneron) VEGF-C −0.778 VGX-100 (Circadian) Angiopoeitin-2 −0.805 PF-04856884 (Pfizer) PDGF-D 0.899 CR-002 (CuraGen) Jagged-1 −0.739 RO4929097 (Roche) ADAM-10 −0.870 INCB3619 (InCyte) FGF-2 −1.732 Gal-F2 (Galaxy/Roche) CXCR4 1.169 BMS-936564 (Bristoll Myers Squibb) S1PR1 −1.108 Fingolimod (Novartis) S1PR3 −1.035 Fingolimod (Novartis) NRP1 −0.801 MNRP1685A (Genentech) antibody IPA was also used to identify biological processes that are regulated by miR-7 (Supplementary Table S3).

The results demonstrate that targeted systemic delivery of miR-7 inhibited tumor angiogenesis and growth.

miR-7 mediated differential expression of genes that are currently clinically explored as anti-angiogenic drug targets.

After transfection of HUVEC with miR-7 mimic cell proliferation was inhibited in a concentration dependent manner, up to 50% inhibition compared to a negative control miRNA with a scrambled sequence (miR-Scr).

Indirect evidence for systemic delivery of miR-7 to the tumor tissue was provided by the observation that mice treated with the miR-7 mimic -loaded, integrin -targeted nanoparticles had smaller, pale and less vascularized tumors than control mice.

Not only did miR-7 inhibit growth factor induced angiogenesis in vitro but miR-7 also impaired developmental angiogenesis in chicken embryo CAM with potency comparable to sunitinib.

MiR-7 not only inhibited EC proliferation by 50%, but also inhibited migration of EC by nearly 70%.

Downregulation of OGT by miR-7 is not EC specific, but was also confirmed in U-87 MG cells in vitro (Supplementary Fig. S8).

In earlier studies, miR-7 was not picked up as miRNA involved in angiogenesis in an EC specific differential expression screen (13), which may suggest that miR-7 has no physiological role in EC to regulate angiogenesis.

The observed downregulation of OGT in miR-7 transfected HUVEC was confirmed by RT-PCR and Western Blot analysis over a concentration range of 15-100 nM (Fig. 2c and d).

Overlay of strongly down-regulated angiogenesis -associated genes (log2 ratio (miR-7 vs.

Figure 4(a) miR-7 inhibits tumor growth after local delivery.

miR-Scr), p-value <0.05) with predicted miR-7 targets (Supplementary Table S4) pointed to O-linked β-N-acetylglucosamine transferase (OGT) as potentially important mediator of miR-7 action.

Together, these studies demonstrate strong anti-angiogenic activity of miR-7 upon overexpression in EC.

U-87 MG tumor bearing mice were treated with miR-7 mimic using a cyclic Arginine-Glycine-Aspartic acid (cRGD) peptide coupled biodegradable polyamide nanoparticles, targeting integrins αvβ3 and αvβ5.

The observed inhibition of tumor growth can thus be ascribed to a combination of an anti-angiogenic effect of miR-7 delivered to tumor -associated EC and an anti-proliferative effect of miR-7 delivered to tumor cells.

The log2(ratio) was calculated as described in Material and Methods and reflects the differential expression of genes in HUVEC treated with miR-7 and gene expression in HUVEC treated with miR-Scr.

The U-87 MG xenograft tumor mo del was selected because it is highly vascularized and responds well to anti-angiogenic therapy such as bevacizumab or sunitinib [40, 41], making it a suitable mo del to study the anti-angiogenic property of miR-7. Naturally, this mo del does not reflect the nature of glioblastomas in the clinic and recently it was shown that angiogenesis inhibition in glioblastoma patients may induce glioblastoma migration and invasion [42, 43].

To explain the anti-angiogenic properties of miR-7, gene expression analysis was performed using RNA-seq.

Indeed, miR-7 inhibited wound closure while miR-Scr transfected cells did not (Fig. 1h).

This is indicative of a strong anti-angiogenic activity of miR-7. This was supported by the observation that treatment of CAM with a clinically approved multikinase anti-angiogenic drug, sunitinib, showed a similar inhibitory effect on vascularization.

Athymic Nude-Foxn1 [nu] mice bearing U-87 MG tumors were injected intravenously with αvβ3/αvβ5 targeted miR-7 nanoparticles (3 mg/kg miRNA).

Indeed, miR-7 inhibits proliferation of not only HUVEC, but also of U-87 MG cells in vitro.

Anti-angiogenic activity of miR-7 mimic in vivo– systemic tumor neovasculature targeting.

This suggests that inhibition of miR-7 mediated tumor growth is caused by a combined effect on both tumor cells and tumor EC.

miR-7 modifies endothelial gene expression.

To confirm effective delivery of miR-7 into the tumor tissue we performed IHC staining of OGT, one of the target genes of miR-7 (Fig. 5g).

Inhibitory effect of miR-7 on tumor growth by local delivery.

In a functional screen with a lentiviral miRNA library miR-7 was identified as inhibitor of EC proliferation.

Figure 2(a) miR-7 changes the expression of 2500 HUVEC genes after transfection.

Our observation that miR-7 inhibits proliferation of U-87 MG cells confirms the anti-tumorigenic effect of miR-7 in tumor cells.

Delivery of miR-7 using this novel formulation demonstrated inhibition of tumor growth in a human glioblastoma xenograft mo del.

Inhibitory effect of miR-7 on tumor growth by systemic delivery.

Figure 1Anti-angiogenic property of miR-7 in vitro(a) miR-7 inhibits HUVEC cell viability.

The decrease in luciferase activity in the presence of miR-7 indicates a direct interaction between miR-7 and the 3′UTR of OGT (Fig. 2e).

This study has identified miR-7 as a prominent regulator of angiogenesis.

The mechanism through which miR-7 regulates distinct pathways in EC and tumor cells will be subject of future research.

To proof that OGT is a direct target gene of miR-7, we measured luciferase activity in Hela cells transfected with a plasmid containing the 3′UTR sequence of OGT.

Eight out of 11 genes that are associated to novel anti-angiogenic drugs are down regulated Mature miR-7 is conserved between chicken, human, and mouse (Fig. 3a).

In more complex in vitro angiogenesis assays, miR-7 inhibited the ability of HUVEC to form two-dimensional tubules on matrigel and three-dimensional sprouts in collagen.

Tumors of the miR-7 treatment group were pale and less vascularized compared to those in control groups (Fig. 5a) and systemic delivery of miR-7 mimic inhibited tumor growth by 42% after two weeks of treatment (Fig. 5b).

miR-7 treated mice showed significant tumor growth inhibition compared to vehicle treated mice.

Caspase-3 cleavage in HUVEC treated with miR-7 suggests that part of the anti-proliferative activity results in cell apoptosis (Supplementary Fig. S2b).

Tumor treatment with intratumoral injection of miRNA delivered by electroporation showed that the miR-7 mimic decreased angiogenesis and reduced tumor growth.

Therefore, this approach was selected for systemic delivery of miR-7. Hereto, a novel biodegradable neovasculature targeted nanoparticles formulation was developed and used to investigate the anti-tumor activity of miR-7 following intravenous administration in human glioblastoma U-87 MG bearing mice.

Systemic delivery of miR-7 nanoparticles in tumor bearing mice was a randomized, blinded study which was performed in an AALAC certified vivarium (Biomedical Research Institute, Rockville, MD, USA).

Gene modulation by miR-7 mimic – HUVEC culture.

Stem-Loop RT-PCR showed that the pre-miRNA-7 hairpin is processed into mature miR-7 (hsa-miR-7-5p, Supplementary Table S2).

The anti-proliferative effect of miR-7 was comparable to that of a positive control, i. e. siRNA against Polo-like kinase 1 (Plk1), a cell proliferation kinase.

Cells were transfected with increasing concentrations of miR-7, miR-scr or siPLK-1. siPLK-1 and miR-scr were used as positive and negative control.

Stem-loop RT-PCR was used to determine the relative tumor amounts of miR-7 in the different treatment groups.

The anti-angiogenic property of miR-7 was also observed microscopically with a statistically significant reduction of immunohistochemical staining of CD31 in tumor tissue from the miR-7 treated mice (Fig. 5c and d).

Hela cells seeded in 24-well plated, were co -transfected with 10 nM miR-7 or miR-Scr and 100ng/well 3′UTR psiCHECK [TM]-2 construct using Lipofectamine 2000 (Invitrogen) according to manufacturers protocol.

This was confirmed by in vitro studies that demonstrated reduced proliferation of U-87 MG cells upon transfection with miR-7 mimic (Supplementary Fig. S7).

Moreover, tumor tissue from the miR-7 treated mice contained considerable amounts of necrotic lesions, which is an indication of hypoxia from reduced angiogenesis.

We developed a novel αvβ3/αvβ5-integrin targeted nanoparticles for systemic delivery of miR-7 mimic to tumor EC and tumor cells and evaluated it in a human glioblastoma xenograft tumor mo del.

Glioblastoma xenograft tumor in vivo mo del: systemic deliverySystemic delivery of miR-7 nanoparticles in tumor bearing mice was a randomized, blinded study which was performed in an AALAC certified vivarium (Biomedical Research Institute, Rockville, MD, USA).

Chick CAMs were treated locally within a nitrocellulose ring with 300 picomol miR-7 or miR-Scr using Lipofectamine 2000 or with 200 picomol sunitinib.

Mutagenesis of the 3′UTR sequence of the predicted binding site of miR-7 (Fig. 2f) restored luciferase activity, thereby confirming the specificity of the interaction between miR-7 and the OGT 3′UTR (Fig. 2e).

Anti-angiogenic activity of miR-7 mimic in vitro.

The effect of miR-7 on tumor cell proliferation was determined by Ki-67 staining (in brown).

AJ mice bearing tumors with Neuro2A cells were treated locally with 10 μg miR-7 or 10 μg miR-Scr or PBS by intratumoral injection and electroporation.

The biochemical process underlying tumor growth inhibition by miR-7 mimics was investigated using immunohistochemical (IHC) detection of CD31, an endothelial cell marker for microvessel density (Fig. 4c).

Figure 3(a) Seed sequence of miR-7. Illustration of conserved seed sequence of miR-7 among different species.

Anti-angiogenic property of miR-7 in vitro.

HUVEC, seeded in a 6-well plate (8×10 [4] cells/well), were transfected at different concentrations of miR-7 using X-tremeGENE (see above).

Systemic delivery of miR-7 mimic not only reduced tumor angiogenesis but also reduced tumor proliferation as demonstrated by the statistically significant reduction in Ki-67 staining of miR-7 treated tumor tissue (Fig. 5e and f).

HUVEC were transfected with 50 nM miR-7 or miR-scr.

Subsequently, the rings were loaded with 300 picomol miR-Scr or miR-7 mimics complexed with Lipofectamine 2000 in 20 mM Hepes buffered glucose (pH 7,4).

Apart from the here described newly discovered anti-angiogenic effect, human miR-7 was first described by Lim et al. in 2003 [28] and later this miRNA was found to be associated with anti-tumorigenic effects in glioma, hepatocellular carcinoma cells, and head and neck cancer cells [29- 34].

In this work, the U-87MG mo del was used to explore the feasibility of miR-7 as anti-angiogenic agent and other therapeutically relevant tumor mo dels will be explored in the near future.

The most potent miRNA, miR-7, was validated for anti-angiogenic activity in vitro.

miR-7 treated mice showed a statistically significant reduction in OGT levels (Fig. 5h).

Anti-angiogenic activity of miR-7 mimic in vivo– local tumor administration.

A reduction in vascular density in the regions between large blood vessels was visible in CAM treated with miR-7 mimic while vascular density was not reduced in untreated or miR-Scr treated CAM (Fig. 3b).

Relative expression was calculated as the ratio of reads mapping to a gene in the miR-7 transfected sample and the reads mapping to a gene in the miR-Scr transfected sample.

Figure 5(a) miR-7 treated animals show pale and less vascularized tumors.

Delivery of miR-7 by electroporation into the tumor tissue was determined by stem loop RT-PCR.

HUVEC were transfected with increasing concentrations of miR-7 or miR-Scr.

We therefore selected miR-7 for further validation as an anti-angiogenic miRNA candidate.

Anti-proliferative effect of systemically delivered miR-7 was determined by Ki-67 staining, indicated as brown spots.

HUVEC were transfected with increasing concentration of miR-7 or miR-Scr.

HUVEC were transfected with either miR-7 or miR-Scr and the transcriptome was quantified by RNA-Seq.

To elucidate the mechanism of action by which miR-7 exerts the anti-angiogenic effects, transcriptional analysis of miR-7 mimic transfected HUVEC was performed.

HUVEC were transfected with 50 nM miR-7 or miR-scr and seeded on matrigel at 48hrs after transfection.

2

By contrast, in the up-regulated genes of 23DSI, the predicted target genes of miR-1-miR-71-miR-7-miR-7-5p appeared to regulate the ribonucleoprotein complex assembly, cellular protein complex assembly, microtubule -based process, response to oxidative stress, multicellular organismal aging, respiratory electron transport chain, pyrimidine ribonucleoside triphosphate biosynthetic process, positive regulation of epithelial cell differentiation, positive regulation of cell proliferation, apoptosis, energy coupled proton transport, electron transport chain, ATP synthesis-coupled proton transport, anatomical structure formation involved in morphogenesis, ribonucleoprotein complex biogenesis, mitotic cell cycle, larval development, microtubule polymerisation or depolymerisation, female gamete generation, regulation of transcription from RNA polymerase II promoter, and imaginal disc development, among others (Table  2 and Additional file 6: Table S4).

In 23 DSI, the high level of bantam and low levels of miR-1, miR-71, miR-7, and miR-7-5p possibly regulated and organised a specific gene expression profile for sexual maturation and egg production by inhibiting and strengthening specific gene expression and metabolic processes.

In unpaired females (23SSI), bantam was notably not up-regulated, whereas miR-1, miR-71, miR-7, and miR-7-5p were significantly up-regulated.

Furthermore, among all samples, bantam was distinctly up-regulated in 23 DSI, and miR-1, miR-71, miR-7-5p, and miR-7 were distinctly up-regulated in 23SSI.

To analyse the effect of the differential expression of miRNAs on female development after pairing, we sequenced the libraries of 23DSI and 23SSI, predicted the target genes of miRNA-1-miRNA-71-miRNA-7-miR-7-5p (Additional file 3: Table S1) and bantam (Additional file 4: Table S2), and analysed the differential expression of these genes in 23DSI compared with 23SSI.

Although miRNAs do not regulate all genes in organisms, evidence provided by miRNA analyses in the present study indicated that pairing likely limited the expression of non-essential genes through increasing the expression of bantam and specific genes by maintaining miR-1, miR-71, miR-7, and miR-7-5p at relatively low levels.

Click here for file Predicted target genes of miR-1-miR-71-miR-7-miR-7-5p in up-regulated genes in 23DSI.

Predicted target genes of miR-1-miR-71-miR-7-miR-7-5p in up-regulated genes in 23DSI.

By contrast, in paired females (23DSI), the above mentioned miRNAs were not up-regulated, suggesting that the functions of the target genes of miR-1-miR-71-miR-7-miR-7-5p were required in paired females.

We found that the target genes of miR-1-miR-71-miR-7-miR-7-5p, such as ribosomal protein genes (CAX72037.1, CAX71939.1, CAX78482.1, CAX77178.1, AAP06483.1, CAX77387.1, CAX72859.1, CAX70956.1, CAX71543.1, CAX83047.1, CAX70121.1) (Additional file 6: Table S4), thioredoxin peroxidase (CAX75860.1), tubulin (XP_002580033.1, CAX75788.1, CAX75500.1, CAX71989.1, CAX76110.1), ATP synthase- H + transporting (CAX75390.1, CAX76063.1), and cytochrome c oxidase (CAX74747.1, CAX76589.1), among others, were significantly up-regulated.

Out of the 50 genes, 33 were the predicted target genes of bantam (Figure  3B), whereas only 2 were predicted target genes of miR-1-miR-71-miR-7-miR-7-5p.

revealed that in unpaired females, the highly-expressed miRNA-1, miRNA-71, miRNA-7, and miR-7-5p only inhibited the limited pathways, such as proteasome and ribosome assembly.

For example, the higher expression of bantam was observed only in 23DSI, whereas higher expression of miR-1, miR-71, miR-7-5p, and miR-7 manifested only in 23SSI (Figure  1B).

The predicted target genes of bantam hardly participated in the proteasome, porphyrin metabolism, ribosome, whereas more predicted target genes of miR-1-miR-71-miR-7-miR-7-5p were involved in these process.

For instance, in ribosome assembly, 15 of 49 detected genes in this metabolic process were predicted as the target genes of miR-1-miR-71-miR-7-miR-7-5p, whereas only 1 of 49 genes was the predicted target gene of bantam (Figure  3A).

Differential expression of the predicted target genes of bantam and miRNA-1-miRNA-71-miRNA-7-5p- miR-7 between samples from 23 DSI and 23SSI.

Moreover, few of the predicted target genes of miR-1-miR-71-miR-7-miR-7-5p participated in the peroxisome, RNA degradation, mRNA surveillance pathway, axon guidance, basal transcription factors, apoptosis, glycerophospholipid metabolism, insulin signalling pathway, lysosome, regulation of actin cytoskeleton, and endocytosis.

Click here for file Predicted target genes of miR-1-miR-71-miR-7-miR-7-5p in Schistosoma japonicum.

Predicted target genes of miR-1-miR-71-miR-7-miR-7-5p in Schistosoma japonicum.

However, none of the predicted target genes of miR-1-miR-71-miR-7-miR-7-5p are involved the citrate cycle, gastric acid secretion, glycolysis/gluconeogenesis, protein digestion and absorption, aminoacyl-tRNA biosynthesis, fatty acid biosynthesis, and the pentose phosphate pathway.

The transcriptomes of 23DSI and 23SSI revealed that the predicted target genes of miRNA-1, miRNA-71, miRNA-7, and miR-7-5p were associated with the ribonucleoprotein complex assembly and microtubule -based process.

Only several high-abundance miRNAs differentially expressed between 23DSI and 23 SSI, such as bantam, miR-1, miR-71, miR-7, and miR-7-5p.

In particular, various ribosomal protein genes were regulated by miR-1-miR-71-miR-7-miR-7-5p.

Furthermore, the low abundance of miR-1, miR-71, miR-7, and miR-7-5p in 23DSI compared with 23SSI was likely capable of promoting specific gene expression.

These results suggested that miR-1, miR-71, miR-7, and miR-7-5p played an essential role in regulating ribosomal assembly.

3

However, more functional analysis of naturally expressed circRNAs within the CNS will provide useful information regarding the precise role of circRNAs and other long-ncRNAs in the regulation of gene expression required through the different developmental stages of the CNS and also, whether miRNAs other than miR-7 are regulated by long-ncRNAs.

This observation suggest that miR-671 might function as an indirect regulator of miR-7 activity by targeting and reducing ciRS-7 levels; however, the exact function of the ciRS-7:miR-671 interaction during the development of the CNS is still unknown.

Moreover, due to the high degree of conservation of miR-7, the binding sites in the human CDR1as are functional when it is expressed in zebrafish resulting in impaired midbrain development which is similar to the phenotype of knocking-down miR-7 (Memczak et al., 2013).

Interestingly, the concentration gradient of a single miRNA is capable of determining specific zones of neuronal differentiation as it is the case of miR-7 that maintains the proper localization of dopaminergic neuronal differentiation regions within the mouse olfactory bulb by having an opposite concentration/expression gradient to that of its target gene, Pax6 (De Chevigny et al., 2012).

Silencing QKI in the U343 glioblastoma cell line, results in miR-7 expression and cell cycle arrest, through a mechanism involving miR-7 negative regulation of epidermal growth factor (EGF) receptor (EGFR) protein levels, thus blunting the EGF -dependent ERK activation (Wang et al., 2013).

In particular, the human circRNA antisense to the cerebellar degeneration-related protein 1 transcript (CDR1as) contains 63 conserved binding sites for miR-7 and specifically regulates miR-7 expression in neuronal tissues (Memczak et al., 2013).

ciRS-7 contains more than 70 conserved binding sites for miR-7 and when miR-7 binds to it, AGO is recruited and binds to ciRS-7:miR-7 complexes however, ciRS-7 is resistant to miR-7 -mediated destabilization resulting in miR-7 activity blockage and derepression of miR-7 target genes (Hansen et al., 2013).

Moreover, knock-out mice for MSI2 present higher levels of mature miR-7 without a change in pri-miR-7 abundance confirming that RBPs are key players in the regulatory mechanism controlling miRNA biogenesis (Choudhury et al., 2013).

A recent report, demonstrated that the processing of the miR-7 pre-miRNA generated from the heterogeneous nuclear ribonucleoprotein K (hnRNP K) pre-mRNA transcript, is inhibited in non-brain human and mouse cells due to the binding of the RNA binding proteins (RBPs) Musashi homolog 2 (MSI2) and Hu antigen R (HuR) to the terminal loop of the pri-miR-7 and the stabilization of the pri-miRNA structure (Choudhury et al., 2013).

Absence of QKI-5 and QKI-6 results in increased mature miR-7 levels due to the fact that these proteins negatively regulate pri-miR-7 to miR-7 processing by maintaining the pri-miR-7 at the nucleus and tightly bounded by Drosha (Wang et al., 2013).

In addition, miR-7 biogenesis regulation also occurs via MSI2 and HuR binding during the in vitro neuronal differentiation of the SH-SY5Y cell line (Choudhury et al., 2013).

Another study showed that the control of miR-7 biogenesis by the quaking (QKI) RBPs, isoforms QKI-5 and QKI-6 that are localized at the nucleus and throughout the cell respectively, contribute to regulate the proliferation rate of glioblastoma cells cultures (Wang et al., 2013).

An independent study, described ciRS-7, another circRNA, as a miR-7 sponge in the human brain and in mouse neocortical and hippocampal neurons (Hansen et al., 2013).

Tissue-specific control of brain-enriched miR-7 biogenesis.

4

We used the miRDB[91, 92] to identify novel miRNA targets (Additional file 2), and we found that the 9 different miRNAs that increased in CD30 [hi] lymphocytes target several genes associated with neoplastic processes (Additional file 2): gga-mir-204 targets FAS apoptosis inhibitory molecule 2, RAB22A (a RAS oncogene family member) and HDAC 9; gga-mir-489 targets FAS associated factor 1 (FAF1) and gga-mir-7 targets RAS related viral oncogene homolog 2. Except FAF1 (which was unchanged) none of these proteins were identified and so we cannot confirm the upregulated miRNA’s potential effects on neoplasia in CD30 [hi] cells.

Of these, nine (gga-mir-1b, gga-mir-7, gga-mir-7b, gga-mir-10b, gga-mir-31, gga-mir-130b, gga-mir-204, gga-mir-215, gga-mir-489) are increased, and five (gga-mir-223, gga-mir-124b, gga-mir-140, gga-mir-183, gga-mir-222a) are decreased in CD30 [hi] cells.

5

Our co -expression network analysis showed that ALV has highly correlated expression with two miRNAs, miR-7 and novel miRNA 51.

In one of the two smaller networks identified, network 28, an ALV gene (env) had direct interaction with 2 miRNAs (miR-7 and novel_51) and 8 protein-encoding genes (Fig. 5d).

6

MicroRNA-7-5p inhibits melanoma cell proliferation and metastasis by suppressing RelA/NF-κB.

NF-κB promotes melanoma cell proliferation via miR-7-5p (Giles et al., 2016).

7

The SDE miRNAs with high fold-changes were all down-regulated in L30, including miR-146b-5p (−8.50-fold), miR-24-3p (−7.39-fold), miR-146a-5p (−5.96-fold), miR-221-5p (−5.85-fold), miR-7b (−5.35-fold), miR-147 (−5.11-fold), miR-20-5p (−4.59-fold), and miR-140b-5p (−4.57-fold).

8

Interestingly, some nearby genes of solo-LTR have already been involved in embryonic or extraembryonic development such as Klf-6 [39], teneurin-4 fragment [40] or the conserved microRNA miR-7b involved in the inhibition of Fos [41] that is required for extraembryonic endoderm epithelial organization [42].

9

Several sex-enriched miRNAs were also previously identified as differentially expressed including sja-miR-7, sja-miR-61, and sja-miR-219 in male worms and sja-bantam in female worms [45, 50].

In S. japonicum, Cai et al demonstrated miR-7-5p, miR-61, miR-219-5p, miR-125a, miR-125b, miR-124-3p, and miR-1 were dominant in males, while bantam, miR-71b-5p, miR-3479-5p and miR-Novel-23-5p were predominantly found in the female parasites [45].

10

Second, some of miRNAs such as miR-36, miR-71, bantam, miR-7, and others, have been shown to specifically express in a specific stage of S. japonicum (Xue et al., 2008; Hao et al., 2010; Cai et al., 2011).

11

The most representative example is that circRNA CDR1as, also known as ciRS-7, affects brain function in zebrafish and mouse by sponging miR-7 efficiently (Hansen et al., 2013; Memczak et al., 2013; Piwecka et al., 2017).

Since circRNA CDR1as was reported to affect brain function in zebrafish or mouse by sponging miR-7 (Hansen et al., 2013; Memczak et al., 2013; Piwecka et al., 2017), more and more circRNAs have been found to play various functions by serve as miRNAs sponge (Yamamura et al., 2017).

12

Furthermore, gga-miR-7, gga-miR-148a-3p, gga-miR-146c-5p, gga-miR-125b-5p, gga-miR-30d, gga-miR-153-3p and gga-miR-126-3p were abundant in our sequencing libraries and have been shown to occur in other animals [15, 69, 73, 74].