Chi-miR-324-3p Regulates Goat Granulosa Cell Proliferation by Targeting DENND1A.
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ABSTRACT:
SUBMITTER: Liu Y
PROVIDER: S-EPMC8636700 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:MicroRNAs (miRNAs) are endogenously expressed small noncoding RNAs and play critical roles in the regulation of post-transcriptional gene expression. Our previous study uncovered that chi-miR-487b-3p is widespread in different goat tissues, which is significantly higher in muscle, especially in lamb. Here, we demonstrate the role of chi-miR-487b-3p as a myogenic miRNA that regulates skeletal muscle development. chi-miR-487b-3p overexpression was demonstrated to significantly inhibit goat myoblast proliferation and differentiation, whereas chi-miR-487b-3p inhibition resulted in the opposite effects. Next, chi-miR-487b-3p was predicted to target the 3'UTR of insulin receptor substrate 1 (IRS1) gene by Target-Scan and miRDB. The results of dual-luciferase assay, RT-qPCR, and western blot all confirmed that IRS1 might be a direct target of chi-miR-487b-3p as its expression was negatively regulated by chi-miR-487b-3p. siRNA silencing of IRS1 further demonstrated significant inhibition on goat myoblast proliferation and differentiation, confirming the effect of IRS1 downregulation by chi-miR-487b-3p in myogenesis. In addition, chi-miR-487b-3p knockout goat myoblast clones were generated using CRISPR/Cas9 technology, and we further illustrated that chi-miR-487b-3p regulates goat myoblast growth through the PI3K/Akt signaling pathway by targeting IRS1. Collectively, our work demonstrated that chi-miR-487b-3p is a potent inhibitor of skeletal myogenesis and provided new insights into the mechanisms of miRNA on the regulation of goat growth.
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Project description:We previously found that chi-miR-99b-3p was highly expressed in the skeletal muscle of 7-month-old (rapid growth period) goats and speculated that it may be associated with muscle development. To further investigate the role of chi-miR-99b-3p in goats, we found that chi-miR-99b-3p acted as a myogenic miRNA in the regulation of skeletal muscle development. Dual-luciferase reporter assays, qRT-PCR, and Western blot results confirmed that Caspase-3 and nuclear receptor corepressor 1 were direct targets for chi-miR-99b-3p as their expression was inhibited by this miR. Cell proliferation and qRT-PCR assays showed that chi-miR-99b-3p promoted proliferation through relevant targets and intrinsic apoptosis-related genes in goat skeletal muscle satellite cells (SMSCs), whereas inhibition of chi-miR-99b-3p had the opposite effect. Furthermore, integrative transcriptomic analysis revealed that overexpression of chi-miR-99b-3p induced various differentially expressed (DE) genes mainly associated with the cell cycle, relaxin signaling pathway, DNA replication, and protein digestion and absorption. Notably, most of the cell-cycle-related genes were downregulated in SMSCs after miR-99b-3p upregulation, including the pro-apoptosis-related gene BCL2. In addition, 47 DE miRNAs (16 upregulated and 31 downregulated) were determined by Small RNA-sequencing in SMSCs after chi-miR-99b-3p overexpression. Based on the KEGG enrichment analysis, we found that these DE miRNAs were involved in the biological pathways associated with the DE genes. Our study demonstrated that chi-miR-99b-3p was an effective facilitator of goat SMSCs and provided new insights into the mechanisms by which miRNAs regulate skeletal muscle growth in goats.
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Project description:The proliferation and apoptosis of granulosa cells (GCs) affect follicle development and reproductive disorders, with microRNAs playing a crucial regulatory role. Previous studies have shown the differential expression of miR-128-3p at different stages of goat follicle development, which suggests its potential regulatory role in follicle development. In this study, through the Cell Counting Kit-8 assay, the EDU assay, flow cytometry, quantitative real-time polymerase chain reaction, Western blot, and the dual-luciferase reporter assay, we used immortal human ovarian granulosa tumor cell line (KGN) cells as materials to investigate the effects of miR-128-3p and its predicted target gene growth hormone secretagogue receptor (GHSR) on GC proliferation and apoptosis. The results show that overexpression of miR-128-3p inhibited the proliferation of KGN cells, promoted cell apoptosis, and suppressed the expression of proliferating cell nuclear antigen (PCNA) and B-cell lymphoma-2 (BCL2) while promoting that of Bcl-2 associated X protein (BAX). The dual-luciferase reporter assay revealed that miR-128-3p bound to the 3' untranslated region sequence of GHSR, which resulted in the inhibited expression of GHSR protein. Investigation of the effects of GHSR on GC proliferation and apoptosis revealed that GHSR overexpression promoted the expression of PCNA and BCL2, enhanced GC proliferation, and inhibited cell apoptosis, whereas the opposite effects were observed when GHSR expression was inhibited. In addition, miR-128-3p and GHSR can influence the expression of extracellular signal-regulated kinase 1/2 protein. In conclusion, miR-128-3p inhibits KGN cell proliferation and promotes cell apoptosis by downregulating the expression of the GHSR gene.
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Project description:BackgroundDuring goat embryonic morphogenesis and postnatal initiation of hair follicle (HF) regeneration, dermal papilla (DP) cells play a vital role in hair formation. Growing evidence shows that microRNAs (miRNAs) participate in HF development and DP cell proliferation. However, the molecular mechanisms have not been thoroughly investigated.ResultIn this study, we utilized miRNA sequencing (miRNA-Seq) to identify differentially expressed miRNAs at different HF cycling stages (anagen and telogen). MiRNA-Seq has identified 411 annotated miRNAs and 130 novel miRNAs in which 29 miRNAs were up-regulated and 32 miRNAs were down-regulated in the anagen phase compared to the telogen phase. Target gene prediction and functional enrichment analysis indicated some major biological pathways related to hair cycling, such as Wnt signaling pathways, ECM-receptor interaction, VEGF signaling pathway, biosynthesis of amino acids, metabolic pathways, ribosome and oxidative phosphorylation. Also, we explored the function of chi-miR-30b-5p in regulating hair growth cycle. Similar to the HF cycling, DP cells were isolated from skin and used to investigate miRNA functions. The MTT and EdU assays showed that the viability and proliferation of DP cells were inhibited or promoted after the transfection of chi-miR-30b-5p mimic or inhibitor, respectively. Bioinformatics analysis revealed CaMKII? as a candidate target gene of chi-miR-30b-5p, and the dual-luciferase and western blot assay demonstrated that chi-miR-30b-5p bound to the 3'UTR of CaMKII? and further inhibited its translation.ConclusionChi-miR-30b-5p was found to be highly expressed in the telogen than that in the anagen phase and could inhibit the proliferation of DP cells by targeting CaMKII?. Our study provides new information on the regulatory functions of miRNAs during HF development.
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Project description:This study found that miR-27 is expressed in muscle and regulates muscle proliferation and differentiation. We explored the function and regulatory mechanism of miR-27b in goat muscle proliferation and differentiation. Compared with the Boer goat, higher expression of miR-27b was observed in all of the collected muscle tissues of Anhuai goat, excluding the kidney, whereas the opposite expression pattern was observed for Pax3, which showed lower expression in Anhuai goat. Expression of miR-27b decreased gradually during the proliferation of skeletal muscle satellite cells in Anhuai goat and increased during differentiation; however, the expression pattern of Pax3 was opposite. The regulatory activity of miR-27b demonstrated that miR-27b inhibited the proliferation of skeletal muscle satellite cells, but promoted their differentiation. Moreover, function research demonstrated that Pax3 negatively regulated myogenic differentiation of goat skeletal muscle satellite cells, but accelerated their proliferation. The results of a dual-luciferase reporter analysis showed that miR-27b directly targeted the 3'-untranslated regions of Pax3 mRNA, and western blot and immunofluorescence staining analyses showed that miR-27b inhibited expression of the Pax3 protein. In goats, miR-27b can regulate myogenic proliferation and differentiation by targeting Pax3.
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Project description:Adipocyte differentiation, which plays an important role in fat deposition, involves a complex molecular mechanism. MicroRNAs (miRNAs) are essential in this progress. Here, we showed that miR-25-3p expression had increased during goat intramuscular preadipocyte differentiation, which peaked at day 3. Using liposome transfection and qRT-PCR techniques, we found that knocking down miR-25-3p reduced the accumulation of lipid droplets by downregulating or upregulating the expression of LPL, PPAR γ , AP2, SREBP1, and C/EBP β but upregulating the expression of KLF4. Overexpression of miR-25-3p results in the opposite. Furthermore, the dual luciferase assay showed that overexpression of miR-25-3p significantly inhibited luciferase activity of KLF4. These results showed that miR-25-3p has a binding site within the 3 ' -UTR of KLF4 mRNA. Together, these findings indicate that miR-25-3p is a positive regulator of intramuscular preadipocyte differentiation via targeting to KLF4 in goats.
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Project description:Background: Increasing evidence suggested that microRNA and kinesin superfamily proteins play an essential role in ovarian cancer. The association between KIF4A and ovarian cancer (OC) was investigated in this study.Methods: We performed bioinformatics analysis in the GEO database to screen out the differentially expressed miRNAs (DEmiRNAs) associated with ovarian cancer prognosis. Upstream targeting prediction for KIF4A was acquired by using the mirDIP database. The potential regulatory factor miR-29c-3p for KIF4A was obtained from the intersection of the above all miRNAs. The prognosis of KIF4A and target-miRNA in OC was obtained in the subsequent analysis. qRT-PCR and Western blot detected KIF4A expression level in IOSE80 (human normal ovarian epithelial cell line). In the meantime, the gene expression level was detected in A2780, HO-8910PM, COC1, and SKOV3 cell lines (human ovarian carcinoma cell line). MTT and colony formation assays were used to detect cell proliferation of SKOV3 cell line. The following assays detected cell migration through the use of transwell and wound heal assays. Targeted binding relationship between KIF4A and miRNA was detected by using the dual-luciferase reporter assay.Results: Both high expression of KIF4A and lower expression of miR-29c-3p could be used as biomarkers indicating poor prognosis in OC patients. Cellular function tests confirmed that when KIF4A was silenced, it inhibited the proliferation and migration of OC cells. In addition, 3'-UTR of KIF4A had a direct binding site with miR-29c-3p, which indicated that the expression of KIF4A could be regulated by miR-29c-3p. In subsequent assays, the proliferation and migration of OC cells were inhibited by the overexpression of miR-29c-3p. At the same time, rescue experiments also confirmed that the promotion of KIF4A could be reversed by miR-29c-3p.Conclusion: In a word, our data revealed a new mechanism for the role of KIF4A in the occurrence and development of OC.
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Project description:Non-coding RNAs play a regulatory role in the growth and development of skeletal muscle. Our previous study suggested that gga-mir-133a-3p was a potential candidate for regulating myoblast proliferation and differentiation in skeletal muscle. The purpose of our study was to reveal the regulatory mechanism of gga-mir-133a-3p in the proliferation and differentiation of chicken myoblasts. Through the detection of cell proliferation activity, cell cycle progression and EdU, we found that gga-mir-133a-3p can significantly inhibit the proliferation of myoblasts. In the process of myogenic differentiation, gga-mir-133a-3p is up-regulated, while gga-mir-133a-3p can significantly promote the up-regulation of differentiation-related muscle-derived factors, indicating that gga-mir-133a-3p can promote the differentiation of myoblasts. Validation at the transcriptional level and protein level proved that gga-mir-133a-3p can inhibit the expression of PRRX1, and the dual-luciferase assay also showed their direct targeting relationship. Correspondingly, PRRX1 can significantly promote myoblast proliferation and inhibit myoblast differentiation. In our study, we confirmed that gga-mir-133a-3p participates in the regulation of proliferation and differentiation of myoblasts by targeting PRRX1.
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Project description:MicroRNAs (miRNAs), a class of 22 nucleotide (nt) noncoding RNAs, negatively regulate mRNA posttranscriptional modification in various biological processes. Morphogenesis of skin hair follicles in cashmere goats is a dynamic process involving many key signaling molecules, but the associated cellular biological mechanisms induced by these key signaling molecules have not been reported. In this study, differential expression, bioinformatics, and Gene Ontology/Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed on miRNA expression profiles of Inner Mongolian cashmere goats at 45, 55, and 65 days during the fetal period, and chi-miR-370-3p was identified and investigated further. Real-time fluorescence quantification (qRT-PCR), dual luciferase reporting, and Western blotting results showed that transforming growth factor beta receptor 2 (TGF-βR2) and fibroblast growth factor receptor 2 (FGFR2) were the target genes of chi-miR-370-3p. Chi-miR-370-3p also regulated the expression of TGF-βR2 and FGFR2 at mRNA and protein levels in epithelial cells and dermal fibroblasts. DNA staining, Cell Counting Kit-8, and fluorescein-labelled Annexin V results showed that chi-miR-370-3p inhibited the proliferation of epithelial cells and fibroblasts but had no effect on apoptosis. Cell scratch test results showed that chi-miR-370-3p promoted the migration of epithelial cells and fibroblasts. Chi-miR-370-3p inhibits the proliferation of epithelial cells and fibroblasts by targeting TGF-βR2 and FGFR2, thereby improving cell migration ability and ultimately regulating the fate of epithelial cells and dermal fibroblasts to develop the placode and dermal condensate, inducing hair follicle morphogenesis.
| S-EPMC8104936 | biostudies-literature
Project description:Several studies have suggested that long intergenic noncoding RNAs are involved in the progression of diabetic nephropathy (DN). However, the exact role and regulatory mechanism of long noncoding RNA (lncRNA) NR_038323 in diabetic nephropathy (DN) remain largely unclear. In the present study, we found that lncRNA NR_038323 overexpression ameliorated the high glucose (HG)-induced expression levels of collagen I, collagen IV, and fibronectin, whereas lncRNA NR_038323 knockdown exerted the opposite effects. Moreover, the results of bioinformatic prediction, luciferase assay, and fluorescence in situ hybridization (FISH) demonstrated that lncRNA NR_038323 directly interacted with miR-324-3p. Additionally, miR-324-3p mimic aggravated the HG-induced expression levels of collagen I, collagen IV, and fibronectin by dual-specificity protein phosphatase-1 (DUSP1) expression to activate p38 mitogen-activated protein kinase (MAPK) and ERK1/2 pathways. In contrast, overexpression of DUSP1 attenuated the HG-induced expression levels of collagen I, collagen IV, and fibronectin via inactivation of p38 MAPK and ERK1/2 pathways. In addition, lncRNA NR_038323 knockdown increased the expression levels of collagen I, collagen IV, and fibronectin by upregulating DUSP1 expression during HG treatment, which were markedly reversed by miR-324-3p inhibitor. Furthermore, these molecular changes were verified in the human kidney samples of DN patients. Finally, overexpression of lncRNA NR_038323 ameliorated the interstitial fibrosis in STZ-induced diabetic nephrology (DN) rat via miR-324-3p/DUSP1/p38MAPK and ERK1/2 axis. In conclusion, our data indicate that overexpression of lncRNA NR_038323 may suppress HG-induced renal fibrosis via the miR-324-3p/DUSP1/p38MAPK and ERK1/2 axis, which provides new insights into the pathogenesis of DN.
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