| Many members of the TGF-βsuperfamily are indicated to play important roles in mammalian reproduction, such as affecting the early folliculogenesis, granulosa cell proliferation and differentiation, oocyte maturation and embryo implantation and the maintanence of pregnancy. The perturbation of the TGF-βsignaling transduction could result in female infertility. MicroRNAs (miRNAs), as small non-coding RNAs, were recently found to regulate gene expression at the post-transcriptional levels, either inhibit or promote. However, little is known about the role of miRNAs in TGF-β-mediated granulosa cell proliferation,hormone release and COC expansion. In this study, the miRNA expression profiling was identified from TGF-β1-stimulated mouse preantral granulosa cells (GCs), and 3 miRNAs were found to be significantly up-regulated and 13 miRNAs were significantly down-regulated. Among the up-regulated miRNAs, miR-224 was the second most significantly elevated miRNA. This up-regulation was attenuated by treatment of GCs with SB431542 (an inhibitor of TGFβsuperfamily type I receptors, thus blocking the phosphorylation of the downstream effectors Smad2/3), indicating that miR-224 expression was regulated by the canonical TGF-β1/Smads pathway. By a series studies of luciferase analysis and western blot, we idendified that smad4 is one of the target of miR-224. The ectopic expression of miR-224 by transfecting with miR-224 mimics can enhance TGF-β1-induced GC proliferation through targeting smad4. Inhibition of endogenous miR-224 partially suppressed GC proliferation induced by TGF-β1. In addition, both miR-224 and TGF-β1 can promote estradiol release, one of the most significant roles of GC in reproduction. By using the real-time PCR analysis, we propose that the functions of estradiol release may partly through increasing the cyp19a1 mRNA levels. This is the first demonstration that miRNAs can control reproductive functions by promoting TGF-β1-induced GC proliferation and ovarian estrogen release.By analyzing the mRNA expression profile in GCs treated by TGF-β1, we found there many genes were de-regulated, of which 463 mRNAs were found to be significantly up-regulated and 440 mRNAs were to be significantly down-regulated. Furthermore, ptx3 is found to be the most significantly up-regulated gene. Considering that ptx3 is the marker gene of cumulus-oocyte complexes (COCs) expansion, we suppose that TGF-β1 may participated in the later follicular development by regulating the expression of ptx3. Therefore, ptx3 is focused in our study. Intersteingly, we found that miR-224 has conserved binding sites in the 3'UTR of ptx3. By luciferase analysis and elisa assay, we verified that ptx3 is indeed the another target of miR-224. In mouse mural granulosa cells and cumulus cells, we found that TGF-β1 promote the expression of ptx3, but inhibit the expression of miR-224. By in vitro culture of COCs, the four COC expansion marker gene, including ptx3, ptgs2, tnfaip6, and has2, were all up-regulated by TGF-β1 stimulation, indicating that TGF-β1 may participate in COC expansion. Thus, we suppose that TGF-β1 may regulate follicular development by fine tuning the expression of miR-224 and ptx3. Whether miR-224 may affect COC expansion, fertilization and subsequent embryo development by targeting ptx3 needs further study.By summarizing the above results, it is demonstrated that miR-224 is involved in TGF-β1-mediated follicular development by targeting various target gene. Such miRNA-mediated effects could be potentially used for the regulation of reproductive processes or for treatment of reproductive disorders.
|
PDF Full Text Request