Research Article: Chi-miR-4110 promotes granulosa cell apoptosis by targeting Sma- and Mad-related protein 2 (Smad2) in the caprine ovary

Date Published: July 13, 2017

Publisher: Public Library of Science

Author(s): Xiaopeng An, Yuxuan Song, Jinxing Hou, Yue Zhang, Kaiwen Chen, Haidong Ma, Xinyan Zhao, Guang Li, Kexin Gao, Shan Wang, Binyun Cao, Yueyu Bai, Thomas Arendt.


Follicular atresia mainly results from the apoptosis of granulosa cells (GCs). Whilst our previous investigations examined the role of chi-miR-4110 in regulating ovarian function, the present study detected the role of chi-miR-4110 in GC development. We transfected caprine GCs cultured in vitro with chi-miR-4110 mimics. Results revealed that chi-miR-4110 decreased mRNA and protein levels of Smad2 by targeting its 3′-untranslated region (3′UTR). FoxC1 and Sp1 mRNA and protein levels markedly increased, whereas those of bHLHe22 significantly decreased (P<0.01 or 0.05) in GCs transfected with the chi-miR-4110 mimics. Further studies revealed a significantly higher number of apoptotic cells in GCs transfected with the chi-miR-4110 mimics (P< 0.05) than in GCs transfected with mimics negative control. GCs transfected with the chi-miR-4110 mimics exhibited significantly increased mRNA and protein levels of the pro-apoptotic gene Bax (P<0.01) and significantly decreased expression levels of the anti-apoptotic gene BCL-2 (P<0.01). Smad2 interference (Si-1282) results were consistent with those of the chi-miR-4110 mimics. Previous reports and our results showed that chi-miR-4110 increases Sp1 expression by repressing Smad2. The increase in Sp1 induces p53-upregulated modulator of apoptosis, which increases the relative abundance of Bax and causes caprine GC apoptosis. Our findings may provide relevant data for the investigation of miRNA-mediated regulation of ovarian functions.

Partial Text

Proliferation of ovarian granulosa cells (GCs) is essential in follicular development, maturation, atresia, ovulation and luteinisation [1]. Folliculogenesis is a complex process during which oocytes increase in size and develop into a mature form, accompanied by the proliferation and differentiation of the surrounding GCs [2]. This process is regulated by various extra- and intra-ovarian factors, such as Anti-mullerian hormone (AMH), Smads, Activins, Inhibins, Bone morphogenetic proteins (BMP) and Growth differentiation factor (GDF9) [3, 4].

Cell proliferation is critical in normal reproductive processes [24], including ovarian development, follicle progression from the primordial stage to the primary, secondary and tertiary or Graafian stages and ovulation [25]. However, because more than 99% of ovarian follicles undergo degeneration and atresia, less than 1% of the ovarian follicles reach the ovulation stage in mammals [26]. Previous studies demonstrated that GC apoptosis triggers follicular atresia [27], but the molecular mechanisms involved in this process are still unclear. In domestic animals, promoting follicular development, maturation and ovulation, whilst inhibiting GC apoptosis and follicular atresia may improve animal fertility, specifically ovulation and litter size. Therefore, GC apoptosis and follicular atresia analyses have become hotspots in animal science research [28–30]. Zhou et al. (2015) demonstrated that let-7g regulates GCs apoptosis in pig ovaries by targeting TGFBR1 and down regulating the TGF-β signalling pathway [20]. MiR-320 regulates GC proliferation and the production of testosterone, progesterone and estradiol by targeting E2F1 and SF-1 during follicular development [25]. Grossman et al. (2015) demonstrated that human chorionic gonadotropin (hCG) decreases miR-125a-3p expression towards ovulation, enabling mural GC migration. This process is partly mediated by Fyn and may be important for proper ovulation [31]. In this study, we demonstrated that chi-miR-4110 promotes GC apoptosis by targeting Smad2 in the caprine ovary.

We demonstrate that chi-miR-4110 promoted GC apoptosis in the caprine ovary by directly targeting and down regulating the Smad2 gene, showing therapeutic implications in the control of fertility. We note that our results do not exclude the possibility that other ovarian functions, such as folliculogenesis, oocyte maturation and ovulation, are regulated by chi-miR-4110. However, our results show that ovarian GC apoptosis is sensitive to chi-miR-4110. Although the precise mechanism regulating GC apoptosis is not elucidated, our findings may provide relevant data for those investigating miRNA-mediated regulation of ovarian functions.




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