miR-215-5p调控YY1通过TGF-β1/Smad信号通路对PCOS大鼠卵巢颗粒细胞凋亡的影响
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四川省医学会专项科研项目(2019HR65)


Influence of miR-215-5p on apoptosis of ovarian granulosa cells in rats with polycystic ovary syndrome by regulating YY1 through TGF-β1/Smad signaling pathway
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    摘要:

    探究MicroRNA-215-5p(miR-215-5p)在多囊卵巢综合征(PCOS)大鼠中的表达及其对卵巢颗粒细胞(GC)凋亡的影响,并分析潜在机制。方法 采用脱氢表雄酮(DHEA)建立PCOS大鼠模型,分离并培养原代PCOS大鼠卵巢GC,分为对照组(control组)、sh-NC组、sh-YY1组、sh-YY1+LY2109761组、miR-NC组、miR-215-5p mimic组、miR-215-5p mimic+pc-NC组、miR-215-5p mimic+pc-YY1组。TUNEL法检测PCOS大鼠卵巢组织GC凋亡;实时荧光定量PCR(RT-qPCR)检测卵巢组织/GC中miR-215-5p、阴阳-1(YY1)mRNA表达;CCK-8法和平板克隆形成实验检测细胞增殖能力;流式细胞仪检测细胞凋亡率;Western blot检测PCOS大鼠卵巢组织/GC中YY1、转化生长因子-β1(TGF-β1)、Smad4蛋白表达。结果 miR-215-5p在PCOS大鼠卵巢组织中低表达,YY1 mRNA和蛋白水平在PCOS大鼠卵巢组织中显著升高(P<0.05);敲低YY1或上调miR-215-5p可显著增强PCOS大鼠卵巢GC增殖能力,抑制GC凋亡,升高TGF-β1、Smad4蛋白水平(均P<0.05);TGF-β/Smad4信号通路抑制剂LY2109761可显著减弱敲低YY1对卵巢GC凋亡的抑制作用(P<0.05);在过表达miR-215-5p的基础上,上调YY1可抑制TGF-β1/Smad信号通路的激活,显著减弱miR-215-5p mimic对PCOS大鼠卵巢GC凋亡的抑制作用(P<0.05)。结论 miR-215-5p在PCOS中呈低表达,过表达miR-215-5p可能通过下调YY1,激活TGF-β1/Smad信号通路,促进PCOS大鼠卵巢GC增殖并抑制其凋亡

    Abstract:

    To investigate the expression of MicroRNA-215-5p (miR-215-5p) in polycystic ovary syndrome (PCOS) rats and its influence on ovarian granulosa cell (GC) apoptosis, and analyze the underlying mechanism. Methods Dehydroepiandrosterone (DHEA) was used to establish a PCOS rat model, and primary PCOS rat ovarian GCs were isolated and cultured. They were grouped into control group, sh-NC group, sh-YY1 group, sh-YY1+LY2109761 group, miR-NC group, miR-215-5p mimic group, miR-215-5p mimic+pc=NC group, and miR-215-5p mimic+pc-YY1 group. The GC apoptosis in PCOS rat ovarian tissue was detected by TUNEL method; the mRNA expression of miR-215-5p and Yin-Yang-1 (YY1) in ovarian tissue/GC was measured by real-time quantitative PCR (RT-qPCR); cell proliferation ability was detected by CCK-8 method and plate colony formation assay; cell apoptosis rate was measured by flow cytometry; the protein expression of YY1, transforming growth factor-β1 (TGF-β1) and Smad4 in ovarian tissue/GC of PCOS rats was detected by Western blot. Results The expression of miR-215-5p was low in the ovarian tissue of PCOS rats, and the mRNA and protein levels of YY1 were significantly increased in the ovarian tissue of PCOS rats (P<0.05). Knockdown of YY1 or up-regulation of miR-215-5p was able to significantly enhance the proliferation of ovarian GCs in PCOS rats, inhibit GC apoptosis, and increase the levels of TGF-β1 and Smad4 proteins (all P<0.05). TGF-β/Smad4 signaling pathway inhibitor LY2109761 was able to significantly attenuate the inhibitory effect of knockdown of YY1 on ovarian GC apoptosis (P<0.05); on the basis of overexpression of miR-215-5p, up-regulation of YY1 was able to inhibit the activation of TGF-β1/Smad signaling pathway, and significantly attenuate the inhibitory effect of miR-215-5p mimic on ovarian GC apoptosis in PCOS rats (P<0.05). Conclusion MiR-215-5p is lowly expressed in PCOS. Overexpression of miR-215-5p may promote the proliferation of ovarian GC and inhibit apoptosis in PCOS rats by downregulating YY1 and activating the TGF-β1/Smad signaling pathway

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  • 在线发布日期: 2023-05-18
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