microRNA181a直接靶向acvr2a抑制小鼠卵巢颗粒细胞的增殖
发布时间：2019-06-04 07:47
【摘要】：颗粒细胞的增殖与分化对卵泡的发育至关重要,颗粒细胞功能异常导致卵泡发育障碍是引发卵巢内分泌异常的重要原因。越来越多的证据显示microRNA参与卵巢颗粒细胞功能的调节。我们研究发现miR-181a在小鼠卵巢生长发育与卵泡生长过程中表达逐渐降低,提示miR-181a在卵泡发育过程中具有重要调节作用,但具体分子机制不清。我们通过荧光定量PCR与细胞增殖活力检测结果证实,随着外源性miR-181a表达量的增加,小鼠卵巢颗粒细胞(mGCs)的增殖活力明显降低；腺病毒介导的小鼠颗粒细胞中外源性miR-181a的表达以梯度依赖的方式抑制mGCs中细胞周期蛋白D2 (cyclin D2) mRNA和蛋白的表达以及增殖细胞核抗原(PCNA)蛋白的表达。miR-181a的功能缺失实验进一步表明mGCs转染50nM miR-181a inhibitor时,可显著增加mGCs的增殖活力以及mGCs中cyclin D2的表达。在进一步的机制研究中,经荧光素酶报告基因实验证实miR-181a通过与激活素受体2a (acvr2a) mRNA 3'UTR区种子序列(TGAATGT)结合抑制荧光素酶报告基因活性；Q-PCR和Western Blot实验结果进一步证实腺病毒介导的miR-181a高表达可以明显抑制mGCs中内源性acvr2a mRNA和蛋白的表达；给予niR-181a inhibitor处理后则显著促进mGCs中acvr2a mRNA和蛋白的表达；在小鼠发育过程中,其卵巢acvr2a的表达呈现逐渐升高的趋势,同时伴随卵泡生长至窦前与窦卵泡期,卵泡中acvr2a mRNA的表达明显增多,与卵泡中同期miR-181a的表达呈负相关。Q-PCR结果显示activin A以浓度依赖的方式显著抑制小鼠颗粒细胞中miR-181a表达；腺病毒介导的颗粒细胞中高表达miR-181a显著抑制activin A (50ng/mL)促进mGCs的增殖(P0.05)以及cyclin D2的表达。Western Blot实验进一步证实miR-181a通过降低细胞中内源性acvr2a的表达而抑制activinA诱导的细胞内Smad2蛋白的磷酸化和CYP19Al、P450scc和ESR1基因的表达。临床样本调查结果初步显示,卵巢早衰病人血浆中miR-181a的表达明显增多。综上所述,我们首次发现卵巢早衰病人血浆中miR-181a明显增高。在研究miR-181a增高可能的病生理意义时,我们通过小鼠试验进一步发现miR-181a通过与acvr2a mRNA 3'UTR区种子序列结合降低内源性acvr2a的表达,并抑制activin A诱导的mGCs的增殖。这为下一步研究卵巢早衰的分子机制提供了新线索。
[Abstract]:The proliferation and differentiation of granulosa cells is very important to the development of follicles. The abnormal function of granulosa cells leads to the disorder of follicular development, which is an important cause of ovarian endocrine abnormalities. More and more evidence suggests that microRNA is involved in the regulation of ovarian granulosa cell function. Our study found that the expression of miR-181a decreased gradually during ovarian growth and follicular growth in mice, suggesting that miR-181a plays an important regulatory role in follicular development, but the specific molecular mechanism is unclear. The results of fluorescence quantitative PCR and cell proliferation activity showed that the proliferation activity of mouse ovarian granulosa cells (mGCs) decreased significantly with the increase of exogenous miR-181a expression. The expression of exogenous miR-181a in mouse granulosa cells mediated by adenovirus inhibited the expression of cell cycle protein D2 (cyclin D2) mRNA and protein and the expression of proliferating cell nuclear antigen (PCNA) protein in mGCs in a gradient-dependent manner. The functional deletion test of miR-181a further showed that when mGCs was transferred into 50nM miR-181a inhibitor, It could significantly increase the proliferation activity of mGCs and the expression of cyclin D2 in mGCs. In further studies, luciferase reporter gene assay confirmed that miR-181a inhibited luciferase reporter gene activity by binding to the seed sequence (TGAATGT) of activin receptor 2A (acvr2a) mRNA 3 'UTR region. The results of Q-PCR and Western Blot further confirmed that the overexpression of miR-181a mediated by adenoviruses could significantly inhibit the expression of endogenous acvr2a mRNA and protein in mGCs, and the expression of acvr2a mRNA and protein in mGCs was significantly promoted after treatment with niR-181a inhibitor. During the development of mice, the expression of acvr2a in ovaries increased gradually, and the expression of acvr2a mRNA in follicles increased significantly with the growth of follicles to the antral and antral follicles. It was negatively correlated with the expression of miR-181a in follicles at the same time. Q-PCR results showed that activin A significantly inhibited the expression of miR-181a in mouse granulosa cells in a concentration-dependent manner. The overexpression of miR-181a in granulosa cells mediated by adenoviruses significantly inhibited the proliferation of mGCs (P 0.05) and the expression of cyclin D2. Western Blot assay further confirmed that miR-181a decreased the intrinsic acvr2a in cells. Inhibition of intracellular Smad2 protein phosphorylation and CYP19Al, induced by activinA Expression of P450scc and ESR1 genes. The results of clinical sample investigation showed that the expression of miR-181a in plasma of patients with premature ovarian failure was significantly increased. To sum up, for the first time, we found a significant increase in plasma miR-181a in patients with premature ovarian failure. In order to study the possible pathophysiological significance of miR-181a, we further found that miR-181a decreased the expression of endogenous acvr2a by binding to the seed sequence of acvr2a mRNA 3 'UTR region, and inhibited the proliferation of mGCs induced by activin A. This provides a new clue for the next step in the study of the molecular mechanism of premature ovarian failure.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R321.1
[Abstract]:The proliferation and differentiation of granulosa cells is very important to the development of follicles. The abnormal function of granulosa cells leads to the disorder of follicular development, which is an important cause of ovarian endocrine abnormalities. More and more evidence suggests that microRNA is involved in the regulation of ovarian granulosa cell function. Our study found that the expression of miR-181a decreased gradually during ovarian growth and follicular growth in mice, suggesting that miR-181a plays an important regulatory role in follicular development, but the specific molecular mechanism is unclear. The results of fluorescence quantitative PCR and cell proliferation activity showed that the proliferation activity of mouse ovarian granulosa cells (mGCs) decreased significantly with the increase of exogenous miR-181a expression. The expression of exogenous miR-181a in mouse granulosa cells mediated by adenovirus inhibited the expression of cell cycle protein D2 (cyclin D2) mRNA and protein and the expression of proliferating cell nuclear antigen (PCNA) protein in mGCs in a gradient-dependent manner. The functional deletion test of miR-181a further showed that when mGCs was transferred into 50nM miR-181a inhibitor, It could significantly increase the proliferation activity of mGCs and the expression of cyclin D2 in mGCs. In further studies, luciferase reporter gene assay confirmed that miR-181a inhibited luciferase reporter gene activity by binding to the seed sequence (TGAATGT) of activin receptor 2A (acvr2a) mRNA 3 'UTR region. The results of Q-PCR and Western Blot further confirmed that the overexpression of miR-181a mediated by adenoviruses could significantly inhibit the expression of endogenous acvr2a mRNA and protein in mGCs, and the expression of acvr2a mRNA and protein in mGCs was significantly promoted after treatment with niR-181a inhibitor. During the development of mice, the expression of acvr2a in ovaries increased gradually, and the expression of acvr2a mRNA in follicles increased significantly with the growth of follicles to the antral and antral follicles. It was negatively correlated with the expression of miR-181a in follicles at the same time. Q-PCR results showed that activin A significantly inhibited the expression of miR-181a in mouse granulosa cells in a concentration-dependent manner. The overexpression of miR-181a in granulosa cells mediated by adenoviruses significantly inhibited the proliferation of mGCs (P 0.05) and the expression of cyclin D2. Western Blot assay further confirmed that miR-181a decreased the intrinsic acvr2a in cells. Inhibition of intracellular Smad2 protein phosphorylation and CYP19Al, induced by activinA Expression of P450scc and ESR1 genes. The results of clinical sample investigation showed that the expression of miR-181a in plasma of patients with premature ovarian failure was significantly increased. To sum up, for the first time, we found a significant increase in plasma miR-181a in patients with premature ovarian failure. In order to study the possible pathophysiological significance of miR-181a, we further found that miR-181a decreased the expression of endogenous acvr2a by binding to the seed sequence of acvr2a mRNA 3 'UTR region, and inhibited the proliferation of mGCs induced by activin A. This provides a new clue for the next step in the study of the molecular mechanism of premature ovarian failure.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R321.1
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