热激蛋白HSP27在多囊卵巢综合征卵巢颗粒细胞增殖分化异常中的作用及分子机制

Heat shock protein HSP27 is ubiquitously expressed in human cells and is critically important in maintaining the cellular homeostasis. The function of HSP27 in the ovary and its association with polycystic ovary syndrome remains poorly known. Our preliminary results suggested that HSP27 might be involved in the regultion of proliferation of granulosa cells by follicle stimulating hormone (FSH), however, the functional mechanisms need to be further explored. Therefore, we hypothesize that HSP27 might have critical roles in the regulation of proliferation and differentiation of granulosa cells by FSH, and the dysfunction of HSP27 would result in the development of polycystic ovary syndrome. By utilizing the primary human/rat granulosa cells and steroidogenic human granulosa cell line KGN, combined with research methods, including CRISPR/Cas9 system, lentivirus-mediated interference and overexpression, pull-down, in vitro phosphorylation assay, in vitro methylation/demethylation assay, and mass spectrometry, this project sets out to explore the function and molecular mechanisms of HSP27 in the regulation of proliferation and differentiation of granulosa cells at molecular, cellular, and tissue levels. This study will contribute to the understanding of the pathogenesis of polycystic ovary syndrome from the novel perspective of HSP27 regulating proliferation and differentiation of granulosa cells, and provide new thoughts for clinical therapies of this disease.

热激蛋白HSP27广泛存在于人体细胞，对维持细胞内稳态至关重要。迄今对HSP27在卵巢中的功能及其与多囊卵巢综合征的关系仍知之甚少。我们的预实验结果提示HSP27可能参与卵泡刺激素FSH对卵巢颗粒细胞的增殖调控，但其作用机制还有待进一步探讨。因此，我们提出假说：HSP27可能在FSH调控颗粒细胞增殖分化中发挥重要作用，其异常将诱导多囊卵巢综合征的发生。为了验证这一假说，本研究拟以人/大鼠原代颗粒细胞和人卵巢颗粒细胞系KGN为研究模型，采用CRISPR/Cas9系统、慢病毒干扰和过表达、pull-down、体外磷酸化实验、体外甲基化/去甲基化实验和质谱等研究方法和手段，从分子、细胞和组织水平探讨HSP27在颗粒细胞增殖分化调控中的作用及分子机制。本研究将从HSP27调控颗粒细胞增殖和分化这个新视点出发，为揭示多囊卵巢综合征的发生机制奠定理论基础，为多囊卵巢综合征的临床防治提供新的思路。

子痫前期（Pre-eclampsia, PE）是妊娠期导致母体和胎儿死亡的主要原因之一，其中尤以重度子痫前期（Severe pre-eclampsia, sPE）的危害最重。sPE发病的具体分子机理目前尚不完全明了，深入研究sPE胎盘发病过程中的分子机制有助于我们了解sPE胎盘的发病机理，并对其临床预防和治疗提供有力的理论支撑。我们的前期研究发现miR-181a-5p异常高表达于重度子痫前期病人血浆，迄今对其在胎盘发育中功能及与重度子痫前期关系仍知之甚少。本项目对miR-181a-5p在胎盘滋养层细胞侵润和迁移中的功能和机制进行了深入研究：我们首先验证了miR-181a-5p异常高表达于重度子痫前期病人胎盘组织，这在一定程度上提示了sPE病人血浆中异常高表达的miR-181a-5p的可能来源；接着我们通过Matrigel transwell实验发现miR-181a-5p显著抑制人胎盘滋养层细胞系HTR-8/SVneo细胞侵润和迁移；我们进一步通过生物信息学筛选分析、荧光素酶实验（Luciferase assay）、qRT-PCR和Western blot等实验鉴定了miR-181a-5p的新底物IGF2BP2，并鉴定了miR-181a-5p在IGF2BP2 mRNA 3'-UTR上的主要结合位点；我们还通过IGF2BP2基因的特异siRNA模拟了miR-181a-5p通过降调IGF2BP2蛋白水平抑制HTR-8/SVneo细胞侵润和迁移的功能；最后我们利用Rescue实验进一步明确miR-181a-5p至少是部分通过对于IGF2BP2基因的转录后调控抑制了人胎盘滋养层细胞的侵润和迁移。后续我们将进一步探讨miR-181a-5p通过降调IGF2BP2蛋白水平来抑制HTR-8/SVneo细胞侵润和迁移的下游分子机制。这些研究将较完整地阐明miR-181a-5p和IGF2BP2在sPE胎盘发育异常过程中的可能关键作用。