HuR/ELAVL1在大鼠卵巢颗粒细胞增殖调控中的作用及机制研究

RNA-binding protein ELAVL1, also named HuR, is involved in the control of various cell proliferation processes by post-transcriptional regulation of cell cycle related genes expressions. HuR is a candidate biomarker for the diagnose of ovarian cancer and other carcinomas. However, the effects and mechanisms of HuR on ovarian granulosa cell proliferation haven’t been reported. Recent studies indicated that the expression and histological localization of HuR protein were related to the phases of cell cycle during rat ovarian granulosa cells proliferation, and these phenomena were influenced by both FSH and PKA. In our previous studies, it has been demonstrated that PKA was involved in FSH-stimulated rat ovarian granulosa cell proliferation. Based on those findings, a hypothesis has been proposed that the post-transcriptional effects of HuR on the expression of cell cycle related genes may be involved in the regulation of ovarian granulosa cell proliferation. Thus, rat models will be taken to demonstrated the hypothesis. Firstly, overexpression and RNA interference experiments will be utilized to investigate the effects of HuR on the processes of rat ovarian granulosa cell proliferation. Secondly, to address the mechanisms underlying HuR-mediated ovarian granulosa cell proliferation, the effects of PKA on the phosphorylation of HuR and post-transcriptional regulation of cell cycle related genes expressions in granulosa cell will be studied by RIP and Gene-directed mutagenesis. These findings will advance the current understanding on the regulation mechanism of ovarian granulosa cell proliferation and follicle development, and also provide new insights into finding a new drug target for ovarian diseases.

RNA结合蛋白ELAVL1，又名HuR，通过调控细胞周期相关基因的转录后表达参与多种细胞增殖过程，是卵巢癌等肿瘤疾病的潜在标志物，然而关于HuR在卵巢颗粒细胞增殖调控中的研究，迄今国内外尚无报道。我们前期研究证实，大鼠卵巢颗粒细胞中HuR蛋白的表达及核质分布与有丝分裂进程密切相关，并且受FSH和PKA信号影响，而FSH激活PKA信号对颗粒细胞增殖有重要的促进作用。据此我们推测：HuR蛋白可能通过介导细胞周期相关基因的转录后表达参与卵巢颗粒细胞增殖过程。为证实该假说，本项目拟以大鼠为研究对象，首先应用过表达和干扰技术，探究HuR蛋白对卵巢颗粒细胞增殖的影响；其次利用RIP及定点突变技术，研究PKA介导HuR磷酸化修饰对细胞周期相关基因转录后表达的影响，进而揭示HuR影响卵巢颗粒细胞增殖的分子机制。研究结果将进一步阐明哺乳动物卵巢颗粒细胞增殖及卵泡发育机制，为寻找卵巢疾病的药物靶点提供新思路。

HuR，是胚胎致死异常视觉(ELAV)家族的一员，广泛表达在动物机体的各类组织中。它可通过结合mRNA进行转录后调控，参与多种癌症细胞的不良增殖过程，然而其在生理环境下的调控功能却鲜少报道。本课题以大鼠卵巢及卵巢颗粒细胞为研究对象，结合体内外实验，利用免疫荧光化学、免疫印迹、细胞增殖检测、基因过表达和干扰技术等技术，对HuR在卵巢发育及颗粒细胞增殖过程中的功能及机制进行探究。结果发现，（1）卵巢组织内的HuR蛋白表达具有显著的细胞类型与发育时间依赖性；（2）在生长阶段的卵巢卵泡中，HuR蛋白的表达及核质分布与有丝分裂进程密切相关；（3）FSH不仅通过PKA信号影响HuR蛋白的核质分布，还对HuR蛋白的含量具有早期上调和晚期下调的反向功能。（4）HuR干扰却能够显著抑制FSH诱导的卵巢颗粒细胞体外增殖作用，并且该过程与细胞周期相关蛋白cyclin A，cyclin D2、p21以及p27的稳定性并不相关。综上所述，我们初步证实HuR在大鼠卵巢发育尤其是卵巢颗粒细胞增殖过程中具有重要的调控功能，但其具体的作用机制还需要进一步深入探究。本项目已取得的研究结果为阐明哺乳动物卵巢颗粒细胞增殖及卵泡发育机制提供了新的理论数据。此外，在该项目的支持下，项目申请人开展了具有学校及学科特色的野生动物相关生殖研究工作。