外泌体传递的长链非编码RNA作为ceRNA调控MAPK1表达在多囊卵巢综合征卵泡发育异常中的作用机制研究

Abnormal folliculogenesis in polycystic ovary syndrome (PCOS) is a main cause of anovulatory infertility in women. However, the pathogenesis of PCOS is still unclear. It is reported that exosome plays an important role in the intercellular communication of follicular microenvironment. In previous studies, we found that exosomes could be taken up by granulosa cells and promoted the proliferation of cells. We also found that the expressions of two long non-coding RNAs (T027892 and T285945, hereinafter referred to as “candidate lncRNA” ) were significantly increased in exosome of PCOS follicular fluid, and the bioinformatics prediction indicated that the candidate lncRNA could up-regulate MAPK1 expression by acting as a ceRNA. Considering the biological function of MAPK1 in promoting cell proliferation, we hypothesized that the exosome mediated the transport of the candidate lncRNA in different granulosa cells, and then increased the expression of MAPK1, leading to the abnormal folliculogenesis of PCOS. To prove this hypothesis, in this study, we will further analyse the expression and clinical significance of the candidate lncRNA in PCOS. Moreover, by using gene transfection, RNA immunoprecipitation and reporter gene technology, the effect and molecular mechanism of the exosome-transmitted candidate lncRNA in regulating MAPK1 expression during the abnormal folliculogenesis of polycystic ovary syndrome by acting as a ceRNA will be determined in cellular, molecular and in vivo levels. The results of this study are expected to provide a theoretical basis for the research on epigenetic regulation and etiology of PCOS.

多囊卵巢综合征（PCOS）卵泡发育异常是女性排卵障碍型不孕的主要原因，但发病机制不明确。研究显示外泌体是卵泡微环境细胞间通讯的重要方式。我们前期研究发现PCOS卵泡液外泌体可被颗粒细胞摄取并促进细胞增殖，且其中有2个长链非编码RNA（T027892和T285945，以下称“候选lncRNA”）表达显著上调，生物信息学预测其均可作为ceRNA上调MAPK1表达，结合MAPK1促细胞增殖的生物学功能，我们推测外泌体介导候选lncRNA在不同颗粒细胞间传递，进而上调MAPK1表达并导致PCOS卵泡发育异常。为了证明该假说，本研究拟完成：分析候选lncRNA在PCOS中的表达及临床意义；利用基因转染、RIP及报告基因等技术从细胞、分子和体内水平上明确外泌体传递的候选lncRNA作为ceRNA调控MAPK1表达在PCOS卵泡发育异常中的的作用及分子机制，为阐明PCOS表观遗传病理机制提供理论依据。

多囊卵巢综合征（PCOS）卵泡发育异常是女性排卵障碍型不孕的主要原因，但发病机制不明确。研究表明长链非编码RNA（lncRNA）的表达与PCOS卵泡发育异常关系密切。另有研究显示外泌体是卵泡微环境细胞间通讯的重要方式。本项目研究从临床、细胞、分子和体内等多个水平上研究外泌体传递的lncRNA在PCOS中的表达水平以及探索相关lncRNA通过ceRNA机制调控MAPK信号通路影响PCOS卵泡发育的作用及分子机制。. 本项目研究首先通过LncRNA芯片并联合生物信息学方法筛选出一系列可能与PCOS卵泡发育有关的lncRNA，然后在临床样本中验证并确定lncRNA T306197和T027892在PCOS中表达显著上调，而lncRNA NR_123722（PKD1P6）和NR_026812在PCOS中显著下调。线性回归分析发现，PKD1P6在PCOS卵丘颗粒细胞中的表达水平与血清睾酮（T）水平成显著负相关性。本研究还发现MAPK1在PCOS卵泡液外泌体和颗粒细胞中的表达均显著下调，生物信息学分析及体外细胞实验结果显示，PKD1P6可能作为ceRNA参与调控MAPK1表达进而影响PCOS卵泡发育过程。本研究进一步研究发现PKD1P6可通过调控细胞增殖和凋亡相关基因的表达而抑制颗粒细胞的生长；PKD1P6可作为ceRNA吸附miR-135b-5p进而调控颗粒细胞生长。PKD1P6还可抑制MAPK信号通路关键分子ERK1/2的磷酸化水平，而miR-135b-5p过表达可显著上调KGN细胞中p-ERK1/2的表达。体内水平研究发现PCOS大鼠卵巢组织中PKD1P6的表达水平显著降低，而miR-135b-5p的表达则显著升高；PCOS大鼠卵巢卵泡颗粒细胞中p-ERK1/2具有一定水平的表达，而对照组卵泡颗粒细胞中几乎没有检测到p-ERK1/2表达。本研究还发现PKD1P6过表达细胞来源的外泌体可被KGN细胞大量摄取，并抑制KGN细胞的生长活力和增殖活性，同时还可上调KGN细胞G0/G1期比例以及下调S期和G2/M期的比例。综上研究结果，本项目阐明了外泌体及其介导的lncRNA作为ceRNA调控MAPK信号通路在PCOS卵泡发育异常中的作用及分子机制，为揭示PCOS疾病发生的表观遗传调控机制、病因研究和早期诊断提供新的科学依据。