piRNA-34871调控线粒体功能在卵巢储备减退中的作用及机制研究

Premature ovarian aging (POA) is a condition where a woman’s ovaries don’t produce enough eggs and/or only eggs of bad quality relative to the woman’s age. In approximately 10 percent of women suffer from POA; however, the underlying molecular mechanisms of aging remain largely elusive. Many theories of aging have been proposed, including the free-radical and mitochondrial theories of aging. Both theories speculate that cumulative damage to mitochondria caused by reactive oxygen species (ROS) is one of the causes of aging. Piwi-interacting RNAs (piRNAs) are 26-31 nt small noncoding RNAs that are processed from their longer precursor transcripts by Piwi proteins. Localization of Piwi and piRNA has been reported mostly in nucleus and cytoplasm of higher eukaryotes germ-line cells, where it is believed that known piRNA sequences are located in repeat regions of nuclear genome in germ-line cells. However, localization of PIWI and piRNA in mitochondria and the role of mitochondria piRNA in POA yet remain unknown. Using small RNA-Sequence, we have identified specific piRNA from POA patients. Most target sequences of abnormal piRNAs located in mtDNA. Further support for the role of piR-34871 in follicle development, was provided by qRT-PCR, Norther Blot, transfection of in vitro cultured mouse ovary and TUNEL assay. The main purpose of the present study is to explore the role of piRNA in POA and mitochondria function. In this study, we will express or inhibit specific piRNA to investigate that 1) cell proliferation and apoptosis; 2) oxidative stress induced damage; 3) follicle development. These results will provide new insight into the properties of the piRNA and establish the hypothesis that piRNA-associated mitochondria damage is one possible cause of POA.

卵巢提早老化（Premature Ovarian Aging, POA）表现为月经周期正常女性卵巢储备功能降低，人群中约有9%的女性表现POA症状。目前病因尚不明确，氧化压力及其造成的线粒体损伤可能与之相关。本项目前期建立了POA患者卵泡颗粒细胞差异表达piRNA谱，发现并验证了piR-34871在POA患者中表达水平异常上调，体外小鼠卵巢组织培养中的研究发现piR-34871过表达可导致线粒体形态异常、促进细胞凋亡，减少原始和初级卵泡数，干扰卵泡发育。推测piR-34871可能通过影响线粒体功能参与卵巢储备调节。本项目拟通过分子及细胞生物学等手段，在细胞及卵巢组织中调整piR-34871的表达，结合施加氧化压力，分析细胞生长凋亡、线粒体功能、氧化压力耐受度及氧化损伤改变，验证piR-34871对卵泡发育的影响，研究piRNA调节卵巢储备功能的机制，为卵巢储备功能提早减退提供防治的新线索。

卵巢提早老化（Premature Ovarian Aging, POA）表现为月经周期正常女性卵巢储备功能降低，人群中约有9%的女性表现POA症状。目前病因尚不明确，氧化压力及其造成的线粒体损伤可能与之相关。.本项目通过建立POA患者卵泡颗粒细胞差异表达piRNA谱，发现并验证了piR-34871在POA患者中表达水平异常上调。同时发现POA患者卵泡颗粒细胞的DNA、脂质及蛋白质氧化损伤修复能力均明显下降，氧化耐受能力下降同时细胞凋亡水平显著升高。在人类卵巢颗粒细胞株COV434中转染piR-34871 mimic及inhibitor，发现piR-34871 mimic转染显著降低细胞线粒体量（mitochondrial mass）以及ATP量（ATP content）。在氧化压力条件下piR-34871 mimic 转染组细胞线粒体膜电位显著降低；线粒体ROS(Reactive oxygen species)水平显著增加；凋亡水平也显著增加。而piR-34871 inhibitor转染可缓解线粒体功能损伤和压力条件下的细胞凋亡。说明piR-34871通过改变卵巢颗粒细胞的氧化耐受能力损伤线粒体功能，从而影响卵泡储备功能。机制研究提示piR-34871通过转录后调控机制来改变SIRT3的表达水平，通过SIRT3-FOXO3-SOD2这条信号通路来调节ROS水平和线粒体功能，这种调控不涉及更上游的PGC1-α和Sirt1。项目组更进一步发现SOD2的一种类似物GC4401可显著提升piR-34871处理组细胞的ROS修复功能，降低细胞凋亡水平，提示GC4401可有望应用于临床治疗POA。在体外培养小鼠卵巢模型中，piR-34871 异常表达可导致因细胞凋亡增加引发的原始卵泡、初级卵泡丢失，导致卵泡发育异常，卵巢储备降低。而GC4401的处理可以拯救这种异常表型，进一步验证GC4401应用于临床治疗POA潜力。.本项目通过分子及细胞生物学等手段，在细胞及体外培养卵巢组织中，验证piR-34871异常表达对卵泡发育的影响，阐释piR-4871调节卵巢储备功能的机制，发现可修复卵巢储备功能的化合物，为卵巢储备功能提早减退提供防治的新线索。.