调控卵巢低反应中颗粒细胞增殖的新分子：miR-1271-5p的功能和机制研究

miR-1271-5p is one of miRNAs significantly elevated in granulosa cells in patients with POR in our previous miRNA array finding. Preliminary data showed miR-1271-5p overexpression significantly inhibited granulosa cell proliferation in mouse preantral follicles in vitro. Further bioinformatics analysis and experiments indicated that mTOR which played a key role during the granulosa cell cycle progression was a candidate target gene of miR-1271-5p. Forced expression of mTOR in granulosa cells can promote granulosa cell proliferation as well as follicular development. Hence, we hypothesized that miR-1271-5p inhibited granulosa cell proliferation was mediated by mTOR, which involved in the pathogenesis of follicular development in POR. Using a mouse preantral follicle and granulosa cell culture model coupled with adenoviral gene manipulation procedures, we will examine the function of miR-1271-5p in granulosa cell proliferation and the possible involvement of mTOR. The current study will elucidate the regulatory mechanism, which may provide a theoretical basis for the targets of the treatment and prevention of POR.

miR-1271-5p是我们前期应用miRNA芯片筛选发现的差异高表达于卵巢低反应（POR）患者卵泡颗粒细胞中功能未知的miRNA。前期研究发现：过表达miR-1271-5p可显著抑制颗粒细胞增殖和卵泡生长；生物信息学和预实验初步证实mTOR可能为miR-1271-5p靶基因；文献报道mTOR是促进颗粒细胞增殖和卵泡发育的关键基因。据此，我们提出“miR-1271-5p通过mTOR调控颗粒细胞增殖，影响卵泡发育，进而参与POR发生”的科学假说。本研究拟通过超表达和沉默实验，从正反两方面评判miR-1271-5p对颗粒细胞增殖和卵泡生长的影响；借助荧光素酶报告系统，明确miR-1271-5p对mTOR的调控作用；以mTOR为线索，论证miR-1271-5p调控颗粒细胞增殖的分子机制。本研究有望为阐明POR病理机制提供新的线索，为POR防治提供新的靶标。

miR-1271-5p是我们前期在IVF治疗中卵巢低反应（POR）患者卵泡颗粒细胞中筛选发现的，在POR患者中显著高表达的一条功能未知的microRNA。通过构建miR-1271-5p病毒表达/沉默载体，在卵泡颗粒细胞中转染miR-1271-5p过表达病毒载体（空载为对照），定量PCR验证miR-1271-5p过表达提示升高miR-1271-5p的表达水平会使小鼠颗粒细胞增殖能力减弱，影响卵泡生长发育。结合生物信息学分析，发现mTOR可能是miR-1271-5p下游靶基因。定量PCR和Western结果显示，miR-1271-5p过表达后，mTOR的表达水平显著下调，以上结果表明mTOR是miR-1271-5p下游调控的靶基因。mTOR在调控卵巢卵泡生长发育及颗粒细胞的增殖过程中发挥着重要作用，mTOR表达下降或沉默，能够通过抑制卵泡颗粒细胞的增殖显著影响卵泡的生长发育。通过挽救实验表明，miR-1271-5p通过调控mTOR的表达，影响卵泡生长发育过程中颗粒细胞的增殖。综上所述，miR-1271-5p能够调控卵泡生长发育过程中颗粒细胞的增殖，有可能为阐明POR病理机制提供新的线索，为临床POR防治提供潜在的干预靶标。此外，我们通过tRF&tiRNA高通量测序技术检测了卵巢低反应患者与正常反应患者卵泡液中差异表达的tRFs&tiRNAs，并选择潜在靶基因进行进一步的功能研究，有望为进一步的机制研究提供必要的数据。