lincRNA-p21通过结合ZFP38调控RAD51蛋白在精子发生异常中的作用及机制研究

Abnormal spermatogenesis may lead to male infertility, and meiosis plays an important role in spermatogenesis. Previous studies have shown that lincRNA-p21 can regulate the G1/S phase in the process of meiosis and spermatogenesis, which suggests that lincRNA is closely related to meiosis of lincRNA-p21. In our previous studies, we found that the ZFP38 regulates the expression of RAD51 through the transcriptional level, but the specific molecular mechanism is not clear. At the same time, we can find that lincRNA-p21 can be combined with ZFP38 by bioinformatics prediction and pre experimental results. On this basis, we supposed that lincRNA-p21 is crucial in the regulation of RAD51 in spermatogenesis abnormality by ZFP38. In order to provide a basis for the prevention and treatment of male infertility, this project studies the effect and mechanism of lincRNA-p21 in abnormal spermatogenesis through ZFP38 by regulating RAD51 protein at the cellular level by knockdown or overexpression of lincRNA-p21, carry on RNA pulldown experiments and knockout mice under the condition of ZFP38 .

精子发生异常易致男性不育，精母细胞减数分裂在精子生成中发挥重要作用。研究指出，lincRNA-p21在减数分裂和精子生成中起调控作用，lincRNA-p21可增强G1/S期检测点。我们前期研究发现，ZFP38是减数分裂的关键，可通过调控RAD51发挥作用，但其上游分子机制尚不明确。同时，我们经过生物信息学预测及预实验发现，lincRNA-p21可与ZFP38结合。基于此，我们推测：lincRNA-p21可能是通过结合ZFP38调控RAD51蛋白在精子发生异常中发挥重要作用。因此，本项目拟从lincRNA-p21与ZFP38结合出发，在细胞水平通过敲低或过表达lincRNA-p21，RNA pull down实验，以及在ZFP38条件敲除鼠水平，研究lincRNA-p21通过结合ZFP38调控RAD51蛋白在精子发生异常中的作用及分子机制。以期为精子发生异常以及男性不育的防治提供研究基础。

精子发生异常易致男性不育，精母细胞减数分裂在精子生成中发挥重要作用。研究指出，lincRNA-p21在减数分裂和精子生成中起调控作用，lincRNA-p21可增强G1/S期检测点。本项目通过Western blot、qRT-PCR等多种技术手段，检测了不育患者标本中lincRNA-p21 mRNA表达水平，结果显示：不育患者精子中lincRNA-p21表达显著高于正常人。为了探索其可能作用机制，我们构建了稳定过表达和敲低lincRNA-p21的精母细胞株模型，Western blot与qRT-PCR法检测相关基因ZFP38、RAD51、DMC1、PALB2表达水平。结果显示：在lincRNA-p21表达量改变后，ZFP38 mRNA表达量未受到显著影响，提示lincRNA-p21可能不直接调控ZFP38表达。而已知的ZFP38下游因子，包括RAD51、DMC1及PALB2的mRNA与蛋白水平则随着lincRNA-p21表达升高显著降低，而当lincRNA-p21下调时其表达显著升高，呈反比关系。随后我们运用RNA pulldown实验方检测发现lincRNA-p21可直接结合到ZFP38蛋白上，其可能是通过影响ZFP38蛋白功能进一步影响下游蛋白的表达。我们运用ZFP38特异性shRNA干扰序列转染精母细胞，qRT-PCR与Western blot法检测干扰ZFP38表达后下游蛋白RAD51、DMC1、PALB2 的表达，结果显示：RAD51、DMC1、PALB2蛋白的表达水平随着ZFE38表达的下调而降低。以上结果进一步的明确了lincRNA-p21在精子生成中的重要作用，该研究结果为精子发生异常以及男性不育的防治提供了一定的研究基础。

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