DNA甲基化参与电压依赖性阴离子通道蛋白2（VDAC2）调控精子活力的机制研究

Voltage dependent anion channel 2(VDAC2) plays an important role in the biologic behavior, such as apoptosis and transport function. However, little research has been reported on the mechanism of VDAC2 distributed on the sperm head region and sperm flagellum mediated the sperm motility. Our previously case-control studies confirmed that VDAC2 mRNA expression was decreased in the idiopathic asthenozoospermia(IAS), and its expression was regulated by the DNA methylation status. DNA methylation always silences gene expression by inhibiting the binding of transcription factors to core promoter, we then compared the VDAC2 promoter methylation states of IAS with normozoospermia and found that the methylation pattern of CpG island was significantly increased in IAS. With literature review and preliminary results, we propose the hypothesis that abnormal hypermethylation of VDAC2 promoter CpG island may disrupt the combination of transcription factors and core promoter, depress the expression of VDAC2 mRNA and protein, and decrease the Ca2+ content in sperm, which result in the decline of sperm motility and male infertility. To verify the above hypothesis, we perform the molecular mechanism study in two aspects, including abnormal DNA hypermethylation inhibit VDAC2 expression and VDAC2 regulates the sperm motility, to reveal the potential causes of IAS disease from epigenetics.

电压依赖性阴离子通道蛋白2(VDAC2)在调节细胞凋亡以及物质转运等生物学行为中发挥重要作用。然而关于精子头部和鞭毛区VDAC2蛋白调控精子活力分子机制的研究鲜有报道。我们前期已证实，特发性弱精子症患者（IAS）精子VDAC2表达降低,并且VDAC2表达受甲基化调控。DNA甲基化阻止转录因子结合抑制基因的表达，我们比较正常人精子和IAS精子VDAC2启动子区CpG岛甲基化，发现VDAC2表达受甲基化调控；同时IAS VDAC2启动子区CpG岛甲基化明显增高。结合查阅文献和我们前期获得的结果，我们提出假设：VDAC2启动子区CpG岛高甲基化抑制转录因子与核心启动子结合，抑制VDAC2表达，精子内Ca2+含量降低，导致精子活力下降。为验证此假说，本项目拟从DNA甲基化调控VDAC2基因表达和VDAC2蛋白调控精子活力的分子机制两个层面，阐述特发性弱精子症患者在表观遗传层面的发病原因。

本项目我们进一步回答了DNA甲基化调控VDAC2基因转录表达，以及VDAC2调控精子活力的具体分子机制。前期研究已经明确特发性弱精子症患者中VDAC2表达与VDAC2启动子区CpG岛（−1337bp ~ −1059 bp）异常高甲基化修饰有关。我们进一步通过5-AZA-杂氮胞苷去甲基化处理工具细胞293T及HK-2，甲基化特异性PCR证实甲基化干预成功，实时定量PCR及琼脂糖凝胶电泳（WB）技术证实VDAC2表达明显增加；同时，利用补丁甲基化技术证将CpG岛（ −1337 bp ~ −1059 bp）进行甲基化修饰后，荧光素酶报告基因实验证实VDAC2转录活性明显降低。我们进一步利用JASPER数据库预测ZNF384、SNAI2，以及ZEB1 三个转录因子可能参与DNA甲基化修饰调控VDAC2转录表达，通过干扰及过表达技术发现转录因子ZEB1参与VDAC2基因的转录调控。染色质免疫共沉淀技术（ChIP）揭示转录因子ZEB1与VDAC2启动子区CpG岛（ −1337 bp ~ −1059 bp）结合，凝胶迁移率实验（EMSA）结果表明经补丁甲基化修饰后，ZEB1与 VDAC2启动子区CpG岛（ −1337 bp ~ −1059 bp）结合力与对照组相比显著降低。由此表明，在DNA甲基化酶DNMT3b的作用下，VDAC2启动子区CpG岛（ −1337 bp ~ −1059 bp）甲基化水平增加，抑制了转录因子ZEB1与启动子区的结合，从而抑制了下游VDAC2的表达。同时，我们利用VDAC2单克隆抗体体外研究表明VDAC2可能通过影响精子内ATP含量调控精子活力；依托于南京医科大学生殖国家重点实验室，采用睾丸网微注射技术构建VDAC2低表达精子模型，研究结果表明VDAC2主要通过干预精子内ATP及钙离子含量，调控精子活力。相比较钙离子前后浓度的改变，ATP对精子活力的影响更加直接，受VDAC2调控的影响也更加大。由此表明，在特发性弱精子症患者中VDAC2 基因启动子区 CpG 岛异常的高甲基化抑制转录因子与核心启动子内转录因子结合位点结合，引起 VDAC2 mRNA 和 VDAC2 蛋白表达降低，精子内ATP含量明显降低，致精子活力减弱，最终引起男性不育。