X染色体连锁miR-507遗传变异在精子生成障碍中的作用及其机制

X-linked pre-miR-507 rs5951785 was significantly rich in whole blood of non-obstructive azoospermia (NOA) identified by next generation sequencing and validated in a large sample of people. Our previous study found that X-linked pre-miR-507 rs5951785 decreased the expression level of mature miR-507 and miR-507 inhibitor induced the germ cell apoptosis and inhibited cell proliferation. Moreover, the results from bioinformatics analysis and preliminary experiment demonstrated that Glycogen Synthase Kinase 3 beta (GSK-3β) was the target of miR-507. GSK-3β, expressed during meiosis stage, is vital in the process of spermatogenesis, all of these suggested that miR-507/ GSK-3β signaling pathway might play an important role in spermatogenesis failure. The study will verify the relationship between miR-507 and spermatogenesis failure. We will explore the role of miR-507 in germ cell biological function via overexpression and knock down strategies. Then we will use rescue experiment strategy to analyze the downstream regulation mechanism of miR-507 modulating GSK-3β signaling pathway. The study will provide new clue and potential intervention target of male infertility.

X染色体连锁miR-507 rs5951785是我们通过二代测序及大样本独立人群验证所筛选到的、在非梗阻性无精子症全血中显著富集的遗传变异位点。前期已发现：X染色体连锁miR-507 rs5951785遗传变异位点负调控成熟体miR-507的表达；miR-507沉默表达可诱导生殖细胞凋亡、抑制生殖细胞的增殖；生物信息学及实验均证实GSK-3β为其可能结合靶标，而GSK-3β表达于生殖细胞的减数分裂期，在精子生成中发挥着重要作用，提示miR-507可能通过调控GSK-3β的机制发挥其生物学作用。研究拟进一步论证 miR-507与精子生成的关系；以过表达、沉默的策略，从正反两方面论证miR-507对生殖细胞生物学功能的作用；以挽救实验策略，论证 miR-507通过调控GSK-3β发挥其生物学作用的分子机制。本研究可为男性不育的防治提供新线索、新靶标。

TBL1XR1是一种保守性蛋白，在癌症和精子生成中发挥重要作用。为了探讨TBL1XR1在弱精症中的作用，我们采用了体内外联合实验。首先，我们发现TBL1XR1在汉族人群中弱精症者中的表达水平显著降低。以TBL1XR1敲除小鼠为模型，我们发现小鼠的精子活力减弱。随后，我们采用CHIP测序和CHIP实时荧光定量分析，发现TBL1XR1的下游基因的表达量减低。为了研究TBL1XR1在组蛋白和鱼精蛋白交互的作用，我们采用了蛋白免疫印迹和荧光免疫分析，发现 TBL1XR1的缺失导致组蛋白和鱼精蛋白交互的失败。最后，荧光报告实验分析发现， TBL1XR1是miR-507的靶基因。总之，我们的数据显示TBL1XR1在弱精症中发挥重要作用，TBL1XR1为改善精子活力提供了新型分子机制。