睾丸源性miR-29c对精子DNA损伤调控的研究

Sperm DNA damage is an important factor that contribute to male infertility，and is also closely related to abortion and birth defects. It is confirmed that the miRNAs are indispensable in spermatogenesis, and participate in the regulation of cellular DNA damage and repair. Therefore, we speculated that miRNA might be a crucial part in the regulation process of sperm DNA damage. In our preliminary studies, we screened the testicular source miRNAs which were stably in the seminal plasma by high-throughput sequencing in patients with serious sperm DNA damage. The differentially expressed miRNAs were verified in mice and miR-29c was found to be associated with sperm DNA damage. Therefore, we hypothesize that miR-29c involves in the regulation process of sperm DNA damage in the testis. To confirm this hypothesis and explore its mechanism, we apply this project to study the localization and expression of miR-29c in testis in mice, the identification of target genes, and pathways and mechanism of regulation of miR-29c expression in sperm DNA damage. In addition, we use the seminal plasma, which is easy to access, to replace testicular biopsy in our research, which will provide an important means for noninvasive method to study the physiological and pathological processes in the testis.

精子DNA损伤是引起男性不育的常见原因，与流产和出生缺陷也有密切关系。研究证实miRNA在精子发生中不可或缺，并参与体细胞中DNA损伤修复的调控，因此我们推测miRNA可能是精子DNA损伤调控的重要因子。本课题组前期通过高通量测序筛选出在精浆中能稳定检测到的睾丸来源miRNAs，将其在精子DNA损伤严重的患者中筛选，再将筛选出差异性表达的miRNAs在动物实验中验证，发现miR-29c与精子DNA损伤相关。因此我们推断：miR-29c可能在睾丸中参与调控精子DNA损伤。为证实此推断并研究其作用机制，本项目拟开展miR-29c在人睾丸和精浆中表达的相关性研究，并检测miR-29c在睾丸中的定位和表达，鉴定其靶基因后进行信号通路研究和其下调的原因，来揭示miR-29c对精子DNA损伤的机制。此外本项目利用精浆的易获得性取代睾丸活检，从而为无创性方法研究睾丸内生理和病理过程提供重要手段。

人精子染色质不仅是将父亲的遗传信息传递给后代的载体，而且其完整性直接影响到受精、卵裂、流产等生殖的各个方面。精子DNA损伤被认为是一个新的评价精子质量的检测指标，对男性生殖力的评估和选择何种辅助生殖技术具有重要的临床意义，而miRNA可能是精子DNA损伤调控的重要因子。.本项目围绕精子DNA损伤的表观遗传调控机制，在1090名男性不育患者中筛选了94例精子DNA损伤高的患者，而后从18个睾丸来源的精浆miRNA中筛选，并验证了2个与人精子DNA损伤相关的睾丸特异性miR-29c和miR-424。在不同时间点、不同温度和反复冻存情况下证实游离miRNA在精浆中稳定存在，在物种间有高度保守性。在小鼠睾丸中对候选miRNA进行定位和表达检测，发现其在不同生精细胞中的表达谱。进一步成功制备了用于抑制miR-322和miR-29c表达的慢病毒载体，利用睾丸显微注射在小鼠睾丸中下调miR-322的表达，发现可引起小鼠精子DNA损伤，证实与睾丸中γH2AX的表达增加相关。通过生物信息学预测并实验鉴定出了候选miRNA的两个靶基因chk1和Ddx3x，精原细胞系中实验发现miR-322可抑制chk1的表达，抑制miR-322的表达可显著降低GC-2细胞的增值率，增加了GC-2的凋亡。最后在DNA损伤高患者人群中通过高通量测序发现其与精子DNA甲基化的异常相关。.本项目的完成，对miRNA参与调控精子DNA损伤的机制有了新的认识，对候选miRNA的靶基因的机制研究和miRNA受甲基化调控的相关研究，为解决男性生育力问题拓宽视野，同时作为无创检测手段有利于临床疾病标记物的建立。