环状RNA circSTRBP靶向调控miR-744-5p在精子发生异常中的作用机制研究

circRNAs are found to be involved in the genesis and development of many diseases, however its role in spermatogenesis remains unclear. Our previous study found that there is a differential expression profile of circRNA-miRNA in the patients with non-obstructive azoospermia (NOA) compared with normal control, the expression of circSTRBP and miR-744-5p were dysregulated in the testis of NOA patients, and circSTRBP has the ability to compete endogenously with miR-744-5p. Moreover, overexpression of miR-744-5p or knockout of circSTRBP in mouse testis resulted in spermatogenic failure. On the basis of previous work, this study is to explore the relationship between the abnormal expression of circSTRBP/miR-744-5p and spermatogenic failure. Another aim is to determine the expression, localization and interaction of circSTRBP/miR-744-5p and its downstream genes, the mouse model of testicular specific miR-744-5p overexpression or circSTRBP knockout was made to observe various stage of spermatogenesis, the rescue experiments were conducted to observe the recovery of spermatogenesis. Finally, we explore the relationship between the expression of circSTRBP in serum or seminal plasma in NOA patients and their clinical characteristics. This study will help to better understand the role of non-coding RNA in spermatogenesis, and provide theoretical basis for the diagnosis and treatment of male infertility.

circRNA在多种疾病的发生发展过程中起重要作用，然而其在男性不育中的作用尚未见报道。申请人前期通过ceRNA芯片检测建立了非梗阻性无精子症（NOA）患者睾丸circRNA-miRNA-mRNA差异表达谱，发现circSTRBP与miR-744-5p在NOA患者中表达异常且存在靶向调控，小鼠睾丸中过表达miR-744-5p或敲除circSTRBP均可导致精子发生异常。本项目拟进一步深入研究：在人和小鼠精子发生中circSTRBP/miR-744-5p及其下游基因的表达定位及调控关系；构建circSTRBP敲除及miR-744-5p过表达的小鼠和细胞模型，并进行挽救实验，检测精子发生各时相重要事件变化；将NOA患者精浆和血清中circSTRBP表达与临床特征进行相关性分析，确定它能否作为新的男性不育标志物。本项目将有助于深入了解非编码RNA在精子发生中的作用，为男性不育的诊治提供理论基础。

本项目通过比较非梗阻性无精子症患者（NOA）与正常对照的睾丸组织ceRNA表达水平，构建了circRNA-miRNA-mRNA差异表达谱及共表达互作网络，并在独立大样本量中验证了多个circRNA和miRNA的变化趋势。我们通过睾丸网显微注射构建了在NOA中高表达的多个miRNAs的睾丸过表达模型，发现miR-744-5p mimic组睾丸生精小管中生精过程破坏严重，生精上皮细胞凋亡比例明显增加，而小鼠血清及睾丸内激素水平未受明显影响。同时我们发现miR-370和miR-143等miRNAs可呈剂量依赖性影响精子发生。为了进一步探索精子发生障碍中导致miR-744-5p表达异常的机制，我们通过荧光素酶报告基因系统发现了在候选circRNAs中circSTRBP与miR-744-5可能存在直接相互作用，利用RNA pull down技术结合生物素探针标记miR-744-5p发现miR-744-5p可与circSTRBP存在大量结合，细胞RNA原位杂交实验也显示circSTRBP和miR-744-5存在共定位。在小鼠睾丸中敲减circSTRBP基因也能导致类似miR-744-5p的精子发生障碍的病理表现，细胞水平circSTRBP表达下降可导致miR-744-5p表达上调和细胞增殖能力下降。我们利用软件预测、RNA-seq等手段，并结合分子生物学实验验证，确定了circSTRBP/miR-744-5p调控下游靶基因TEX14影响精子发生。此外，我们还在人睾丸组织中明确了包括circSTRBP在内的多个circRNAs的表达定位规律，发现了其表达量与与睾丸Johnson评分具有明显的正相关性；利用Logistic回归和ROC曲线检测发现了精浆中多个circRNA和miRNA对于NOA具有良好的预测准确性。本研究为无精子症病因学的阐明和诊断新方法的建立提供了新的靶点和理论参考。