lncRNA AC000061.1/miR-1199调控CFTR在非梗阻性无精子症发病机制中的研究

非梗阻性无精子症（NOA）是严重危害男性生育力的疾病，发病机制尚不清。项目组前期已筛选出NOA及正常对照睾丸组织中lncRNA和mRNA差异表达谱。选择差异表达极为显著的LncRNA AC000061.1为研究对象，并预测筛选出与其结合的miR-1199及下游靶基因纤维化跨膜转运调节因子（CFTR）。已知CFTR与NOA发病密切相关,故以其为切入点进行上游调控机制的研究。本项目拟通过目的lncRNA高、低表达载体干预和荧光素酶实验证实lncRNA对miR-1199的靶向调控；其次采用miR-1199模拟物，抑制物及阴性对照转染NTERA-2细胞，明确miR-1199与CFTR基因的结合位点；最后动物在体实验探究目的LncRNA对小鼠生精细胞功能的调控机制和对小鼠生育力的影响。探讨LncRNA/miR-1199-CFTR分子机制在NOA发病中的意义，并为寻找NOA诊断分子标志物和治疗新思路。

Non-obstructive azoospermia (NOA) is the most serious disease that affecting male fertility, and its pathogenesis is unclear. we have identified the lncRNA and mRNA expression profiles in testis tissues of NOA patients and the normal testis tissues through high-throughput sequencing. lncRNA AC000061.1 was selected as the research object for its significantly high expression in NOA. Besides, we identified the potential binding sites between lncRNA and miR-1199, CFTR and miR-1199 using microarray analysis. CFTR is a gene well known for having close relationship with the pathogenesis of NOA, therefore we applied CFTR as the entry point to study the mechanism of lncRNA/mir1199 in NOA. We adopt high and low expression vector intervention of lncRNA and luciferase assay to verify the targeted regulation of miR-1199 by lncRNA. Then we verify the binding site of miR-1199 and CFTR gene through transfection of miR-1199 mimic, miR-1199 inhibitor and the negative control to NTERA-2 cells. Finally, we apply animal experiments in vivo to determine the effect of lncRNA on the mouse’s spermatogenic function and fertility. This study will explore the molecular mechanism of LncRNA/miR-1199-CFTR in the pathogenesis of NOA, which may provide new perspective for searching NOA molecular markers and therapeutic targets.