miR-195-5p通过Smad7调控阴茎海绵体平滑肌细胞表型转化的机制研究

ED (Erectile Dysfunction, ED) is a common disease which seriously harassed men. MiR-195-5p was a highly homologoused and conserved miRNA identified from differential miRNA expression profile between aged-ED and normal rat. MiR-195-5p is highly homologous and conserved in mammals, so it will contribute to the treatment of ED by clarifying its function and mechanism. Our previous studies revealed overexpression of miR-195-5p in corpora cavernosa could reduce erectile function and increase collagen fiber. Bioinformatics analysis and experiments suggested Smad7 was its candidate target gene. Based on these data, we will confirm the role of miR-195-5p in the pathogenesis of ED aspects from function, organization and molecular from the perspective of Smad7, and reveal the regulatory of the miR-195-5p. So far there is no report of miR-195-5p, and our studies may open up a world in ED research. This study may provide a theoretical basis for the targets of the diagnosis and treatment of ED.

勃起功能障碍（Erectile Dysfunction, ED）是严重困扰男性的常见疾病。miR-195-5p是前期研究发现的差异高表达于老年ED大鼠阴茎组织的miRNA。鉴于miR-195-5p在哺乳动物高度同源和保守，因此阐明其功能与机制将有助于解决男性ED的治疗难题。前期生物信息学及实验发现miR-195-5p过表达可导致勃起功能下降，阴茎海绵体胶原纤维增多，其下游机制与Smad7有关。文献证实Smad7是海绵体平滑肌细胞表型转化的关键因子之一，与勃起功能密切相关。本项目将在已有研究基础上，从功能、组织、分子等多方面明确miR-195-5p在ED发病中作用，并以Smad7为机制研究切入点，揭示miR-195-5p的可能作用机制。miR-195-5p在ED中尚无报道，具有源头创新性，可能为ED提供新的病因机制线索和新的诊断治疗靶标。

背景:表型转换是指平滑肌细胞由收缩型向合成型转变并获得增殖能力的过程。表型转化涉及许多细胞功能的改变，如胶原沉积和纤维化，影响阴茎的正常勃起功能。.目的:探讨miR-195-5p在调节海绵体平滑肌(CCSM)细胞表型转换中的作用。方法:将一个小型的Smad7病毒载体和一个miR-195-5p模拟物或si- Smad7病毒载体和一个miR-195-5p抑制剂转染到CCSM细胞中。细胞来自大鼠海绵体平滑肌组织细胞原代培养物。采用实时聚合酶链反应(Real-time polymerase chain reaction, PCR)实验、Western blotting、苏木精-伊红(hematoxylin-eosin, HE)染色、transwell实验、MTT法、流式细胞术检测CCSM细胞的miR-195-5p、Smad7、表型切换标记物及相关蛋白的表达，以及细胞形态、迁移、增殖和凋亡。主要观察指标:通过过表达和沉默策略研究miR-195-5p在CCSM细胞中的调控作用。结果:过表达miR-195-5p可促进CCSM细胞由收缩型向合成型转化。同时，CCSM细胞的迁移能力和增殖能力增强，凋亡率降低。miR-195-5p的表达沉默则产生相反的效果。挽救实验的结果表明，过表达Smad7可以单独挽救过表达miR-195-5p引起的CCSM细胞表型从收缩型向合成型转换的抑制。此外，对CCSM细胞迁移能力和增殖能力的增强作用也被消除，细胞凋亡率增加。同时沉默miR-195-5p和Smad7会产生相反的效果。结论:miR-195-5p可能通过靶向Smad7调控CCSM细胞表型转换。