MiR-185-5p靶向调控REG1A表达对膀胱肿瘤发生和侵袭的影响

Effect of miR-185-5p on proliferation and development of bladder cancer through targeted regulation of REG1A expression

关键词:  MiR-185-5p  REG1A  膀胱肿瘤

Key words:  MiR-185-5p  REG1A  bladder cancer

摘要:

目的　探讨REG1A基因和微小RNA miRNA-185-5p对膀胱癌细胞增殖、侵袭、血管形成和上皮间充质转化的影响，并探讨其可能存在的分子调控机制。方法　应用荧光实时定量PCR（qRT-PCR)分别检测膀胱癌细胞系中REG1A和miR-185-5p的mRNA表达水平，应用免疫组化技术检测24对膀胱癌临床样本中REG1A的蛋白表达水平。应用荧光素酶报告基因验证miR-185-5p和REG1A的特异性结合，进一步应用细胞转染技术和qRT-PCR明确miR-185-5p和REG1A的调控关系。应用细胞转染技术分别研究miR-185-5p抑制物(inhibitor)和模拟物(mimic)对膀胱癌T24细胞功能的影响。利用小RNA干扰技术敲低REG1A后进行T24细胞功能回复实验。结果　与正常尿路上皮细胞系SV-HUC-1相比，T24、J82和UM-UC-3细胞系中REG1A显著高表达（P<005），miR-185-5p显著低表达(P<005)；与癌旁组织相比，膀胱癌组织中REG1A蛋白显著高表达（P<005）。荧光素酶报告基因验证miR-185-5p和REG1A存在特异性结合，qRT-PCR结果表明，细胞转染后miR-185-5p靶向调控REG1A在T24细胞的表达。miR-185-5p mimic和inhibitor能分别抑制和增强T24细胞的增殖、侵袭、血管形成和上皮间充质转化（epithelial-mesenchymal transition, EMT），差异有统计学意义（P<005）。REG1A-siRNA能减弱miR-185-5p inhibitor对T24细胞的增殖、侵袭、血管形成能力和EMT的促进作用。结论　miR-185-5p靶向调控REG1A表达影响膀胱癌的增殖、侵袭、血管形成和EMT发生，miR-185-5p/REG1A调控通路是潜在的膀胱癌治疗靶点。

Abstract:

Objective　To investigate the effects of miRNA-185-5p on proliferation, invasion, angiogenesis and epithelial-mesenchymal transition（EMT) of bladder cancer cells, and its molecular regulatory mechanisms. Methods　The mRNA expression levels of REG1A and miR-185-5p in bladder cancer cell lines were detected by qRT-PCR; the protein expression of REG1A in 24 pairs of bladder cancer and adjacent bladder tissue samples was detected with immunohistochemistry. The luciferase reporter gene assay was used to verify the specific binding between miR-185-5p and REG1A; cell transfection and qRT-PCR assay were conducted to investigate the targeted regulatory relationship between miR-185-5p and REG1A. Cell transfection was used to study the effects of miR-185-5p inhibitor and mimic on the function of bladder cancer T24 cells; and small RNA interference was applied to knock down REG1A in T24 cells. Results　Compared with the normal urothelial cell line SV-HUC-1, the expression level of REG1A was significantly higher in bladder cancer T24, J82 and UM-UC-3 cells（P<005), and the expression level of miR-185-5p was significantly lower（P<005). Compared with adjacent tissues, the expression level of REG1A protein in bladder cancer tissues was significantly higher（P<005). The targeted regulatory relationship between miR-185-5p and REG1A was verified by the luciferase reporter gene, cell transfection and qRT-PCR assay. The transfection of miR-185-5p mimic or inhibitor suppressed or enhanced the proliferation, invasion, angiogenesis and EMT of T24 cells, respectively（P<005). REG1A siRNA attenuated the promotion of miR-185-5p inhibitor on the proliferation, invasion, angiogenesis and EMT of T24 cells. Conclusion　The miR-185-5p affects the proliferation, invasion, angiogenesis and EMT of bladder cancer through targeted regulation of REG1A expression, indicating that the miR-185-5p/REG1A regulatory pathway might be a potential therapeutic target for bladder cancer.