heparanase通过调控EMT促进人膀胱移行细胞癌细胞T24迁移及侵袭的作用研究

目的 探讨乙酰肝素酶（heparanase）是否通过上皮-间充质转化（EMT）影响膀胱移行细胞癌细胞的侵袭及迁移能力.方法 利用荧光定量PCR来检测人膀胱移行细胞癌细胞T24、EJ、MGH-U1以及BIU-87中heparanase的表达水平;设计并合成靶向heparanase的特异性shRNA,通过脂质体转染法转染heparanase表达最高的膀胱移行细胞癌细胞T24以构建稳定低表达heparanase细胞株,通过Western blot法检测和定量PCR来验证shRNA的干扰效率;通过transwell法检测干扰后细胞的迁移及侵袭能力,Western blot法检测EMT相关指标E-cadherin和N-cadherin以及其上游信号分子Snail和WNT-5a的变化.结果 heparanase-shRNA转染后,能够有效抑制T24细胞中heparanase的表达;Transwell法结果显示,heparanase干扰组细胞侵袭及迁移能力显著低于阴性对照组（P ＜0.001）.Western blot法检测结果发现,heparanase干扰组细胞的E-cadherin表达增加,N-cadherin的表达降低;此外,heparanase干扰组细胞的Snail以及WNT-5a表达较对照组显著下降.结论 运用RNA干扰技术能够有效沉默T24细胞的heparanase基因,并诱导其迁移侵袭能力的下降,其可能的机制是通过调节EMT的上游信号分子Snail、WNT-5a的表达来调控EMT,从而影响膀胱移行细胞癌细胞的迁移及侵袭.提示heparanases可能在膀胱移行癌T24细胞的发生发展中起重要作用,抑制heparanase的表达可能成为一种治疗膀胱移行细胞癌的新方法.

Down-regulating of Heparanase Suppresses Invasion and Migration of Human Bladder Transitional Cell Carcinoma T24 via Inhibiting EMT

Objective To investigate the effect of down-regulation of Heparanase on the invasion and migration of bladder transitional cell carcinoma cells T24.Methods shRNA targeting Heparanase was designed and synthesized,and then transfected into the T24 cells via Lipofectamine 2000 mediation.The migration ability and invasion ability of T24 was detected using transwell assay.Expression of E-cadherin,N-cadherin,WNT-5a and Snail was detected by using western blot.Results Heparanase-targeted shRNA could down -regulate the Heparanase expression of T24.Compared with control group,cell migration ability and invasion ability was significantly inhibited in shRNA interference group （P ＜ 0.01）.In addition,the expression of E-cadherin was increased,while that of N-cadherin,WNT-5a and Snail was decreased in shRNA interference group.Conclusion Down-regulation of heparanase can induce inhibition of invasion and migration in bladder transitional cell carcinoma cells T24 via regulating epithelial mesenchymal transitions（EMT）.It could be regarded as a novel target for clinical diagnosis and gene therapy for bladder transitional cell carcinoma.