抑制COL6A1基因表达对膀胱癌T24细胞增殖和侵袭能力的影响

Effect of down-regulated COL6A1 on proliferation and invasion of bladder carcinoma T24 cells

目的:研究小干扰RNA抑制COL6A1基因表达对膀胱癌T24细胞增殖和侵袭能力的影响。方法:设计合成靶向COL6A1基因的siRNA,采用阳离子脂质体试剂瞬时转染膀胱癌细胞株T24,利用实时荧光定量PCR和Western blot检测RNA干扰后COL6A1基因的沉默效果,评价siRNA设计的合理性及RNA干扰抑制COL6A1表达的有效性,分别在RNA干扰后第1、2、3天应用噻唑蓝(MTT)比色法检测细胞增殖状态,并绘制生长曲线,应用Transwell法检测各组膀胱癌侵袭能力。结果:靶向COL6A1的siRNA成功抑制了膀胱癌细胞株T24中COL6A1 mRNA及蛋白表达,COL6A1-siRNA组蛋白相对表达量为阴性对照组的14.1%,mRNA相对表达量为空白对照组的20.5%;MTT比色法显示COL6A1-siRNA组细胞增殖能力显著减弱(P<0.05),在第1、2、3天后重复检测,分别达到了空白对照组的75.6%、58.2%和49.4%;Transwell细胞迁移实验显示COL6A1-siRNA组细胞迁移能力明显减弱(P<0.05),穿膜细胞数为空白对照组的45.3%。结论:利用siRNA技术能有效降低COL6A1基因的表达,同时有效抑制T24细胞的体外增殖和侵袭的能力。

Objective:To examine the influence of COL6 A1 on proliferation and invasion of bladder carcinoma cell.Method:The expression of COL6 A1 gene was knocked down using RNA interference(RNAi)in the bladder carcinoma cell T24.The transcription level of COL6 A1 was tested by RT-PCR.The expression of COL6 A1 protein was examined by Western blotting.The cell proliferation was detected by MTT.The cell’s invasion ability was performed on Matrigel transwell assay.Result:The results showed that the level of COL6 A1 mRNA and protein expression were significantly reduced after RNAi with 20.5%of control group on mRNA expression and 14.1%of control group on protein expression.MTT assay showed that the proliferation of T24 cell was decreased due to RNA interference,with OD values reaching 75.6%,58.2%and 49.4%of control group at 1 d,2 d and 3 d respectively.Transwell assay showed that invasion abilities of T24 cells were reduced obviously due to COL6 A1 RNAi(P<0.05),the transmembrane cells were 54.3%of control group.Conclusion:Our findings revealed that down-regulated COL6 A1 could inhibit abilities of proliferation and invasion in T24 cells.COL6 A1 can serve as a potential target for gene therapy of human bladder carcinoma.