极低密度脂蛋白受体基因促进膀胱癌细胞迁移的作用及机制

Very low-density lipoprotein receptor gene promotes the migration of bladder cancer and its mechanism

目的探讨极低密度脂蛋白受体(VLDLR)对人膀胱癌细胞迁移活性的影响及机制。方法采用实时荧光定量反转录-聚合酶链反应(RT-qPCR)检测人源膀胱癌细胞(J82、UMUC3与T24)与正常尿路上皮细胞(SV-HUC-1)VLDLR的表达水平;采用小干扰RNA(siRNA)建立VLDLR敲低T24细胞模型,通过划痕实验与Transwell实验评估细胞的迁移活性;采用蛋白印迹法测定波形蛋白(Vimentin)、蛋白激酶B(Akt)与磷脂酰肌醇-3-激酶(PI3K)的蛋白含量。采用独立样本t检验进行统计。结果VLDLR在T24(2.70±0.55,t=5.294,P<0.05)、UMUC3(6.51±0.99,t=9.596,P<0.05)及J82中的含量(10.54±1.87,t=8.802,P<0.05)均显著高于SV-HUC-1(1.00±0.06);VLDLR敲低T24细胞模型建立成功,VLDLR核酸水平在siVLDLR-1(0.22±0.01,t=18.573,P<0.01)和siVLDLR-2(0.07±0.02,t=21.511,P<0.01)显著低于对照组;细胞相对迁移速率和发生迁移细胞数量在siVLDLR-1组(0.61±0.07,t=3.368,P<0.05;107.00±14.53,t=14.865,P<0.01)和siVLDLR-2组(0.63±0.04,t=3.305,P<0.05;87.00±29.60,t=11.716,P<0.01)均显著低于对照组,差异均有统计学意义。蛋白印迹结果显示,VLDLR敲低后PI3K和Akt含量未见明显变化,p-PI3K、p-Akt及Vimentin的蛋白水平明显下调。结论敲低VLDLR可能通过PI3K/Akt通路抑制人膀胱癌细胞迁移活性。

Objective To explore the effect of very low-density lipoprotein receptor(VLDLR)on the migration of bladder cancer and the underlying molecular mechanism.Methods Cultivated and extracted RNA from human bladder cancer cell lines(T24,UMUC3 and J82)and normal urothelial cells(SV-HUC-1),and quantitative real-time fluorescent quantitative real-time polymerase chain reaction(qRT-PCR)was performed to detect the transcription level of VLDLR in cells.Selected VLDLR high-expressed T24 cells and utilized siRNA to interfere with the expression of VLDLR,and performed the wound healing and the transwell assay to observe the effect of VLDLR on cell migration capacity.Then,Western blot were applied to detect the protein levels of phosphatidylinositol-3-kinase(PI3K),protein kinase B(Akt)and Vimentin.The SPSS version 23.0 and independent sample t test were employed to analyze.Results Compared with SV-HUC-1(1.00±0.06),VLDLR was up-regulated in T24(2.70±0.55,t=5.294,P<0.05),UMUC3(6.51±0.99,t=9.596,P<0.05)and J82(10.54±1.87,t=8.802,P<0.05).The expression level of VLDLR in T24 cells was significantly reducedin siVLDLR-1(0.22±0.01,t=18.573,P<0.01)and siVLDLR-2(0.07±0.02,t=21.511,P<0.01).The relative migration rate and the number of invaded cells in the siVLDLR-1(0.61±0.07,t=3.368,P<0.05;107.00±14.53 cells/filed,t=14.865,P<0.01)and the siVLDLR-2(0.63±0.04,t=3.305,P<0.05;87.00±29.60 cells/filed,t=11.716,P<0.01)showed a significantly decline.Western blot indicated that the contents of PI3K and Akt showed no significant changes when VLDLR knockdown,and the protein levels of p-PI3K,p-AKT and Vimentin were obviously downregulated.Conclusion Knockdown of the VLDLR mayinhibit the migration ability of human bladder cancer cells through the PI3K/AKT pathway.