胞质分裂蛋白调节因子1通过激活黏附斑激酶调控膀胱癌细胞的迁移和侵袭

Protein regulator of cytokinesis 1 regulates the migration and invasion of bladder cancer cells by activating focal adhesion kinase

目的探讨胞质分裂蛋白调节因子1(PRC1)对膀胱癌细胞迁移和侵袭的影响及黏附斑激酶(FAK)在其中的作用。方法设计合成靶向抑制PRC1的小干扰RNA(siRNA),分别转染人膀胱癌细胞系EJ和T24,两种细胞系各分为对照组(转染对照siRNA)和敲低组(转染PRC1 siRNA),通过实时荧光定量聚合酶链反应(PCR)和蛋白质免疫印迹法检测两组PRC1、FAK、桩蛋白(Paxillin)的mRNA和蛋白质表达水平以及FAK和Paxillin的磷酸化水平。通过细胞迁移和侵袭实验分析两组的迁移和侵袭能力。采用t检验进行组间比较。结果敲低组EJ、T24细胞PRC1 mRNA表达量(0.43±0.11、0.55±0.07)低于对照组(1.00±0.19、1.00±0.20,t=4.533、3.699,P<0.05)。敲低组FAKmRNA表达量(0.95±0.05、1.09±0.14)与对照组差异无统计学意义(1.00±0.20、1.00±0.20,t=0.423、0.622,P>0.05)。敲低组PaxillinmRNA表达量(1.12±0.01、1.02±0.10)相较对照组差异无统计学意义(1.00±0.08、1.00±0.04,t=2.521、0.360,P>0.05)。敲低组EJ、T24细胞FAK和Paxillin蛋白质表达量(0.90±0.02、0.99±0.02和0.93±0.01、1.10±0.02)相较对照组差异无统计学意义(0.88±0.12、0.99±0.01和0.91±0.30、1.08±0.02,t=0.714、0.147和0.806、1.019,P>0.05)。敲低组EJ、T24细胞FAK和Paxillin蛋白质磷酸化水平(0.25±0.02、0.20±0.01和0.47±0.02、0.84±0.04)显著低于对照组(2.22±0.30、2.73±0.15和0.86±0.02、1.13±0.04,t=11.510、28.390和20.450、8.576,P<0.01)。迁移实验中敲低组穿越的细胞数[(38.33±3.21)、(34.67±2.52)个]显著少于对照组[(114.00±7.55)、(71.33±4.04)个,t=15.970、13.340,P<0.01],差异有统计学意义。侵袭实验中敲低组穿越的细胞数[(24.67±3.21)、(29.00±3.00)个]显著少于对照组[(84.00±5.56)、(69.33±3.21)个,t=15.980、15.890,P<0.01]差异有统计学意义。结论下调PRC1的表达可通过抑制FAK的磷酸化降低膀胱癌细胞的迁移和侵袭能力.

Objective To investigate the effect of protein regulator of cytokinesis 1(PRC1)on the migration and invasion of bladder cancer cells and the role of focal adhesion kinase(FAK).Methods Small interfering RNA(siRNA)targeting inhibition of PRCI was designed,synthesized and transfected in-to bladder cancer cell lines EJ and T24 respectively.The two cell lines were divided into control group(transfected with control siRNA)and knockdown group(transfected with PRC1 siRNA).The mRNA and protein expression levels of PRC1,FAK and Paxillin and the phosphorylation levels of FAK and Paxillin in the two groups were detected by real-time quantitative PCR and Western blotting.The migration and inva-sion abilities of the two groups were analyzed by cell migration and invasion experiments.Comparisons be-tween groups were performed using t-tests,and P<0.05 was considered statistically significant.Results The expression of PRC1 mRNA in EJ and T24 cells in knockdown group(0.43±0.11,0.55±0.07)was lower than that in control group(1.00±0.19,1.00±0.20.t=4.533,3.699,P<0.05).There was no significant difference in FAK mRNA expression between the knockdown group(0.95±0.05,1.09±0.14)and the control group(1.00±0.20,1.00±0.20,t=0.423,0.622,P>0.05).There was no significant difference in Paxillin mRNA expression between the knockdown group(1.12±0.01,1.02±0.10)and the control group(1.00±0.08,1.00±0.04,t=2.521,0.360,P>0.05).There was no significant difference in the protein expression of FAK and Paxillin in EJ and T24 cells between the knockdown group(0.90±0.02,0.99±0.02 and 0.93±0.01,1.10±0.02)and the control group(0.88±0.12,0.99±0.01 and 0.91±0.30,1.08±0.02,t=0.714,0.147 and 0.806,1.019,P>0.05).The protein phosphorylation levels of FAK and Paxillin in EJ and T24 cells in knockdown group(0.25±0.02,0.20±0.01 and 0.47±0.02,0.84±0.04)were significantly lower than those in control group(2.22±0.30,2.73±0.15 and 0.86±0.02.1.13±0.04,t=11.510,28.390 and 20.450,8.576,P<0.01).In the migration experiment,the number of migrating cells in the knockdown group[(38.33±3.21),(34.67±2.52)cells]was significantly less than that in the control group[(114.00±7.55),(71.33±4.04)cells,t=15.970,13.340,P<0.01],and the difference was statistically significant.In the invasion experiment,the number of invasive cells in the knockdown group[(24.67±3.21),(29.00±3.00)cells]was significantly less than that in the control group[(84.00±5.56),(69.33±3.21)cells,t=15.980,15.890,P<0.01].Conclusion Downregulation of PRC1 expression can reduce the migration and invasion ability of bladder cancer cells by inhibiting the phosphorylation of FAK.