下调ETS1对人膀胱癌细胞侵袭能力的影响

Effect of down-regulation of ETS1 on invasion of bladder cancer cells

目的:探究E26转录因子1(ETS1)在膀胱癌(BCa)组织中的表达以及下调ETS1对人膀胱癌T24细胞侵袭能力的影响。方法:通过免疫组织化学方法检测人正常膀胱上皮组织和BCa组织(组织均来源于我院泌尿外科)中ETS1蛋白的表达;体外实验,将人工合成的靶向ETS1的小干扰RNA(siRNA)转染至T24细胞中,根据细胞转染情况和siRNA序列,将T24细胞分为阴性对照组(NC组)和敲低组(siETS1-1和siETS1-2组);采用Transwell小室实验检测T24细胞系NC组、siETS1-1组和siETS1-2组细胞侵袭能力;采用蛋白印迹法(Western blot法)和荧光定量PCR法检测NC组、siETS1-1组和siETS1-2组中ETS1、基质金属蛋白酶2(MMP2)和基质金属蛋白酶9(MMP9)等蛋白及mRNA的表达;采用Pearson相关性分析膀胱癌肿瘤基因组图谱(TCGA)临床数据库中ETS1与MMP2和MMP9的相关性。结果:ETS1在人正常膀胱上皮组织中相对表达水平为3.36±0.53,低于BCa组织的5.52±0.39,差异有统计学意义(P<0.01);转染后,荧光定量PCR结果显示,NC组、siETS1-1组和siETS1-2组ETS1相对表达水平分别为1.00±0.05、0.32±0.04和0.28±0.02,差异均具有统计学意义(P<0.05);Transwell结果显示,NC组、siETS1-1组和siETS1-2组侵袭细胞数分别为25.67±3.48、12.67±1.86、9.67±2.60,NC组高于siETS1-1、siETS1-2组,差异均具有统计学意义(P<0.05);Western blot结果显示,siETS1-1、siETS1-2组与NC组比较,MMP2和MMP9都明显下调;Pearson相关性分析结果显示,在BCa组织中,ETS1的表达与MMP2(P<0.0001)和MMP9(P<0.0001)的表达均呈正相关,且差异均具有统计学意义。结论:通过免疫组织化学实验和体外细胞实验检测显示,ETS1在膀胱癌组织中表达上调,且下调ETS1可以抑制膀胱癌细胞的侵袭特性。

Objective:To investigate the expression of ETS1 in bladder cancer(BCa)and its effect on invasion of T24 cell line.Methods:Immunohistochemical staining method was used to evaluate the expression of ETS1 protein in human normal bladder epithelia and BCa tissues which were from the department of urology of our hospital.In vitro,siRNA strategy was used to knock down ETS1 in T24 cell lines which was divided into NC(negative control)group,siETS1-1(knockdown 1)group and siETS1-2(knockdown 2)group.Transwell assay was performed to explore invasion ability of NC group,siETS1-1 group and siETS1-2 group.Western blot analysis and real-time quantitative PCR were used to detect the protein and mRNA expression of ETS1,MMP2 and MMP9 in NC group,siETS1-1 group and siETS1-2 group.Pearson correlation was used to analyze the correlation between ETS1 mRNA and MMP2 mRNA or MMP9 mRNA in BCa of TCGA database.Results:The expression of ETS1 protein was significant higher in BCa tissues than that in normal bladder epithelium[(5.52±0.39)vs(3.36±0.53)](P<0.01).Real-time quantitative PCR showed that the expression of ETS1 were 1.00±0.05,0.32±0.04 and 0.28±0.02,respectively in NC group,siETS1-1 group and siETS1-2 group after transfection,and there were statistical differences among the three groups(P<0.05).The results ofTranswell showed that the invasion cells were25.67±3.48,12.67±1.86 and 9.67±2.60,respectively in NC group,siETS1-1 group and siETS1-2 group,and there were statistical differences among the three groups(P<0.05).The results of Western blot showed that the expression of MMP2 and MMP9 were significant lower in siETS1-1 and siETS1-2 group than NC group.The Pearson correlation showed that ETS1 was positively correlated with MMP2(P<0.0001)and MMP9(P<0.0001)in BCa.Conclusion:ETS1 was upregulated in BCa tissues and downregulation of ETS1 could inhibit BCa cell invasion.