siRNA抑制S100A4蛋白对人胃腺癌细胞增殖和凋亡及化疗敏感性的影响

目的 研究应用小干扰RNA沉默S100A4蛋白后对人胃腺癌细胞BGC-823增殖、凋亡及化疗敏感性的影响.方法 人胃腺癌BGC-823细胞系转染siRNA,RT-PCR检测转染效果后将对数期胃腺癌细胞分为干扰组、阴性对照组、正常对照组,MTT检测不同浓度奥沙利铂对胃腺癌细胞中效浓度IC50值;绘制细胞生长曲线;TUNEL法检测细胞凋亡;RT-PCR检测各组细胞mRNA变化;Western blot检测蛋白水平的变化,计量资料选用t检验,SPSS17.0分析RT-PCR检测各组细胞mRNA的变化,Western blot检测蛋白水平的变化,细胞生长曲线描述细胞增殖,TUNEL法检测细胞凋亡,MTT法检测不同浓度奥沙利铂对胃腺癌细胞中效浓度IC50值.计量资料以((x)±s)表示,多个样本比较采用单因素方差分析和LSD检验方法.P＜0.05为差异有统计学意义.结果 RT-PCR结果显示BGC-823细胞转染S100A4siRNA 48 h后,S100A4mRNA的表达量分别为:(0.674±0.011)、(0.652±0.021)、(0.345±0.040),正常对照组与干扰组相比差异有统计学意义(P =0.012,P＜0.05),阴性对照组与干扰组差异均有统计学意义(P =0.000,P＜0.05),正常对照组与阴性对照组差异无统计学意义(P =0.380,P＞0.05);Western blot结果显示BGC-823细胞转染S100A4 48 h后表达量明显下调分别为:(0.654±0.025)、(0.642 +0.014)、(0.317 +0.061),正常对照组与干扰组相比差异有统计学意义(P=0.01,P＜0.05),阴性对照组与干扰组差异均有统计学意义(P=0.000,P＜0.05),正常对照组与阴性对照组无统计学差异(P =0.341,P＞0.05).S100A4siRNA转染后人胃腺癌BGC-823细胞增殖下降;TUNEL法结果显示转染后凋亡明显增加;MTT结果表明人胃腺癌BGC-823单用奥沙利铂中效浓度IC50分别为56.31 μmol/L,转染后奥沙利铂中效浓度IC50为0.654 μmol/L.结论 本研究结果表明,siRNA沉默S100A4蛋白后抑制胃腺癌细胞增殖、诱导凋亡并提高奥沙利铂化疗敏感性,提示S100A4可能是治疗胃腺癌的有效靶点.

Effect of small interference RNA silenced S100A4 protein in human gastric carcinoma cells on proliferation,apoptosis and chemotherapy sensitivity in vitro

Objective To study the application of small interfering RNA silencing S100A4 protein in human gastric cancer cell BGC-823 proliferation,apoptosis and the effect of chemotherapy sensitivity.Methods Human gastric carcinoma cell line BGC-823 transfection siRNA,RT-PCR detected the changes of mRNA after transfection.Groups divided into interference group,negative control group and normal control group.MTT test determined different concentrations of oxaliplatin in gastric cancer cells and calculated IC50,then draw cell growth curve,TUNEL method to detect apoptosis,RT-PCR tested each cell mRNA changed,Western blot detected the change of the S100A4 protein.All data analysis by SPSS17.0,t test applied,RT-PCR and Western blot results analysis by SPSS17.0,comparing multiple samples by using single factor analysis of variance and LSD test.P ＜ 0.05 was statistically significant.Results RT-PCR results showed that BGC-823 cell transfection,S100A4mRNA expression quantity respectively after 48 hours:(0.674+0.011),(0.652+0.021),(0.345 + 0.040),the interference group and normal control group were statistically significant (P =0.012,P ＜ 0.05) and the negative control group with interference group differences were statistically significant (P =0.000,P ＜ 0.05),and normal control group was no statistically significant difference with the negative control group (P =0.380,P ＞ 0.380);Western blot results showed BGC-823 cell transfection S100A4 expression significantly lowered respectively after 48 hours,there were (0.654 + 0.025),(0.642 + 0.014),(0.317 ± 0.061),the interference group and normal control group was statistically significant (P =0.01,P ＜ 0.05),between negative control group and interference group were statistically significant (P =0.000,P ＜ 0.05),normal control group and the negative control group had no significant difference (P =0.341,P ＞ 0.341).After S100A4-siRNA transfection,gastric carcinoma BGC-823 cell proliferation decreased,TUNEL method showed obviously increase apoptosis,MTY showed that IC5o of oxaliplatin was 56.31 μmol/L,after transfection,IC50 was 0.654 μmol/L.Conclusions This study showed that the siRNA silence S100A4 protein inhibit gastric cancer cell proliferation,induced apoptosis and improved chemotherapy sensitivity of oxaliplatin.S100A4 might be prompt targets for the treatment of gastric carcinoma.