siRNA干扰S100A4蛋白引起钙离子信号改变后对人胃癌细胞的影响

The effect of siRNA on human gastric cancer cells after interfering with S100A4 protein caused calcium signal changes

目的:探讨siRNA干扰S100A4蛋白引起钙离子信号改变后对人胃癌细胞的影响。方法:利用lip2000将siRNA-NC和siRNA-S100A4转染进胃癌细胞SGC-7901和MGC-803中。S100A4的基因表达的变化由qRT-PCR检测验证;细胞增殖能力的改变通过MTT、细胞克隆、细胞凋亡实验检测。侵袭和迁移能力的改变利用Transwell实验和细胞划痕实验检测;采用荧光探针Fura-2乙酰羧甲酯(Fura-2/AM)测定细胞内钙离子浓度。Western blot检测蛋白激酶PKC-α、钙调蛋白CaM和S100A4蛋白的表达变化。结果:与siRNA-NC组相比,siRNA-S100A4组S100A4基因的水平显著降低;细胞增殖、侵袭和迁移能力也降低;siRNA-S100A4组细胞的凋亡增加,细胞内PKC-α、CaM和S100A4蛋白以及游离钙离子浓度降低。结论:下调S100A4的表达使细胞内钙离子浓度降低,从而导致需要钙离子激活才能发挥作用的PKC-α和CaM蛋白表达量降低,进而诱发一系列生物功能改变。

Objective:To explore the effects of siRNA on human gastric cancer after interfering with S100A4 protein caused calcium signal changes.Methods:The siRNA-NC and siRNA-S100A4 were transfected by Lipofectamine 2000 Ttansfection Reagent(lip2000)into human gastric cancer SGC-7901 and MGC-803 cells.The mRNA of S100A4 was measured by real-time quantitative PCR.Effects of S100A4 gene silencing on the proliferation of gastric cancer cells were measured by MTT assay,plate clone formation assay and flow cytometry.The migration and invasion ability of the cells were detected by scratch assay and Transwell assay.Concentrations of cytosolic Ca^2+was measured by fluorescent probe Fura-2/AM.Western blot was used to measure the expression of protein PKC-α,CaM and S100A4.Results:The mRNA levels of S100A4 in gastric cancer cell lines was lower than that in siRNA-NC cells.The survival of cells was decreased,and number of cells migrating and invading were decreased in the siRNA-S100A4 group.The siRNA-S100A 4 group induced apoptosis of gastric carcinoma.And the expression level of protein PKC-α,CaM,S100A4 and the level of[Ca^2+]in gastric cancer cell lines was lower than that in siRNA-NC cells.Conclusion:Down-regulation of S100A4 reduced the intracellular calcium ion concentration,which led to a decrease in the expression levels of PKC-αand CaM proteins that needed calcium ion activation to function,and thus induced a series of biological functional changes.