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- 2018
沉默精氨酸琥珀酸合成酶对SMMC-7721细胞增殖、凋亡的影响
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Abstract:
目的:探讨shRNA靶向沉默精氨酸琥珀酸合成酶(ASS1)基因后对人肝癌细胞株SMMC-7721细胞增殖、凋亡的影响。方法:应用RT-PCR及Western blot检测5种肝癌细胞系中ASS1的表达情况; 将shRNA-ASS1转染入SMMC-7721细胞(shRNA-ASS1组),同时设空白对照组和阴性对照组,观察转染后细胞的生长变化,运用Western blot鉴定其对ASS1基因的沉默效果; 采用CCK-8法检测各组转染3 d后的细胞增殖情况; 流式细胞术检测各组转染24 h后细胞凋亡的情况。结果:ASS1 mRNA和蛋白在Changliver、SMMC-7721、QGY-7703、HepG2和HepG3B细胞中的表达差异有统计学意义(FmRNA=1 129.140,F蛋白=1 114.240,P均<0.001),ASS1 在SMMC-7721和HepG3B肝癌细胞系中呈高表达。CCK-8实验显示,与阴性对照组及空白对照组相比,shRNA-ASS1组细胞增殖率降低(F=7.176,P=0.007)。细胞凋亡实验显示,shRNA-ASS1组细胞凋亡率高于阴性对照组和空白对照组(F=88.120,P<0.001)。结论:沉默ASS1基因可抑制SMMC-7721细胞增殖,诱导细胞凋亡。
Aim: To investigate the silencing effects of argininosuccinate synthetase 1(ASS1)gene via shRNA on the proliferation and apoptosis of hepatocellular carcinoma cell line SMMC-7721.Methods: RT-PCR and Western blot were used to examine the expression of ASS1 in 5 different hepatocellular carcinoma cell lines. SMMC-7721 cells were allocated into transfected group(shRNA-ASS1), negative control group(NC group), and blank group(Mock group), and the proliferation and apoptosis of SMMC-7721 cells were examined. Subsequently, Western blot was performed to detect protein expression of ASS1 in the cells. CCK-8 assay was applied to evaluate the cell proliferation on the 3rd day and flow cytometry assay was conducted to estimate cell apoptosis after being transfected for 24 h.Results: The expression of ASS1 mRNA and protein had significant difference among Changliver, SMMC-7721, QGY-7703, HepG2 and HepG3B cells(FmRNA=1 129.140,Fprotein=1 114.240,P<0.001). SMMC-7721 and HepG3B cells showed high level expression of ASS1 mRNA and protein. CCK-8 assay showed that the proliferation in shRNA-ASS1 group was lower than those in NC group and Mock group(F=7.176,P=0.007).The apoptotic rate in shRNA-ASS1 group was higher than those in NC group and Mock group(F=88.120,P<0.001).Conclusion: Knockdown of ASS1 by shRNA may inhibit cell proliferation and induce apoptosis of SMMC-7721 cells