斯钙素2基因调控肝癌细胞增殖凋亡的机制

Molecular mechanism of stanniocalcin 2 gene regulating proliferation and apoptosis of hepatocellular carcinoma

目的:探讨斯钙素2(STC2)基因对肝癌细胞增殖凋亡的影响并初步分析其分子作用机制。方法:2018年4月至2019年3月,采用慢病毒介导的小干扰RNA(siRNA)技术构建STC2敲减组和对照组SMMC-7721肝癌细胞株(购自中国科学院生化与细胞研究所),应用黄色噻唑蓝(MTT)生长曲线、细胞周期测定、克隆形成实验和膜联蛋白V-别藻蓝蛋白荧光素(Annexin V-APC)染色法检测两组细胞增殖凋亡的差异,采用荧光实时定量PCR阵列(PCR Array)技术检测92个增殖凋亡相关基因在两组细胞中的表达水平差异,并且应用荧光实时定量PCR法(RT-qPCR)验证差异表达的基因,两组比较采用t检验。结果:STC2敲减组SMMC-7721肝癌细胞中的STC2基因表达量明显低于对照组(0.472±0.041比1.003±0.091,t=9.207,P<0.01),STC2敲减组细胞的增殖速率明显低于对照组(3.090±0.045比4.190±0.052,t=27.218,P<0.01),其集落形成数目也明显低于对照组(96.670±4.163比161.000±3.606,t=20.232,P<0.01)。STC2敲减组细胞的凋亡水平明显高于对照组[(17.113±0.350)%比(6.397±0.350)%,t=37.546,P<0.01]。与对照组比较,STC2敲减组细胞处于G 0~G 1期的细胞比例稍增多[(67.183±0.945)%比(64.500±0.341)%,t=4.625,P<0.05],S期的细胞比例则稍减少[(29.360±0.403)%比(30.777±0.640)%,t=3.243,P<0.05],G 2~M期细胞比例未见明显差异[(3.457±0.558)%比(4.723±0.598)%,t=2.684,P>0.05]。PCR Array筛选和RT-qPCR验证的结果显示,STC2敲减组细胞中凋亡蛋白酶激活因子-1(APAF1)、第10号染色体同源缺失性磷酸酶-张力蛋白同源基因(PTEN)、腺瘤性结肠息肉病基因(APC)的表达水平明显高于对照组(APAF1:2.49±0.29比1.00±0.03,t=8.724,P<0.05;PTEN:2.10±0.15比1.00±0.06,t=11.888,P<0.01;APC:2.59±0.14比1.00±0.05,t=18.113,P<0.01)。结论:STC2基因可能通过抑制APAF1、PTEN和APC等基因的表达而发挥其调控肝癌细胞增殖凋亡的作用。

Objective To explore the effect of stanniocalcin 2(STC2)gene on proliferation and apoptosis of hepatocellular carcinoma(HCC)cell line and molecular mechanism.Methods SMMC-7721 HCC cell line was purchased from Shanghai Institute of Biochemistry and Cell Biology and the expression of STC2 gene in this cell line was silenced by lentiviral-mediated small interfering RNA(siRNA)technique.Methyl thiazol tetrazolium(MTT)growth curve,cell cycle assay,colony formation assay and Annexin V-allophycocyanin(APC)staining assay were used to detect the difference of proliferation and apoptosis between the STC2-silenced and control groups of SMMC-7721 cells.The expression levels of 92 proliferation and apoptosis-related genes in two groups of SMMC-7721 cells were detected by polymerase chain reaction(PCR)Array technique,then those differentially expressed genes were verified by real-time quantitative PCR(RT-qPCR).The experimental data were expressed as mean±standard deviation(Mean±SD),and comparisons between the two groups were analyzed by t test.Results The expression level of STC2 gene in STC2-silenced group was significantly lower than in control group(0.472±0.041 vs.1.003±0.091,t=9.207,P<0.01).The growth rate of STC2-silenced group was significantly lower than in control group(3.090±0.045 vs.4.190±0.052,t=27.218,P<0.01).The number of colony formation in STC2-silenced group was less than in control group(96.670±4.163 vs.161.000±3.606,t=20.232,P<0.01).The apoptosis level of STC2-silenced group was significantly higher than in control group[(17.113±0.350)%vs.(6.397±0.350)%,t=37.546,P<0.01].The proportion of STC2-silenced SMMC-7721 cells in G0-G1 phase was slightly higher than in control group[(67.183±0.945)%vs.(64.500±0.341)%,t=4.625,P<0.05],while the proportion in S phase was slightly lower than in control group[(29.360±0.403)%vs.(30.777±0.640)%,t=3.243,P<0.05].There was no significant difference in G2-M phase between the two groups[(3.457±0.558)%vs.(4.723±0.598)%,t=2.684,P>0.05].PCR Array and RT-qPCR validation showed that there were only three differentially expressed genes between STC2-silenced group and control group,including apoptotic protease activating factor-1(APAF1:2.49±0.29 vs.1.00±0.03,t=8.724,P<0.05),phosphatase and tensin homolog deleted on chromosome ten(PTEN:2.10±0.15 vs.1.00±0.06,t=11.888,P<0.01)and adenomatous polyposis coli(APC:2.59±0.14 vs.1.00±0.05,t=18.113,P<0.01).Conclusion STC2 may play a role in regulating proliferation and apoptosis of HCC cells by inhibiting the expression of APAF1,PTEN and APC.