SEMA5A对肝细胞癌增殖及凋亡的影响及可能机制

Effect of SEMA5A on cell proliferation and apoptosis of hepatocellular carcinoma and possible mechanism

目的探讨信号素5A(SEMA5A)对人肝细胞癌(HCC)增殖及凋亡的影响并初步探讨可能的发生机制。方法应用定量逆转录聚合酶链反应(qRT-PCR)方法检测2021-01-01-06-30南通大学附属医院30例新鲜HCC组织及配对癌旁肝组织和2020-06-01-2021-12-31收集30例正常肝组织中SEMA5A mRNA的表达;应用蛋白质印迹法检测人HCC细胞株HepG2、Huh7、SK-HEP1、Hep3B及正常肝细胞株LO2中SEMA5A蛋白的表达;转染SEMA5A siRNA沉默HCC细胞株中SEMA5A的表达,qRT-PCR检测沉默效率;四甲基偶氮唑(MTT)比色实验及克隆形成实验检测细胞增殖;碘化丙啶染色流式细胞术检测细胞凋亡;蛋白质印迹检测沉默后SEMA5A、Cleaved caspase-3及Cleaved caspase-9的蛋白表达。结果新鲜HCC组织中SEMA5A mRNA的表达(0.77±0.26)低于配对的癌旁肝组织(1.46±0.38)及正常肝组织(1.56±0.36),差异有统计学意义,t值分别为8.030和9.574,均P<0.001。SEMA5A mRNA表达既可以区分HCC及癌旁肝组织,灵敏度和特异度分别为83.33%和93.33%;也可以区分HCC及正常肝组织,灵敏度和特异度分别为96.67%和86.67%。LO2、HepG2、Huh7、SK-HEP1和Hep3B细胞株中的SEMA5A的蛋白相对表达量分别为1.02±0.12、0.72±0.06、0.73±0.05、0.37±0.09及0.23±0.05,与LO2细胞株相比,4株HCC细胞株中SEMA5A的蛋白表达水平下降,差异有统计学意义,F=50.230,P<0.001。沉默SEMA5A的HepG2(F_(处理组)=366.400,P<0.001;F_(时间点)=508.100,P<0.001;F_(处理组×时间点)=2.009,P=0.186)及Huh7(F_(处理组)=60.110,P=0.001;F_(时间点)=699.600,P<0.001;F_(处理组×时间点)=9.417,P=0.004)细胞增殖活力高于对照组,差异有统计学意义。与对照组比较,沉默SEMA5A后,HepG2[(46.33±3.51)%]及Huh7[(43.67±4.04)%]细胞的克隆形成率更高,差异有统计学意义,F值分别为28.840和10.640,P值分别为0.008和0.011;沉默SEMA5A后HepG2及Huh7细胞的凋亡率分别为(1.34±0.19)%及(2.45±0.23)%,低于对照组,差异有统计学意义,F值分别为195.500和300.900,均P<0.001。沉默SEMA5A后,HepG2细胞内SEMA5A、Cleaved caspase-3及Cleaved caspase-9的蛋白相对表达水平为0.34±0.04、0.19±0.01及0.33±0.04,低于对照组,差异有统计学意义,F值分别为120.500、84.960和205.100,均P<0.001。Huh7细胞内3种蛋白相对表达水平为0.36±0.05、0.24±0.07、0.27±0.02,低于对照组,差异有统计学意义,F值分别为98.260、27.790和129.700,均P<0.001。结论SEMA5A在HCC中表达下降,沉默SEMA5A表达可促进HCC的细胞增殖,抑制细胞凋亡;该作用可能与Cleaved caspase 3及Cleaved caspase 9的蛋白表达下降相关。

Objective To investigate the effect of semaphorin 5A(SEMA5A)on the proliferation and apoptosis of human hepatocellular carcinoma(HCC)and to preliminarily explore the possible mechanism.Methods Quantitative reverse transcription polymerase chain reaction(qRT-PCR)was used to analyze the expression of SEMA5A mRNA in 30fresh HCC tissues and paracancerous liver tissues from January 1to June 31,2021at the Affiliated Hospital of Nantong University.Western blot was utilized to detect the expression of SEMA5Aprotein in human HCC cell lines(HepG2,Huh7,SK-HEP1,Hep3B)and normal liver cell line LO2.Next the expression of SEMA5A was knocked down by small interfering RNA(siRNA)and the efficiency was determined by using qRT-PCR.MTT assay and colony formation assay were used to detect cell proliferation.Flow cytometry with propidium iodide(PI)staining was used to detect cell apoptosis.The protein expressions of SEMA5A,Cleaved caspase 3and Cleaved caspase 9after silencing were detected by using Western blot.Results The expression of SEMA5A mRNA in fresh HCC tissues(0.77±0.26)was lower than that in paracancerous liver tissues(1.46±0.38)and normal liver tissues(1.56±0.36),and the differences were statistically significant(t=8.030,P<0.001;t=9.574,P<0.001).SEMA5A mRNA expression could not only distinguish HCC and paracancerous tissues with sensitivity and specificity of 83.33%and 93.33%,but also distinguish HCC and normal tissues with sensitivity and specificity of 96.67%and 86.67%,respectively.Relative expression levels of SEMA5Aprotein in the cell lines LO2,HepG2,Huh7,SK-HEP1and Hep3Bwere 1.02±0.12,0.72±0.06,0.73±0.05,0.37±0.09and 0.23±0.05,respectively.Compared with LO2,the relative expression levels of SEMA5Aprotein in the four HCC cell lines were decreased in all four HCC cell lines,and the difference was statistically significant(F=50.230,P<0.001).Cell proliferation viability of HepG2and Huh7cells after silencing SEMA5A were higher than those of the control groups(F_(treated group)=366.400,P<0.001,F_(time point)=508.100,P<0.001,F_(treated group×time point)=2.009,P=0.186;F_(treated group)=60.110,P=0.001,F_(time point)=699.600,P<0.001,F_(treated group×time point)=9.417,P=0.004).Clone formation rates of HepG2[(46.33±3.51)%]and Huh7cells[(43.67±4.04)%]were higher after silencing SEMA5Acompared with the control groups,and the differences were statistically significant(F=28.840,P=0.008;F=10.640,P=0.011).Apoptosis rates of HepG2and Huh7cells after silencing SEMA5A were(1.34±0.19)%and(2.45±0.23)%,respectively,which were significantly lower than those of the control groups,and the differences were statistically significant(F=195.500,P<0.001;F=300.900,P<0.001).After silencing SEMA5A,the relative protein expression levels of SEMA5A,Cleaved caspase-3and Cleaved caspase-9in HepG2cells were 0.34±0.04,0.19±0.01and 0.33±0.04,which were significantly lower than those in the control groups,and the differences were statistically significant(F=120.500,P<0.001;F=84.960,P<0.001;F=205.100,P<0.001),and the levels in Huh7cells were 0.36±0.05,0.24±0.07and 0.27±0.02,which were significantly lower than those in the control groups,with statistically significant differences(F=98.260,P<0.001;F=27.790,P<0.001;F=129.700,P<0.001).Conclusion SEMA5Aexpression decreases in HCC.After silencing SEMA5Ain HCC,the cell proliferation is promoted and the apoptosis is inhibited which may be associated with the decreased protein expressions of Cleaved caspase-3and Cleaved caspase-9.