β-catenin蛋白H36P突变对肝癌细胞的增殖的影响  

Effect of β-catenin H36P Mutation on Proliferation of HepatocellularCarcinoma Cells

作　　者：吴忠坤 宋平辉 张毅 李春博 李勤新 WU Zhongkun;SONG Pinghui;ZHANG Yi;LI Chunbo;LI Qinxin(Department of General Surgery,No.215 Hospital of Shaanxi Nuclear Industry,Xianyan,Shaanxi,712000,China;Department of Pediatrics,Xingping Municipal People’s Hospital,Xingping,Shaanxi,713199,China)

机构地区：[1]陕西省核工业二一五医院普通外科,陕西省咸阳市712000 [2]兴平市人民医院儿科,陕西省兴平市713199

出　　处：《医学分子生物学杂志》2023年第5期450-454,共5页Journal of Medical Molecular Biology

摘　　要：目的探究β-catenin蛋白H36P突变对肝癌细胞的增殖的影响。方法通过PCR扩增法扩增CTNNB1基因,包括WT-CTNNB1、G34R-CTNNB1和H36P-CTNNB1。通过双酶切法克隆至p-CMV5质粒载体,将相对应的质粒瞬转至Huh7、HepG2细胞中,并检测其蛋白表达情况。其次,使用CHX按时间梯度处理表达目的基因的细胞,比较H36P-β-catenin与WT-β-catenin、G34R-β-catenin的泛素化降解情况并分析蛋白稳定性。随后通过质核分离检测H36P-β-catenin核转位能力。最后,通过CCK-8计数法检查H36P-β-catenin对肝癌细胞增殖能力的影响。结果突变体G34R、H36P的蛋白质表达水平,与野生型β-catenin无明显差异。在HepG2细胞株中,CHX作用8 h时WT-β-catenin几乎完全降解,而G34R和H36P只降解了75%和60%。Huh7细胞株中的情况类似,6 hrs时WT-β-catenin几乎完全降解,而G34R和H36P只降解了约40%。以上结果差异均具有统计学意义(P<0.05)。相较于WT-β-catenin,G34R和H36P促使HepG2细胞增殖量提升。在表达β-catenin突变体的肝癌细胞泛素化降解受抑制的情况下,与WT-β-catenin、突变体G34R相比,表达突变体H36P的肝癌细胞核转位明显增加。结论β-catenin蛋白H36P突变蛋白表达稳定、核转位情况增多,泛素化降解过程受阻,并能促进HepG2和Huh7肝癌细胞的增殖。Objective To investigate the effect ofβ-catenin H36P mutation on the proliferation of hepatocellular carcinoma cells.Methods The CTNNB1genes(WT-CTNNB1,G34R-CTNNB1,H36P-CTNNB1)were amplified by PCR amplification.It was cloned into the p-CMV5 plasmid vector with a double digestion method,and the corresponding plasmids were transiently transferred into the Huh7 and HepG2 cells.The protein expression levels were then detected by Western blotting.Cells transfected with the target genes were treated with CHX in a time gradient,the degradation time of the H36P-β-catenin,WT-β-catenin and G34R-β-catenin were compared and their stabilities were analyzed.The nuclear translocation ability of H36P-β-catenin was examined after separation of the nuclear and cytoplasmic fractions.Finally,the effect of H36P-β-catenin on the proliferation of hepatocellular carcinoma cells was examined by CCK-8 method.Results The protein expression levels ofβ-catenin mutants(G34R and H36P)were not significantly different from that of the wild-typeβ-catenin.In HepG2 cell line,WT-β-catenin was almost completely degraded by CHX in 8 hours,while the mutants only degraded 75%and 60%in the same time period.Similarly,in the Huh7 cell line,WT-β-catenin was almost completely degraded in 6 hours,while the mutants were only degraded about 40%in the same time period.The differences in the above results were statistically significant(P<0.05).Compared with the WT-β-catenin,mutantβ-catenin promoted the proliferation of HepG2 cell. The nuclear translocation ability of H36P mutant β-catenin was increased compared with those of WT and G34R mutant β-catenin in the hepatocellularcarcinoma cells. Conclusion β-catenin H36P mutant protein blocks the protein ubiquitination degradationprocess, promotes the proliferation of HepG2 and Huh7 hepatocellular carcinoma cells,and has an increased nuclear translocation.

关 键 词：Β-CATENIN蛋白 突变 肝癌 增殖 

分 类 号：R735.7[医药卫生—肿瘤]