C端截断的HBX突变体通过上调DR4和DR5表达促TRAIL诱导的肝癌细胞凋亡  

C terminal truncated HBX promotes TRAIL-induced apoptosis of hepatocellular carcinoma cells by up-regulating DR4 and DR5 expression

作　　者：胡威[1] 魏肖 周凯[1] 孔凡运[1] 寇艳波 房贤达 李向阳[1] 尤红娟[1] 郑葵阳[1] 汤仁仙[1] 

机构地区：[1]徐州医学院病原生物学与免疫教研室,221004 [2]徐州市第三中学,221004

出　　处：《免疫学杂志》2016年第6期480-485,共6页Immunological Journal

基　　金：江苏省高校自然科学基金(14KJB310023);江苏高校优势学科建设工程;江苏省青蓝工程资助项目

摘　　要：目的探讨不同的HBX截断突变体对TRAIL-R1（DR4）和TRAIL-R2（DR5）表达的影响,以及DR4和DR5在HBX截断突变体增强TRAIL诱导的肝癌细胞凋亡中的作用。方法构建HBX的1~120 aa（C端截断）、51~120 aa（C端和N端共同截断）和51~155 aa（N端截断）的突变体表达载体HBX1、HBX2和HBX3;在HepG2和Huh-7肝癌细胞中,Real-time PCR和Western blot检测稳定转染HBX不同突变体的DR4和DR5的表达;Western blot检测RNA小干扰载体（DR4-siRNA和DR5-siRNA）的HBX突变体的DR4和DR5表达情况,流式细胞术检测TRAIL（200 ng/ml）诱导的肝癌细胞凋亡情况。结果经PCR扩增和双酶切鉴定,3种突变体表达载体HBX1、HBX2和HBX3重组质粒构建成功,测序检测目的基因序列正确;Real-time PCR和Western blot表明,HBX1能显著增加2种肝癌细胞系中的DR4和DR5基因和蛋白的表达;而HBX2和HBX3对DR4和DR5基因和蛋白的表达无显著影响;在Hep G2-HBX1和Huh-7-HBX1细胞中,与对照组相比,DR4-siRNA和DR5-siRNA显著降低了DR4、DR5的表达;流式细胞术检测结果显示HepG2-HBX1和Huh-7-HBX1细胞的凋亡率较对照组细胞均显著提高;RNA小干扰抑制DR4和DR5的表达后,HepG2-HBX1和Huh-7-HBX1细胞的凋亡率下降。结论成功构建了3种不同HBX突变体的真核表达载体;HBX基因C端截断（1~120 aa）的突变体能够上调DR4和DR5的表达,且能够上调DR4和DR5的表达促进TRAIL诱导的肝癌细胞凋亡。To investigate the effect of different truncated HBX mutants on the expression of TRAIL-R1（DR4）and TRAIL-R2（DR5）,and detect the role of DR4 and DR5 on TRAIL-induced cell apoptosis in hepatocellular carcinoma cells transfected with truncated HBX,we constructed three HBX mutant gene fragments,including1-120 aa（C terminal truncated）,51-120 aa（C and N terminal truncated）,51-155 aa（N terminal truncated）,and named as HBX1,HBX2 and HBX3.Real-time PCR and Western blot were used to detect the expressions of DR4 and DR5 in HepG2 and Huh-7 cells transfected with different mutant HBX.Then the cells expressing HBX mutant were transfected with DR4 and DR5 interference vectors（DR4-siRNA and DR5-siRNA）.The down-regulation of DR4 and DR5 were detected by Western blotting,and the apoptosis of hepatocellular carcinoma cells induced by TRAIL（200 ng/ml） was detected by flow cytometry.PCR amplification and double enzyme appraisal revealed that HBX1,HBX2 and HBX3 recombinant plasmid were built successfully.Sequencing demonstrated that the gene sequences of three HBX mutant genes were correct;Real-time PCR and Western blot showed that HBX1 could significantly increase the expression of DR5 and DR4 in HCC cells,whereas HBX2 and HBX3 had no significant effect on gene and protein expressions of DR4 and DR5.In HepG2-HBX1 and Huh-7-HBX1 cells,the expressions of DR4 and DR5 were significantly decreased by DR4-siRNA and DR5-siRNA.The apoptosis rates of HepG2-HBX1 and Huh-7-HBX1 cells were significantly increased compared to control cells,while after transfected with DR4-siRNA and DR5-siRNA plasmids,the apoptosis rates of HepG2-HBX1 and Huh-7-HBX1 cells decreased.In conclusion,the eukaryotic expression vectors of three different HBX mutants are successfully constructed.The C terminal truncated HBX（1-120 aa）could increase the expression of DR4 and DR5,and could promote TRAIL induced apoptosis through DR4 and DR5 in HCC cells.

关 键 词：HBX突变体 肝癌细胞 DR4 DR5 TRAIL 

分 类 号：R735.7[医药卫生—肿瘤]