【摘要】 目的探讨肝癌细胞株SMMC-7721中GTD连接蛋白(GTPBP)4基因的表达下调对细胞体内外生物学行为的影响及其作用机制。方法采用免疫组织化学方法比较肝癌组织和癌旁组织GTPBP4的表达水平差异。运用实时荧光定量PCR技术检测4种人肝癌细胞株(SMMC-7721、HEPG2、HUH-7、HEP3B)中GTPBP4mRNA的表达。利用RNA干扰技术下调肝癌细胞株SMMC-7721中GTPBP4的表达水平,并观察细胞的生物学行为变化。结果在肝癌组织芯片中,GTPBP4蛋白在肝癌组织中的表达水平明显高于癌旁组织。GTPBP4在4种肝癌细胞株中均明显上调。下调GTPBP4的表达后,肝癌细胞增殖能力减弱;细胞凋亡增多;处于S期、G1期的细胞无明显变化,处于G2期的细胞增多;细胞体外克隆形成能力减弱;裸鼠体内成瘤能力减弱。表达谱基因芯片结果显示GTPBP4敲减后SMMC-7721细胞内基因发生改变的有333个,其中上调的有134个,下调的有199个。通路富集分析找到富集的差异基因所在的10条信号通路。Western blot显示GTPBP4表达下调后与细胞周期调控相关的基因CCND1、CCND2、CDK6、MDM2的表达水平发生明显变化。结论 GTPBP4作为一促肝癌基因,其可能通过调控细胞周期关键基因的表达从而影响肝细胞癌的生物学行为。更多还原

【Abstract】 Objective To investigate the influence of GTPBP4 gene expression down-regulation in hepatocellular carcinoma(HCC)cell line SMMC-7721 on the intracellular and extracellular biological behaviors and its action mechanism.Methods The differences of GTPBP4 expression level in HCC tissues and paracancerous tissues were compared by using the immunohistochemical method.The expression of GTPBP4 mRNA in 4 kinds of HCC cell line(SMMC-7721,HEPG2,HUH-7,HEP3 B)was detected by using the real-time fluorescent quantitative PCR(RT-PCR).The expression of GTPBP4 in HCC cell line SMMC-7721 was downregulated by using the RNA interference technique and the cellular biological behavior change was observed.Results In HCC histological chip,the expression level of GTPBP4 protein in HCC tissue was significantly higher than that in para-cancerous tissue.GTPBP4 was also significantly up-regulated in 4 kinds of HCC cell line.After the expression of GTPBP4 was down-regulated,the proliferation ability of HCC cells was weakened and apoptosis was increased.The cells in S phase and G1 phase had no significant changes,but which in G2 phase were increased,the ability of in vitro clone formation was weakened,the nude mouse in vivo tumor formation capacity was weakened.The expressional profiles microarray results showed that 333 genes were changed in SMMC-7721 cells after GTPBP4 knockdown,in which the up-regulated genes were 134,the down-regulated genes were 199.The channel enrichment analysis found 10 signal transduction pathways of the enriched differential genes.Western blot showed that the expression levels of CCND1,CCND2,CDK6 and MDM2 were changed significantly after GTPBP4 expression down-regulation.Conclusion GTPBP4 as a promoting HCC gene may influence HCC biological behavior possibly by regulating the expression of key gene in cell cycle.更多还原