摘要
目的观察新型接头蛋白CARMA3在肝癌HepG2细胞中的表达,以及CARMA3在G蛋白偶联受体诱导核转录因子κB(NF-κB)激活中的作用。方法提取肝细胞肝癌的总mRNA和蛋白,用逆转录PCR、免疫沉淀和免疫印迹方法检测肝脏肿瘤细胞中CARMA3基因和蛋白的表达水平。负显性CARMA3质粒转染肝脏肿瘤细胞24 h后用溶血磷脂酸(LPA)诱导NF-κB的激活,提取胞浆蛋白和核蛋白,用免疫印迹和电泳迁移试验检测负显性CARMA3的表达和IKK及NF-κB的活化。结果肝细胞肝癌表达CARMA3。在肝脏肿瘤细胞中转染负显性CARMA3后,LPA所诱导IKK和NF-κB的激活被显著抑制。结论肝细胞肝癌表达CARMA3。GPCR(LPA)-CARMA3-NF-κB信号传导通路存在于肝细胞肝癌中。抑制CARMA3的功能可以阻遏GPCR(LPA)所诱导的NF-κB的激活,由此为肝细胞肝癌的治疗提供了新的思路。
Objective To observe the expression of a novel adaptor protein,CARMA3,in hepatic cellular carcinoma HepG2 cells,and its role in G protein-coupled receptor-induced NF-κB activation.Methods Total mRNA and protein lysate of hepatic cellular carcinoma cells were harvested to detect the expression level of CARMA3 with RT-PCR,immunoprecipitation,and Western blot respectively.Upon transfection with dominant-negative CARMA3 and stimulation with lysophosphatidic acid（LPA）,the protein lysate and nuclear extraction fraction of the hepatic cellular carcinoma cells were collected and examined for phosphorylation of IKK and NF-κB activation using Western blot and EMSA（Electrophoretic Mobility Shift Assay）.Results Our results demonstrated that CARMA3 mRNA and protein were expressed in the hepatic carcinoma cells.Upon transfection with dominant-negative CARMA3,the LPA-induced NF-κB activation in hepatic cellular carcinoma cells was substantially impaired.Conclusion CARMA3 is expressed in hepatic cellular carcinoma.GPCR（LPA）-CARMA3-NF-κB signaling axis exists in hepatic cellular carcinoma cells.CARMA3 deficiency impairs GPCR（LPA）-induced NF-κB activation,thereby providing a novel strategy for hepatic cellular carcinoma therapy.
出处
《肝胆胰外科杂志》
CAS
2010年第5期363-365,370,共4页
Journal of Hepatopancreatobiliary Surgery
关键词
G蛋白偶联受体
溶血磷脂酸
CARMA3
核转录因子-ΚB
肝细胞肝癌
G protein-coupled receptor lysophosphatidic acid caspase recruit ment domain-containing membrane-associated guanylate kinase protein 3 nuclear factor kappa B hepatic cellular carcinoma
