hAph2β和c-abl在肝细胞癌中的表达及其相互作用

Expressions of hAph2β and c-abl in hepatocellular carcinoma and their interaction

目的:探讨hAph2β蛋白与c-abl蛋白在肝细胞癌(hepatocellular carcinoma,HCC)中的相互作用。方法:RT-PCR和Western印迹法分析c-abl基因和hAph2基因在肝癌细胞株及永生化人肝细胞株中的表达。脂质体转染、免疫共沉淀和Western印迹法检测hAph2β与c-abl在SMMC-7721细胞中的相互作用。激光共聚焦-荧光显微镜观察hAph2β和c-abl在SMMC-7721细胞中的定位。用组织芯片结合免疫组织化学法检测hAph2β和c-abl蛋白在肝癌和相应癌旁组织中的表达差异,统计学分析其表达差异的临床意义。结果:RT-PCR检测结果表明,c-ablmRNA在HepG2、L-02及MHCC-LM3细胞中高表达,在SMMC-7721、BEL-7402、Hep3B和Chang’sliver细胞中中度表达,而在Huh-7、MHCC-97L细胞中低表达。Western印迹法检测结果发现,hAph2总蛋白在HepG2、Huh-7、MHCC-LM3和MHCC-97L细胞中高表达,在BEL-7402、L-02细胞中低表达;c-abl蛋白在Huh-7细胞中低表达。免疫共沉淀观察发现SMMC-7721细胞中过表达的hAph2β-GFP和内源性c-abl相互作用。荧光免疫细胞化学检测发现hAph2β和c-abl在SMMC-7721细胞中部分共定位。免疫组织化学检测结果表明,hAph2β/c-abl蛋白在人正常肝组织、硬化肝组织、癌旁肝及原发性HCC癌组织中的强阳性率分别为:100%/50%、91.7%/100%、100%/89.5%和50.9%/19.3%,其中hAph2β和c-abl在癌旁肝组织中的表达均明显高于肝癌组织(均P<0.001),2者在56.1%(32/57)的肝癌组织中同时高或低表达。结论:hAph2β和c-abl在SMMC-7721细胞中相互作用,在原发性HCC及相关组织中共表达。

Objective：To explore the expressions and interaction of hAph2β and c-abl in hepatocellular carcinoma （HCC）. Methods：The expression levels of c-abl and hAph2 mRNA and protein were analyzed by RT-PCR and Western blotting in HCC cell lines and immortalized hepatic cell lines, respectively. Lipofectamine transfaction, co-immuno-precipitation, and Western blotting were used to detect the interaction between hAph2β and c-abl protein. Co-localization of hAph2β and c-abl in SMMC-7721 cells was observed by laser confocal microscopy. The differential expression and co-expression of hAph2β and c-abl in HCC tissues and adjacent tumor tissues were analyzed by immunohistochemical staining （IHC） combined with tissue microarray technique, and their clinical pathological significance was analyzed by SPSS15.0 software. Results： RT-PCR analysis indicated that c-abl mRNA was highly expressed in HepG2, L-02 and MHCC-LM3 cells, moderately expressed in SMMC-7721, BEL-7402, Hep3B and Chang’s liver, and weakly expressed in Huh-7 and MHCC-97L cells, However, Western blotting demonstrated that hAph2 was highly expressed in HepG2, Huh-7, MHCC-LM3, and MHCC-97L cells and weakly expressed in BEL-7402 and L-02 cells; c-abl was weakly expressed in Huh-7 cells. Co-immuno-precipitation showed that overexpressed hAph2 β -GFP interacted with endogenous c-abl in SMMC-7721 cells. Fluorescence immunocytochemical examination found partial co-localization of hAph2β-and c-abl in SMMC-7721 cells. IHC detection showed that the positive rate of hAph2β/c-abl was 100%/50% in normal liver, 91.7%/100% in cirrhotic liver, 100%/89.5% in noncancerous liver, and 50.9%/19.3% in HCC tissues. The expression levels of hAph2β and c-abl in noncancerous liver tissues were significantly higher than those in their matched HCC tissues （P〈0.001, P〈0.001）. In 56.1% （32/57） of HCC tissues, concurrent overexpression or weak expression of hAph2β and c-abl were identified. Conclusion： hAph2β interacted with c-abl in SMMC-7721 cells. They were co expressed in primary HCC and related tissues.