Objective: To investigate the expression of gap junction protein connexin 26(Cx26) in hepatocellular carcinoma(HCC) and its significance.
Methods: The expression of Cx26 in liver tissue was examined by immunohistochemistry staining in 159 paraffin-embeded liver sections, including 20 samples of normal liver tissue, 30 samples of chronic hepatitis, 33 samples of liver cirrhosis, and 76 samples of HCC. Normal hepatic cell line LO2 and HCC cell line SMMC-7721 were used in vitro to verify the characteristics of gap junction and Cx26 expression pattern. The expression and localization of Cx26 were measured by Western blotting and immunofluorescence assay, respectively. The function of gap junction between adjacent cells was detected by dye transfer assay.
Results: Compared to normal liver samples, the positive rate of Cx26 was markedly decreased in hepatitis, cirrhosis and HCC tissues(all P<0.05). The intensity of Cx26 staining was significantly increased in HCC tissues compared with those in non-carcinomatous liver(NCL) tissues(all P<0.05). In NCL tissues, there was a mild to moderated staining of Cx26 which located mainly on the membranes of hepatocytes at intercellular contacts. The positive staining of Cx26 in HCC tissues was observed mainly in cytoplasm. Positive Cx26 expression was positively associated with tumor size(P=0.036), but not with age, gender, histologic grade, clinical stage, underlying hepatitis and cirhosis, lymph node metastasis and intrahepatic vascular embolism(all P>0.05). Compared with LO2 cells, an aberrant expression and distribution of Cx26 in SMMC-7721 cells was confirmed, which may lead to a decreased function of gap junctions.
Conclusions: The aberrant expression and distribution of Cx26 protein may be associated with hepatocarcinogenesis, and the residual gap junction in HCC may provide a new target for treatment of HCC.
目的: 研究缝隙连接蛋白26(Cx26) 在人肝细胞癌(HCC)组织中的表达程度及定位, 探讨该基因在HCC发生中的作用。
方法: 应用免疫组织化学染色法检测159份肝脏石蜡包埋组织中Cx26蛋白的表达和定位, 其中正常肝组织20份、肝炎30份、肝硬化33份, HCC 76份。另在体外使用人正常肝细胞株LO2和肝癌细胞株SMMC-7721, 采用蛋白质印迹法和免疫荧光法检测Cx26蛋白表达和定位, 细胞接种荧光示踪法检测缝隙连接功能。
结果: 与正常肝组织比较, 肝炎、肝硬化和HCC组织中Cx26蛋白阳性表达率显著下降(均 P < 0.05)。Cx26蛋白表达强度在HCC中明显高于非癌肝组织(均 P < 0.05)。非癌肝组织Cx26多呈轻中度表达, 主要定位于相邻细胞间相互接触的细胞膜上, 偶见于细胞质; 而HCC组织中阳性表达的Cx26染色多较强, 且多数定位于细胞质, 偶见于细胞膜。Cx26蛋白与肿瘤大小呈正相关( P < 0.05), 而与年龄、性别、肿瘤病理分级、TNM分期、伴肝炎肝硬化、淋巴结转移及脉管癌栓无明显相关(均 P>0.05)。体外实验进一步证实, 与LO2细胞比较, SMMC-7721肝癌细胞存在Cx26蛋白表达降低及分布异常, 并且功能性缝隙连接明显下调。
结论: Cx26蛋白的表达及定位异常可能与HCC的发生有关, HCC中残存的缝隙连接可能成为抗肝癌治疗新靶点。