TC 21/R-Ras 2在肝癌中的表达、亚细胞定位及意义

Expression,subcellular localization and clinical implications of TC 21/R-Ras 2 in hepatocellular carcinoma

目的:探讨TC21基因在肝癌细胞、肝癌及癌旁组织中的表达、亚细胞定位及意义。方法:用RT-PCR检测TC 21在肝癌细胞系及人肝癌及癌旁组织中mRNA水平的表达,用免疫组化及Western印迹检测TC21蛋白在肝癌相关组织中的表达差异,用组织芯片技术结合免疫组化检测TC21蛋白的亚细胞定位。结果:RT-PCR检测结果表明TC21 mRNA在SMMC-7721、BEL-7402、Huh-7、Hep3B、HepG2、MHCC-97L、MHCC-LM3 7个肝癌细胞系及L-02和Chang liver 2株永生化人肝细胞系、7对肝癌及对应癌旁肝组织中均有较高表达;Western印迹结果表明上述细胞系中TC21蛋白表达与mRNA表达一致,TC21在4对肝癌及癌旁组织中均呈较高水平的表达;免疫组化检测结果表明TC21在人原发性肝癌、癌旁组织、硬化肝组织与正常肝组织中的表达阳性率分别为84.2%、77.2%、41.7%、0%,肝癌与肝硬化、正常肝相比显著高表达(P<0.05,P<0.01),与癌旁组织比无统计学意义(P>0.05);统计学分析结果表明TC21蛋白表达与肿瘤大小呈正相关(P<0.05),与是否肝硬化呈负相关(P<0.05);肝癌组织中TC21主要定位于肝癌细胞核,部分定位于胞质,膜定位不明显,TC21蛋白在肝癌细胞中的核定位显著高于癌旁肝细胞(P<0.001)。结论:本研究首次揭示TC21蛋白在肝癌组织中的高表达及核定位预示其在肝细胞恶性转化及肝癌发生发展中发挥重要作用。

Objective： To explore the expression, subcellular localization and clinical implications of TC 21/R-Ras 2 gene in hepatocellular carcinoma. Methods： TC21 mRNA transcription was detected by semi-quantitative Reverse Transcription Polymerase Chain Reaction （RT-PCR） in HCC and adjacent liver tissues. Immunohistochemistry （IHC） and Western blot were used to measure the differential expression of TC21 protein in HCC related tissues, and subcellular localization of TC21 was determined with tissue microarray combined with IHC staining. Results： RT-PCR showed that higher TC21 mRNA expression was occurred in 7 HCC cell lines such as SMMC-7721, BEL-7402, Huh-7, Hep3B, HepG2, MHCC-97L, MHCC-LM3, and immortal human liver cell lines L-02 and Chang＇s liver, also 7 HCC tissues and matched noncancerous livers. Western blot indicated an accordant trend of TC21 mRNA and protein expression in 7 HCC cell lines above. TC21 overexpression was detected in 4 HCC and their adjacent liver tissues by Western blot. Furthermore, immunohistochemical staining showed that TC21 positive staining trends： primary HCC 〉 matched noncancerous liver 〉 cirrhotic liver 〉 normal liver, in which the positive staining rates were 84.2% , 77.2%, 41.7% , 0% , respectively. Compared to cirrhotic liver and normal liver tissues, TC21 was significantly over-expressed （ P 〈 0.05, P 〈 0.01 ）. TC21 expression in HCC tissues was appeared higher than that in noncancerous livers, but no significance was found （ P 〉 0.05 ）. Biological statistic analysis revealed that TC21 over-expression in HCC positively correlated to the tumor sizes （ P 〈 0.05 ） , but negatively correlated to cirrhosis or not （ P 〈 0.05 ）. Beside above-mentioned findings, it was found that TC21 protein mainly localized to the nuclei of HCC cells, parts to cytoplasms, but not to plasma membranes. Nuclear staining of TC21 was higher in HCC tissues than that in their adjacent livers （P 〈 0.001 ）. Conclusion： Present study firstly shows that up-regulation and nuclear localization of TC21 protein may play important roles in hepatocellular malignant transformation and hepatocarcinogenesis, although its mechanism is to be elucidated.