MT1E在肝癌形成过程中的表达及其在肝癌细胞中的作用

Expression of MT1E during the formation of hepatocellular carcinoma and its function in hepatocarcinoma cells

目的:探讨MT1E基因在肝癌形成过程中不同阶段的表达及其在肝癌细胞中的生物学功能.方法:DEN诱发大鼠肝癌形成模型,分别观察造模4、8、16、20wk后肝脏组织病理形态学的改变以及MT1E基因表达的差异;针对MT1E基因mRNA序列设计2个siRNA靶点,分别经质粒重组后电转入SMMC-7721肝癌细胞株中,实时荧光定量PCR检测MT1E基因的表达,MTT法检测细胞生长增殖.结果:大鼠肝癌造模4、8wk时肝组织主要表现为炎性病变,而到16wk后呈现典型的增生病变,20wk后已全部发展为肝细胞癌.MT1E基因在造模16wk后表达明显增加,与对照组比较差异显著(芯片读数:11524vs5462).成功筛选到MT1E基因RNA干扰的一个有效靶序列,电转染72h后该基因的表达量较空白对照组与阴性对照组明显降低(0.38vs1.00,0.93,P<0.01).与阴性对照组比较,有效干扰靶点电转染144h后细胞的生长增殖得到了明显抑制(0.1700±0.0313vs0.5748±0.0480,P<0.01).结论:成功应用DEN诱发大鼠肝癌形成MT1E基因在肝癌形成的后期表达明显升高其高表达与肿瘤细胞的恶性增殖有关.

AIM： To investigate the expression of MTIE mRNA at different stages of liver cancer development and its biological functions in hepatocarcinoma cells. METHODS： Rat hepatocellular carcinoma （HCC） models were induced by diethylnitrosamine （DEN）, and the dynamic histological changes of liver tissue and the differential expression of MTIE gene were observed after the 4^th, 8^th, 16^th and 20^th weeks. Two siRNA targets toward MTIE gene were designed, recombinant plasmid was transfected into HCC cell line SMMC-7721, the gene expression of MTIE was determined using real-time quantitative PCR, and the cell viability was determined by MTT assay. RESULTS： At the 4^th week and 8^th week the major pathological changes presented inflammatory changes in liver tissue, after 16^th week presented typical proliferation changes, until 20^th week developed into HCC. According to the gene chip results, the expression of MTIE was increased significantly after 16^th week, with significant difference compared with control group （gene chip reading： 11524 vs 5462）. An effective siRNA target sequence in MTIE was got, the gene expression was decreased greatly at the 72 h after transfection compared with blank control group and negative control group （0.38 vs 1.00, 0.93, both P 〈 0.01）. MTT assay result displayed, the cell growth and proliferation were depressed obviously when interference target was effectively transfected 144 h （0.1700 ± 0.0313 vs 0.5748 ± 0.0480, P 〈 0.01）. CONCLUSION： DEN-induced rat HCC models were established successfully, the expression of MTIE is increased obviously at the later stage of development of HCC, perhaps related to the malignant proliferation of tumor cells.