MT1E在肝癌细胞中的作用及不同中医治法对其表达调控的差异研究

Role of MT1E in Hepatocarcinoma cells and Differential Study on Several TCM Therapeutical Principles on Its Expression

目的:比较常用不同中医治法对MT1E表达调控作用的差异,以及初步探讨MT1E在肝癌细胞中的生物学功能。方法:(1)以DEN诱发大鼠肝癌形成为模型,应用基因芯片检测健脾益气、清热解毒、活血化瘀等常用中医治法对MT1E基因表达的差异;(2)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,用实时荧光定量PCR技术检测MT1E基因的表达情况,用MTT法检测细胞生长增殖情况。结果:(1)芯片结果显示,MT1E基因在肝癌形成后表达明显增加,不同中医治法对其具有不同程度的调控作用,其中健脾益气法的下调作用最明显;(2)成功筛选到MT1E基因RNA干扰的1个有效靶序列,经实时荧光定量PCR技术检测到电转染72h后,该基因的表达水平明显降低;(3)MTT的结果显示,有效干扰靶点电转染144h后细胞的生长增殖得到了明显的抑制。讨论:MT1E基因是中医健脾益气法防治原发性肝癌的有效靶点之一,经生物学功能研究表明,该基因的高表达与肿瘤细胞的恶性增殖有关。

Objective：To compare the different effects of several common TCM formula therapies on the expression of MT1E, and discuss primarily the biological functions of MT1E in hepatocarcinoma cells. Methods ： 1 ） Rat hepatocellular carcinoma （ HCC ） models were induced by diethylnitrosamine （ DEN ）, the different expression of MTIE gene were determined by gene chips caused by different therapeutical methods including Nourishing spleen and enriching qi, Clearing away heat and toxic material, Activating blood circulation;2） Two siRNA targets toward MTIE gene were designed, recombinant plasmid was transfected to Hepatocellular Carcinoma cell line SMMC-7721 ,the gene expression of MTIE were determined using real-time quantitative PCR, and the cell viability were determined by MTT assay. Results ： 1 ） According to the gene chip results, the expression of MT1E increased significantly after formation of Hepatocellular Carcinoma, several TCM therapeutical methods had different effections, and down-regulating of nourishing spleen and enriching qi prescription was obivious; 2） an effective siRNA target sequence in MT1E was got, the gene expression decreased greatly at the 72 hour after transfected; 3 ）MTT assay result displayed, the cell growth and proliferation were inhibited obviously when intefferenced target effectivly was transfected 144 hours. Conclusion： MTIE gene is an effective target which treatmented liver cancer with nourishing spleen and enriching qi, and the high expression of MTIE had relation with the malignant proliferation of tumor cells proved by studies of biological functions.