目的:观察不同中医治法对大鼠肝癌干扰素相关发育调节因子1(IFRD1)表达的调控差异,以及IFRD1在人肝癌细胞增殖中的作用。方法:(1)采用二乙基亚硝胺(DEN)诱发大鼠肝癌,分别经中药健脾益气、清热解毒、活血化瘀等治疗后,Aflymetrixrat230AGene Chiparrays检测肝癌组织IFRD1表达的差异;(2)设计3个人IFRD1基因siRNA靶点,将重组质粒电转入SMMC-7721人肝癌细胞株,MTT法检测细胞生长增殖情况、Realtime—PCR检测该基因的表达差异。结果:(1)芯片结果显示,IFRD1基因在大鼠肝癌形成后表达显著增加,不同中医治法对其具有不同程度的下调作用,其中活血化瘀治法下调作用较明显,对照西药化疗反而具有上调作用;(2)采用MTT、法筛选到1个IFRD1基因RNA干扰有效靶序列,转染144h后细胞的生长增殖得到了明显的抑制(与阴性对照组相比,P〈0.01);(3)实时荧光定量PCR检测表明,转染72h后该基因的表达明显降低。结论:SMMC-7721肝癌细胞恶性增殖有赖于IFRD1基因的高表达,而中药活血化瘀治法对其具有下调作用。
This work aimed to study the regulation effects of several common TCM formula therapies on the expression of IFRD1 gene in rat liver cancer and its role in growth and proliferation of human hepatocarcinoma cells. Rat hepatocellular carcinoma (HCC) models were induced by diethylnitrosamine (DEN). The rats were treated by different therapeutical methods, including nourishing spleen and enriching qi, clearing away heat and toxic material, and activating blood circulation. Then gene chips were used to measure the expression of IFRD1 gene. Two siRNA targets toward IFRD1 gene were designed. Recombinant plasmid was transfected to hepatocellular carcinoma cell line SMMC-7721. The gene expression of IFRD1 was determined using real-time quantitative PCR, and the cell viability was determined by the MTr assay. According to the gene chip results, the expression of IFRD1 was significantly increased after HCC formation. All three methods had therapeutic effects, while nourishing spleen and enriching qi prescription had the significant down-regulation effect. An effective siRNA target sequence in IFRD1 was got by the MTT assay. The cell growth and proliferation were greatly inhibited when the interfered target was transfected for 144 h. The real-time quantitative PCR result showed that the gene expression was greately decreased 72 h after the transfection. Taken together, the proliferation of malignant hepatocarcinoma cells is related with high expression of IFRD1 gene, so it may act as a target for screening and evaluating the curative effect to hepatocarcinoma.