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目的:探索经载体介导的RNA干涉对肝癌细胞cmyc基因表达抑制的可行性。方法:设计并合成2条靶向癌基因cmyc的RNA干涉模板片段,分别将其连接至pSilencer1.0U6载体上构建siRNA真核表达载体;脂质体转染法将上述重组质粒转入BEL7402肝癌细胞株。半定量RTPCR分析cmyc基因的表达抑制效率,以及cmyc基因表达下调细胞中CDK4、hTERT和Gadd45β基因表达的改变。结果:获得2个能在细胞内有效转录生成靶向cmyc基因siRNA的重组载体pSicmyc1和pSicmyc2;其中pSicmyc2可有效介导肝癌细胞BEL7402cmyc基因的表达抑制,抑制率高达90%以上;在cmyc基因经RNAi抑制的细胞中,CDK4和hTERT基因的表达分别下调至85%和57%;而Gadd45β的表达上调约110%。结论:肝癌细胞BEL7402中cmyc基因的表达可被载体介导诱发的RNA干涉有效抑制,进而导致细胞中与增殖和凋亡相关基因的表达改变。
Objective: To explore the feasibility of inhibition of cmyc gene expression by hepatoma cells by vector-mediated RNA interference. METHODS: Two RNA interference template fragments targeting oncogene cmyc were designed and synthesized, and were respectively ligated into pSilencer1.0U6 vector to construct siRNA eukaryotic expression vector. The recombinant plasmids were transfected into BEL7402 hepatoma cells Strain. Semi-quantitative RTPCR analysis cmyc gene expression inhibition efficiency, and cmyc gene expression down-regulated CDK4, hTERT and Gadd45β gene expression changes. RESULTS: Two recombinant vectors pSicmyc1 and pSicmyc2, which could efficiently transcribe the siRNA targeting cmyc gene in cells, were obtained. PSicmyc2 could effectively inhibit the expression of BEL7402cmyc gene in hepatocellular carcinoma cells with the inhibition rate as high as 90%. After cmyc gene was transfected by RNAi Inhibitory cells, CDK4 and hTERT gene expression were down to 85% and 57%; and Gadd45β expression up about 110%. CONCLUSION: The expression of cmyc gene in BEL7402 hepatoma cells can be effectively inhibited by RNA interference mediated by carrier, leading to the change of the expression of genes related to proliferation and apoptosis in hepatocellular carcinoma cells.