肝癌细胞系BEL-7402中siRNA表达载体介导靶向c-myc基因

siRNA expressing vector-based RNA interference targeting c-myc in hepatocellular carcinoma cell line BEL-7402

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 using vector-based c myc specific RNA interference （RNAi） approach to block expression of e myc oncogene in hepatocellular carcinoma cells. Methods：Two RNA interference DNA templates targeting c-myc oncogene were designed and synthesized. The fragments were ligated to pSilencer1. 0-U6 vector to construct the recombinant siRNA expressing plasmids. The identified recombinants were introduced into BEL-7402 hepatocellular carcinoma cells in the presence of LipofaetamineTM. The inhibition of c-myc expression, together with the expression of CDK4, hTERT and Gadd45β in BEL 7402 cells with down-regulated c-myc expression, were analyzed by semi quantitative reversed transcriptase-polymerase chain reaction （RT PCR）. Results：Two c-myc-targeted siRNAs recombinant vectors named as pSic-myc-1 and pSic-myc-2 were constructed successfully. Among which, pSic-myc-2 triggered a RNAi-mediated inhibition of expression of c-mye in BEL- 7402 cells with inhibitory rate of more than 90%. In BEL7402 cells with knockdown of c-myc expression induced by RNAi, the expression of CDK4 and hTERT were down-regulated by 85 % and 57%,respectively, while the expression of Gadd45β was upregulated by up to 110 %. Conclusion： The expression of c-myc in BEL-7402 could be suppressed by vector-based RNA interference efficiently. The knockdown of c-myc in turn caused the changes in gene expression related with cell proliferation and apoptosis.