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目的:通过抑制肺癌细胞DNA甲基转移酶1(DNMT1)的表达,从而部分恢复抑癌基因的表达。方法:采用RNAi技术来抑制DNMT1的活性。应用体外转录的方法,共合成了5个针对DNMT1不同靶位的siRNA序列,并转染人肺癌细胞NCI H157。采用MSP PCR、COBRA等方法对抑癌基因RASSF1A进行甲基化分析,半定量RT PCR检测DNMT1mRNA的敲降与RASSF1A基因的表达。结果与结论:5个靶位的siRNA序列中有两个靶位能够有效地抑制DNMT1的表达,且呈剂量依赖效应。人肺癌细胞NCI H157中抑癌基因RASSF1A发生了高度甲基化,在抑制DNMT1活性后,RASSF1A基因去甲基化而恢复表达。
OBJECTIVE: To partially restore the expression of tumor suppressor gene by inhibiting the expression of DNA methyltransferase 1 (DNMT1) in lung cancer cells. Methods: The RNAi technique was used to inhibit the activity of DNMT1. Using in vitro transcription method, a total of five siRNA sequences targeting different DNMT1 targets were synthesized and transfected into human lung cancer cell line NCI H157. Methylation analysis of tumor suppressor gene RASSF1A was performed by MSP PCR and COBRA. The knockdown of DNMT1 mRNA and the expression of RASSF1A gene were detected by semi-quantitative RT-PCR. RESULTS AND CONCLUSION: Two of the five target siRNA sequences effectively inhibited DNMT1 expression in a dose-dependent manner. The RASSF1A gene was highly methylated in human lung cancer cell line NCI H157. After inhibition of DNMT1 activity, RASSF1A gene was demethylated and restored.