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目的运用siRNA抑制肝门部胆管癌QBC939细胞系中人DNA甲基转移酶1(Human DNA methylation trans-ferase 1,hDNMT1)基因的表达,探讨hDNMT1基因沉默后对QBC939细胞抑癌基因p16和RASSF1A去甲基化的影响,初步阐明其机制。方法根据GenBank中登录的hDNMT1 mRNA的核苷酸序列设计并合成发夹式siRNA序列,并以此为模板构建siRNA质粒,经脂质体转染QBC939细胞,通过RT-PCR检测hDNMT1、p16、RASSF1A基因mRNA转录水平;甲基特异性PCR(MSP)检测p16、RASSF1A基因的甲基化情况;MTT法检测QBC939细胞增殖活力。结果 sihDNMT1可有效抑制QBC939细胞中hDNMT1基因mRNA转录水平,同时上调p16、RASSF1A抑癌基因mRNA转录水平;sihDNMT1可促进p16、RASSF1A抑癌基因的去甲基化,并抑制QBC939细胞增殖,增殖抑制率在转染后72 h达到峰值。结论 hDNMT基因引起的抑癌基因启动子区异常甲基化是促进肝门部胆管癌发生发展的一个重要因素,抑制hDNMT1基因的表达可促进抑癌基因的去甲基化,为肝门部胆管癌的基因治疗提供了新的思路。
Objective To use siRNA to inhibit the expression of human DNA methylation trans-ferase 1 (hDNMT1) gene in human cholangiocarcinoma of the hilar cholangiocarcinoma (QBC939) and to investigate the effect of hDNMT1 gene silencing on the expression of tumor suppressor genes p16 and RASSF1A in QBC939 cells Methylation of the initial clarify the mechanism. Methods According to the nucleotide sequence of hDNMT1 mRNA registered in GenBank, hairpin siRNA sequences were designed and synthesized. The siRNA plasmids were constructed and transfected into QBC939 cells by lipofectamine. The expression of hDNMT1, p16, RASSF1A The mRNA level of mRNA expression of p16 and RASSF1A was detected by methyl-specific PCR (MSP). The proliferation of QBC939 cells was detected by MTT assay. Results sihDNMT1 could effectively inhibit the mRNA transcription of hDNMT1 in QBC939 cells and up-regulate the mRNA transcription level of p16 and RASSF1A. SihDNMT1 can promote the demethylation of p16 and RASSF1A tumor suppressor genes and inhibit the proliferation and proliferation of QBC939 cells 72 h after transfection peaked. Conclusion The abnormal methylation of tumor suppressor gene promoter induced by hDNMT gene is an important factor to promote the occurrence and development of hilar cholangiocarcinoma. Suppression of hDNMT1 gene expression can promote the demethylation of tumor suppressor gene. Cancer gene therapy provides a new way of thinking.