DNMT1干扰对唾液腺腺样囊性癌细胞系ACC-M E-cadherin表达的影响

目的：建立DNA甲基转移酶1（DNMT1）表达稳定抑制的唾液腺腺样囊性癌细胞系ACC-M,探讨DNMT1表达抑制对ACC-M细胞E-cadherin表达的影响。方法：设计靶向干扰DNMT1的shRNA序列,构建携带该序列的慢病毒载体并转导ACC-M细胞,对筛选出的抗性克隆采用RT-PCR、荧光定量PCR、免疫印迹方法分别检测DNMT1mRNA、蛋白质水平,筛选获得DNMT1表达稳定抑制的ACC-M细胞,并通过甲基化特异性PCR检测E-cadherin基因启动子的甲基化状态,荧光定量PCR检测E-cadherin的表达情况。采用SPSS11.0软件包对数据进行t检验。结果：筛选获得DNMT1稳定表达抑制的ACC-M细胞,其mRNA、蛋白相对表达水平（0.156±0.008,0.163±0.013）显著低于空白对照组和空载对照组。进一步检测发现,E-cadherin基因启动子甲基化水平明显降低,E-cadherinmRNA表达水平显著增高,P〈0.05。结论：shRNA慢病毒载体介导的RNA干扰能够有效、稳定地抑制ACC-M细胞DNMT1的表达,并降低ACC-M细胞E-cadherin基因启动子的甲基化水平,从而使E-cadherin基因表达增强。

Effect of RNA interference mediated knockdown of DNMT1 on E-cadherin gene expression in ACC-M line

PURPOSE： To construct ACC-M cell line of RNA interference-mediated knockdown of DNMT1, and to explore the effeets on E-cadhefin gene expression in this cell line. METHODS： The ACC-M cell was transdueted by lentiviral particles containing shRNA targeting DNMT1 mRNA. After the puromycin-resistant colonies had been screened and expanded, the DNMT1 mRNA and protein was analyze by RT-PCR, real-time PCR and Western blot. The results were compared with the blank and empty-loading control. The promoter methylation of E-cadherin gene was detected by methylation specific PCR and mRNA expression of E-cadherin gene was analyzed by real-time PCR. The data were analyzed with SPSS 11.0 software for t test. RESULTS： The relative level of DNMT1 mRNA and protein of KD 3 group （0.156±0.008, 0.163±0.013） was significantly lower than two control group. In the KD 3 group, the E-cadherin promoter demethylated significantly and expression of E-cadherin gene increased greatly, P〈0.05. CONCLUSION： Lentiviral vectorbased DNMT1 RNA interference can effectively and stably reduces the expression of DNMT1 and lead to promoterdemethylation and increased expression of E-cadherin in ACC-M line.