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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。
OBJECTIVE: To establish a murine adenoid cystic carcinoma cell line ACC-M with stable expression of DNMT1 and investigate the effect of DNMT1 expression inhibition on E-cadherin expression in ACC-M cells. METHODS: The shRNA targeting DNMT1 was designed and constructed. The lentiviral vector carrying this sequence was constructed and transfected into ACC-M cells. The selected clones were tested for DNMT1 mRNA by RT-PCR, real-time PCR and Western blotting. Protein level. The ACC-M cells with stable expression of DNMT1 were screened. The methylation status of E-cadherin gene promoter was detected by methylation-specific PCR. The expression of E-cadherin was detected by quantitative PCR. Data was t-tested using SPSS11.0 software package. Results: The relative expression levels of mRNA and protein in ACC-M cells with DNMT1 knockdown were significantly lower than those in control group and blank control group (0.156 ± 0.008, 0.163 ± 0.013). Further examination revealed that promoter methylation of E-cadherin gene was significantly decreased and E-cadherin mRNA expression was significantly increased (P <0.05). CONCLUSION: shRNA lentiviral vector-mediated RNA interference can effectively and stably inhibit the DNMT1 expression in ACC-M cells and reduce the methylation level of E-cadherin gene promoter in ACC-M cells, so that E-cadherin gene expression Enhanced.