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目的验证在乳腺癌上皮细胞系中,miR-217通过直接作用于DNMT1 3′UTR调控DNMT1表达,为后期动物实验和临床试验提供实验基础。方法利用瞬时转染技术,在对应细胞系中,过表达、抑制miR-217,Western blot检测各实验组中DNMT1蛋白表达,并用含有miR-217野生型及突变型识别位点的DNMT1 3′UTR载体质粒分别转染293T细胞,检测相对荧光素酶活性改变。结果过表达miR-217,DNMT1表达下降;抑制miR-217,DNMT1表达增高;双荧光素酶检测实验,DNMT1-UTR-WT的相对荧光素酶活性显著低于DNMT1-UTR-MUT,差异有统计学意义(P<0.05)。结论在乳腺癌上皮细胞系中,miR-217对DNMT1表达具有调控作用,且miR-217通过直接作用于DNMT1 3′UTR调控DNMT1表达。
Objective To verify that miR-217 regulates DNMT1 expression by directly acting on DNMT1 3’UTR in breast cancer epithelial cell lines, providing experimental basis for later animal experiments and clinical trials. Methods The expression of DNMT1 protein in each experimental group was overexpressed, inhibited by miR-217 and Western blotting by transient transfection. DNMT1 3’UTR containing miR-217 wild-type and mutant recognition sites The vector plasmid was transfected into 293T cells respectively to detect the change of relative luciferase activity. Results The overexpression of miR-217 and DNMT1 decreased, while the expression of miR-217 and DNMT1 decreased. In dual luciferase assay, the relative luciferase activity of DNMT1-UTR-WT was significantly lower than that of DNMT1-UTR-MUT Significance (P <0.05). Conclusion miR-217 regulates DNMT1 expression in breast cancer epithelial cell lines, and miR-217 regulates DNMT1 expression by acting directly on DNMT1 3’UTR.