人乳腺癌上皮细胞miR-217对DNMT1调控作用的研究

The research of the regulation of miR-217 to DNMT1 in breast cancer cells

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 validate whether miR-217 could directly regulate the expression of DNA DNA methyltransferasel （DNMT1） by targeting on DNMT1 3＇ untranslated region（3＇UTR） in breast cancer cells,and to lay the foundation for the further research of animal experiments and clinical trials. Methods The expression of miR-217 in human mammary epithelial cell lines of HBL-100,MCF-7 and MDA-MB-231 were detected by real-time polymerase chain reaction（RT-PCR）, miR-217 rninics was trans- fected in the low expression of miR-217 cell line,and miR-217 inhibitor was transfected in the high expression of miR-217 cell line. The expression of DNMT1 protein in the transfected cells was analyzed by Western blot. The psiCHECK-2 reporter vectors contai- ning 312-bp DNMT1 3＇UTR with the wild-type（WT） and mutations（MUT） miR-217 recognizing sites were respectively transfect- ed 293T cells and the relative luciferase activity was measured after 48 h. Results Upregulation of miR-217 expression in MDA- MB-231 cells（low expression of miR-217） could decrease the expression level of DNMT1 protein, the downregulation of miR-217 expression in HBL-100 cells（high expression of miR-217） could increase the expression level of DNMT1 protein. The results of du- al-luciferase activity assay showed that the relative luciferase activity of DNMT1-UTR-WT was significantly lower than DNMT1- UTR-MUT（P〈0.05）. Conclusion In breast cancer cells,miR-217 could directly regulate the expression of DNMT1 by targeting on DNMT1 3＇UTR.