The Research Of The Regulation Of MiR-217to The Methylation Key Genes In Breast Cancer Cells

Objective: To discuss miR-217could directly regulate the expression level aftertranscription of DNA methyltransferase1(DNMT1) and Enhancer of Zeste homolog2(EZH2) by targeting on DNMT1and EZH23′untranslated region (3′UTR) in breast cancercells, and laying the foundation for the further research of animal experiments and clinicaltrials.Methods: The expression level after transcription of miR-217and EZH2in humanmammary epithelial cell lines of HBL-100, MCF-7and MDA-MB-231were detected byreal-time polymerase chain reaction (RT-PCR). The expressions of EZH2protein of threecell lines were tested by Western blotting. miR-217minics was transfected in the lowexpression of miR-217cell line, and miR-217inhibitor was transfected in the highexpression of miR-217cell line. The protein expression of DNMT1and EZH2in thetransfected cells was analyzed by Western blotting. The psiCHECK-2reporter vectorcontaining361bp DNMT13′UTR or312bp EZH23′UTR with the “WT” or “MUT”miR-217recognizing sites were respectively transfected293T cells, then the relativeluciferase activity was measured after48h.Result: PT-PCR results showed that miR-217was significantly higher expression inMCF-7and MDA-MB-231breast cancer cells, compared with in HBL-100humanmammary epithelial cells (P<0.05). And the results of Western blotting showed that EZH2was significantly lower expression in MCF-7and MDA-MB-231, compared with inHBL-100. Upregulation of miR-217expression in MDA-MB-231cells (low expression ofmiR-217) could decrease the expression level of DNMT1and EZH2protein. Anddownregulation of miR-217expression in HBL-100cells (high expression of miR-217) could increase the expression level of DNMT1and EZH2protein. The results ofdual-luciferase activity assay showed that the relative luciferase activity ofDNMT1-UTR-WT was significantly lower than DNMT1-UTR-MUT (P<0.05), the relativeluciferase activity of EZH2-UTR-WT was also significantly lower than EZH2-UTR-MUT(P<0.05).Conclusion: In the breast cancer cells, miR-217could directly regulate the proteinexpression of DNMT1and EZH2through targeting on DNMT13′UTR and EZH23′UTR.