DNA Methylation Mechanisms Of MiR-21 Regulated Proliferation In Breast Cancer Cells

ObjectiveBoth of small RNA 21(miR-21) and DNA methylation play important roles in the process of breast cancer, and are closely related to the proliferation of cancer cells.However, the interaction between the two in breast cancer is poorly understood.Therefore, in this study, we aim to explored the interaction between miR-21 and DNA methylation in breast cancer cells as the starting point, and further by regulating the expression miR-21 and DNA methylation level, to study their combined effect on the proliferation of breast cancer cells.MethodsVirus mediated mi R-21 inhibitor and its negative control were transfected into MCF-7 and MDA-MB-231 cells. The transfection efficiency were observed using fluorescence microscope, and the inhibited expression of miR-21 were detected by Real-time PCR. The genomic DNA methylation level were detected by bisulfite-qMSP, and the RNA and protein level of Dnmts(Dnmt1, Dnmt3 a,Dnmt3b),HRAS and RASGRP1 were detected by Real-time PCR and western blot respectively. On the other hand, cells were treated with 5-Aza(2.5 M) for 72 hours, to detect the effect of DNA methylation on miR-21 expression.BrdU(5- bromodeoxyuridine) assay was used to detect the combined effects of miR-21 inhibitor and 5-Aza on proliferation of breast cancer cells, and the cell cycle and apoptosis were also examined using flow cytometry.ResultsFluorescence microscopy showed that, LV3-AS-miR-21 and LV3-NC can effectively infected cells, and LV3-AS-miR-21 significantly knock down miR-21 expression levels(P<0.05). Knockdown of miR-21 expression, can significantly reduce the genomic DNA methylation level of MCF-7(P<0.05), with the transcriptional levels of Dnmt1 and Dnmt3 a increased(P<0.05) and Dnmt3 b decreased(P<0.05). And in the MDA-MB-231 cells, the genomic DNA methylation level was found significant increased(P<0.05), accompanied by a elevated transcriptional levels of Dnmt1(P<0.05), Dnmt3a(P<0.05) and Dnmt3b(P<0.05).While in the detection of Dnmts protein levels, we found knockdown of miR-21 expression can only induce Dnmt3 b protein level decreased in MCF-7 cell and increased in MDA-MB-231 cell. Meanwhile, significantly reduced expression of RASGRP1(P<0.01) in MCF-7 cell and increased expression level of H-RAS(P<0.01)and RASGRP1(P<0.05) in MDA-MB-231 cell were also observed. Cells treated with5-Aza, which can significantly reduce genomic DNA methylation levels in both two cells(P<0.01), can effectively increase the expression level of miR-21(P<0.01).BrdU assay results showed that, compared to single effects, combined use of miR-21 inhibitor and 5-Aza can synergistically inhibited the proliferation of MCF-7 cells(P<0.01); and in MDA-MB-231 cells, the inhibitory effect of miR-21 inhibitor on cell proliferation can be further enhanced by 5-Aza(P<0.01). The following cell cycle detection results showed that 5-Aza and miR-21 inhibitor in combination may further exacerbate the G1 / S arrest in MCF-7 cells and synergistically reduce protein expression level of the cell cycle regulatory protein Cyclin D1.ConclusionThe regulation effects of DNA methylation by virus-mediated miR-21 downregulation in MCF-7 and MDA-MB-231 cells are almost opposite, in which Dnmt3 b played a major role, and the RAS signaling pathway may involved in this process.Whilst the expression of miR-21 in two cell lines were all upregulated by decreased DNA methylation level. Compared to single effects, DNA hypomethylation can promote the inhibitory effect of miR-21 inhibitor on the proliferation of breast cancer, which may caused by the loss of control of the cell cycle and the dysregulation of Cyclin D1 expression. This study contributes to understanding the mechanism of breast cancer tumorigenesis, which may further provide new theoretical evidences for setting reasonable breast cancer chemotherapy scheme.