The Research Of Methylation Change And Resistance Mechanisms In Breast Cancer Cell Lines With Acquired Drug Resistance

Background:Breast cancer has a high incidence in the world, but the main cause influence the treatment of chemotherapy drug curative effect and prognosis of patients is the multi-drug resistance, however multi-resistant mechanism is still not clear. Studies have shown that breast cancer resistance protein BCRP/ABCG2 differentially expressed in the breast cancer tissue, which is the important cause of acquired drug resistance. The change of methylation pattern may be one of the mechanisms of drug resistance. In the study of drug resistant cell lines, researchers found that the expression of PARP-1 also affect cell drug resistance phenotype, and found that PARP-1 can act on the promoter region of DNMT1, affecting DNMT1’s methylation level. Therefore, this research use the mitoxantrone induce MCF- 7 cells to establish different drug resistance cell lines, and use drug-resistant cell line as the model, detecting the change of genome-wide methylation and methylation binding proteins. Then use RNA interference technique to build PARP-1 lower expression cell lines, by using the bisulfite sequence method to detect specific gene promoter methylation, preliminary discussion on the breast cancer cells induced by mitoxantrone resistant phenotypes, whether PARP-1 will affect the drug resistance of breast cancer cells by changing its DNA methylation patterns. Method:Using a final concentration of 0.005, 0.01 0.02, 0.04μmol/L of mitoxantrone for induce MCF-7 cell for 30 days, screening of resistant cells, and using the untreated MCF-7 as control group. Adopting CCK-8 and flow cytometry to detect the mitoxantrone tolerance of MCF-7 cells which treat with different drug concentrations to identify whether induction success. Western blot to detect MCF-7/mit resistant cells methyl-DNA-binding protein MBD1, MBD2 and MBD4 expression level. We used capillary electrophoresis method to detect genome-wide methylation level in MCF-7/mit cell. Using RNA interference technology to construct PARP-1 lower expression cell lines, and then by using the BSP, detecting ABCG2, MBD4 and DNMT3 b gene promoter region methylation level of control group and PARP-1 low expression cells. Results:Compared with control group of MCF-7 cell, with the mitoxantrone concentration increase, cells gradually increased resistance to mitoxantrone by CCK-8 method and flow cytometry. Western blot analysis of methylation binding protein expression in MCF-7/mit resistant cell lines, we found MBD1 MBD2, MBD4 expression showed a decreasing trend, especially MBD4(P<0.05). Capillary electrophoresis method to detect MCF-7/mit resistant cell lines of genome-wide methylation levels, with the increase of drug concentration, the whole genome methylation level continue to decrease. Tested by western blot, PARP-1deficient cell lines constructed by using lentivirus vector which compared to the control group, the interfering cell line PARP-1 protein levels were significantly lower. With BSP detection of PARP-1 normal expression MCF-7 cell and PARP-1 interference cell lines, compared with control group, the ABCG2, MBD4 and DNMT3 b gene promoter region methylation level all reduced in the lower expression PARP-1 cell lines, the analysis found in the promoter region of ABCG2 227,308,499,520 and 538 bp position, PARP-1 interfere with cell lines happen hypermethylation while normal cells did not occur methylation. The transcription factors TFAP2 A and KDM5 A may be involved in these sites, at the same time in DNMT3 b gene promoter region 195, 288, 312 and 345 bp position, PARP-1 interfere with cell lines occurred high hypermethylation while in normal cells happen hypomethylation. Those might be involved in these sites for transcription factors are EGR3, RARG, YY, NFKB1 CYB5 R, POU2F1, DUSP26 and etc. Conclusion:Successfully established cell lines for different levels of mitoxantrone-resistant MCF-7/mit resistant and PARP-1 deficient cell lines, provided the cell model for the research of breast cancer resistance protein mediated tumor cells resistant. In the MCF-7/mit cell lines, the reduce of genome-wide methylation levels as well as methylation binding protein expression may be one of the epigenetic mechanisms of breast cancer cells to acquired drug resistance. After interference PARP-1, we found in the ABCG2, MBD4, DNMT3 b gene promoter region methylation level have reduced, compared with the control group, suggesting that PARP-1 may be synergistic MBDs and DNMTs, through the action of transcription factors regulate the expression of ABCG2. And the differences of methylation site have selected in the ABCG2 and DNMT3 b gene promoter region, may be the specific methylation sites to promote ABCG2 gene differently express, which providing new targets for reversing the ABCG2 mediated multi-drug resistant.