The Regulating Mechanisms Of Multiple Gene Methylalion Level On Breast Cancer MCF-7 Susceptibility To Ramoxijen

ObjectiveLooking for MCF- 7 tamoxifen resistance of breast cancer(TAM) cell lines related to tumor suppressor promoter Cp G island methylation is unusual, selecting key genes which can predict the susceptibility of MCF- 7 related to tamoxifen, to provide basis for clinical chemotherapy sensitivity prediction method using abnormal tumor suppressor gene expression and promoter regions abnormal methylation.Methods1.With thiazole blue colorimetry method(determined by MTT) contrast detection of MCF 7 / TAM MCF- 7 cell lines and multiple drug resistance and breast cancer drug-resistant strains of MCF 7 / TAM proliferation differentiation.2.Flow cytometry(FCM) contrast differences between the two cell lines growth cycle and apoptosis rate.3.RT-PCR and real- time PCR compared to two cell lines related genes(DAPK、RASSF1A、MGMT、P53、DNMT1、DNM3B、DNMT3A)m RNA expression differences.4.the methylation status of 4 candidate genes(DAPK, MGMT, P53, RASSF1A) in parents and resistant strains using methylation specific PCR(MSP).Results1.Determined by MTT showed: 48 HMCF/TAM IC50 alue concentration of(17.920.03) mu mol/L, the resistance ratio was 2.63(t = 51.20, P < 51.20) MCF/TAM cell line proliferation higher levels(t = 4.472, P < 0.001).2.The flow analysis: MCF- 7 the MCF- 7 / TAM cell lines Gl period part of the block, growth inhibition and apoptosis rate is higher.3.Real- time shows, DAPK(Z = 2.087, P < 0.05), RASSF1A(Z = 4.123, P < 0.001) level is lower than the parent plants;The P53(Z = 10.378, P < 0.001), MGMT(Z = 42.378, P < 0.001), DNMT1(Z = 19.378, P < 0.001) DNM3B(Z = 9.378, P < 0.001) m RNA expression level up from their parent cell lines;Gene DNMT3 A m RNA expression level is low, in the parent and the resistant strains has not been checked out.4.MSP shows, in MCF- 7 cell, DAPK, P53 、 RASSF1 A is unmethylation status,whiletamoxifen resistance cell line for these two genes methylation status.Combining with the real- time PCR results, gene methylation status and methylatransferase(DNMT1, DNMT3B) is closely relative to express;In addition to the P53 gene, gene m RNA expression level and methylation status into contravariant relations.Conclusion1.there is a variety of gene promoter methylation The MCF- 7 / TAM breast cancer cells.2. The MCF- 7 / TAM in breast cancer cell lines, the MGMT gene methylation levels, a significant rise in expression level, tolerance of hormone therapy can be used as a prediction of sensitive genes.3. The MCF- 7 / TAM in breast cancer cell lines, DNMT1, DNMT3 B gene expression quantity increased significantly, to apply for epigenetics reverse drug tolerance research provides new ideas.