| OBJECTIVEIn this paper,we analyzed the results of High-throughput sequencing from the pre-laboratory to find the difference miRNAs between the drug resistance of human breast cancer cells MCF-7/ADR to adriamycin and the wild-type human breast cancer cells MCF-7,combined with relevant literature screening results of domestic and overseas,selected a number of up-regulated and down-regulated of miRNAs from the differential expression results.Detect the cell inhibitory rate by transfected with miRNAs into MCF-7/ADR cells and the results of the rate of miR-200 a mimics and the miR-877 inhibitor were more significant.However,miR-200 a,miR-877 can reverse the drug resistance of MCF-7/ADR cells and it will further affect the related cell function is unclear.Then we will explore the effect of miR-200 a or miR-877 in MDR phenomenon of breast cancer whether it can become a new target for diagnosis and treatment of reference and theoretical basis.METHOD1.MCF-7/ADR cell stable drug resistance testReal-Time RT-PCR,flow cytometry and WST-1 assay were used to detect the drug resistance of MCF-7/ADR cells to adriamycin in laboratory.2.miR-200 a,miR-877 on MCF-7/ADR cell resistanceTo investigate the role of miR-200 a or miR-877 in reversing the drug resistance of MCF-7/ADR cells to adriamycin by Real-Time RT-PCR,flow cytometry.3.miR-200 a,miR-877 on MCF-7/ADR cell functionTo investigate the effect of miR-200 a or miR-877 on the reversal of drug resistance in MCF-7/ADR cells to adriamycin by WST-1 assay,flow cytometry and transwell assay.RESULT1.MCF-7/ADR cells have stable resistance(1)MCF-7/ADR cells were found to be resistant to adriamycin when treated with adriamycin for 24 h.(2)The results of cell proliferation showed that MCF-7/ADR cells were less lethality than MCF-7 cells in the same concentration of adriamycin.(3)Apoptosis results showed that MCF-7/ADR cells were lower apoptosis rate.Real-Time RT-PCR results showed that MDR1 and MRP1 expression of MCF-7/ADR cells was higher than MCF-7 cells.(4)Flow cytometry detection of intracellular adriamycin results showed that MCF-7/ADR is more difficult to take adriamycin than MCF-7 cells.2.High expression of miR-200 a or low expression of miR-877 can reverse the drug resistance of MCF-7/ADR cells to adriamycin(1)miR-200 a and miR-877 were transfected into MCF-7/ADR cells respectively by lipofectamineTM 2000.Real-Time RT-PCR results showed that the genes expression of MDR1 and MRP1 after high pression of miR-200 a or low expression of miR-877 of MCF-7/ADR cells were decreased.(2)Uptake of adriamycin with high expression of miR-200 a or low expression of miR-877 of MCF-7/ADR cells were increased.3.High expression of miR-200 a,low expression of miR-877 on cell function(1)The cell proliferation of high expression miR-200 a or low expression of miR-877 of MCF-7/ADR cells in WST-1 assay showed that the proliferation inhibition rate of MCF-7/ADR cells treated with different concentrations of adriamycin was higher than the control group.(2)The results of flow cytometry showed that high expression miR-200 a or low expression of miR-877 of MCF-7/ADR cells were more likely to occur to apoptosis.The cell cycle was mainly arrested in G0/G1 phase.(3)The results of transwell assay showed that the migration and invasion of MCF-7/ADR cells were inhibited after transfected with miR-200 a or miR-877.CONCLUSION1.The results of cell morphology test,cell proliferation test,apoptosis test,Real-Time RT-PCR test and flow cytometry test of intracellular adriamycin accumulation showed that MCF-7/ADR cell is a stable cell line with drug resistance to adriamycin.2.The results of Real-Time RT-PCR and flow cytometry after high expression miR-200 a or low expression of miR-877 of MCF-7/ADR cells showed that miR-200 a,miR-877 can reverse the drug resistance of MCF-7/ADR cells by decreasing the expression of MDR1 and MRP1,then increasing the concentration of adriamycin in MCF-7/ADR cells through induced P-gp encoded by MDR1.3.Up-regulation of miR-200 a or down-regulation of miR-877 could reverse the drug resistance of MCF-7/ADR cells through MDR1 mechanism.Then inhibit cell proliferation,promote cell apoptosis,arrest cell cycle in G0/G1 phase,inhibit cell migration and cell invasion and other related cell functions. |
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