MiR-106b～25 Cluster Regulates Multidrug Resistance In An ABC Transporter-independent Manner Via Downregulation Of EP300

Background:Chemotherapy,as a pivotal part of therapy of breast cancer,plays a key role in prolonging the disease-free survival and long-term survival after recurrence and metastasis.However,multidrug resistance(MDR),which is shown by tumor cells,affects the effect of comprehensive treatment of tumor.Therefore,chemoresitance has become one of the key problems of cancer therapy.MicroRNAs(mi RNAs)are endogenous,highly conserved,non-coding RNAs of about 18-25 base sequences that are widely found in eukaryotic cells.In recent years,it has been found that microRNAs are combined with 3’UTR of target mRNA sequence through base complementary pairing,so as to regulate the expression of genes,and thus affect cell proliferation and apoptosis,cell cycle.Current studies have shown that microRNAs are involved in the development of tumors,including the process of drug resistance to chemotherapy.In this study,we focused on the microRNA-miR-106 b ~ 25 cluster,which affects the multidrug resistance of breast cancer cells,and explores its function in the process of MDR formation in breast cancer and explores miR-106 b ~ 25 The mechanism of the interaction between the relevant molecules.Method:Minimally transformed mammary epithelial cells(MTMECs)overexpressing the miR-106b～25 cluster by lentiviral transfection(MTMEC-miR-106b～25)or expressing a short hairpin targeting EP300 or E-cadherin(MTMEC-shEP300 and MTMEC-shCDH1,respectively).Paclitaxel,VP-16 and colchicine,and to verify the effect of paclitaxel on the induction of apoptosis in these cell lines.The sensibility of these cell lines are measured by MTT and drug resistance clonogenic assey.The expression level of ABCB1 mRNA in MTMEC-miR-106 b ~ 25,MTMEC-shEP300,NCI-ADR / RES and CAL51 were measured by RT-qPCR technique.The expression levels of P-glycoprotein in MTMEC-ev,MTMEC-miR-106 b ~ 25,MTMEC-shEP300 and NCI-ADR / RES,CAL51 cells,and the cell cycle of them were evaluated by flow cytometry.Apoptosis of MTMEC-ev,MTMEC-miR-106 b ~ 25 and MTMEC-shEP300 cells were analyzed by Annexin V / Propidium staining,caspase-3 / 7 activity test and caspase-9 activity test.The cytotoxicity test(MTT)of MTMEC-shCDH1 cells was applied to MTMEC-shCDH1 cells.The expression of E-cadherin in MTMEC cells was down-regulated by MTT assay.Drug resistance clone formation,Annexin V / Propidium staining,caspase-3/7 activity detection and caspase-9 detection,P-glycoprotein expression levels,intracellular drug accumulation experiments to determine MTMEC-shCDH1 cells and overexpression mi R-106 b ~ 25 cluster or inhibition of EP300 expression of MTMEC cells have similar biological function.Result:1.Overexpression of the miR-106 b ~ 25 cluster or knocked down the expression of EP300 in the MTMEC cell line attenuates the sensitivity of breast cancer cells to chemotherapeutic agents,resulting in a MDR phenotype.MTT assay and drug resistance clonogenic assey showed that chemoresistance of MTMEC-miR-106 b ~ 25 and MTMEC-shEP300 cells were significantly higher than those of MTMEC cells.2.Paclitaxel alterations in the cell cycle are abolished in MTMEC-miR-106b～25 and MTMEC-shEP300 cells.Overexpression of miR-106 b ~ 25 cluster or downregulation of EP300 reduces the ability of apoptosis,induced by paclitaxel.At the same time,the percentage of G2 / M cells in MTMEC-miR-106 b ~ 25 cells and MTMEC-shEP300 cells was significantly lower than that of MTMEC-ev cells under the same concentration of paclitaxel,While the proportion of G1 phase cells increased accordingly.In addition,the apoptotic cells of the first two were significantly decreased,and the activity of caspase-3/7 and caspase-9 were significantly decreased.3.ABC transporters are not responsible for the MDR phenotype of cells either overexpressing the miR-106b～25 cluster or with EP300 downregulated.MTMEC-ev cells,MTMEC-miR-106 b ~ 25 cells and MTMEC-shEP300 cells showed no significant difference in the expression of P-gp protein and the expression of corresponding genes by RT-QPCR and flow cytometry.And in the determination of intracellular drug accumulation level experiments found that,regardless of the presence or absence of cyclosporine,the drug in the three cells in the accumulation of the same level,and its negative control group of cells with the same trend.4.The down-regulation of E-cadherin expression can also induce the multidrug resistance of cells and weaken the ability of paclitaxel-induced apoptosis,and the drug resistance is independent of the expression of P-gp.For MTMEC-shCDH1 cells,the same experiment was repeated under the same experimental conditions,and the results were the same as those of the above experimental results.Conclusion:Overexpression of the mi R-106 b ~ 25 cluster or knocked down the expression of EP300 in the MTMEC cell line attenuates the sensitivity of breast cancer cells to chemotherapeutic agents,resulting in a MDR phenotype.By using drug efflux assay and P-glycoprotirn expression assay,we confirm that ABC transporters are not responsible for the MDR phenotype of cells either overexpressing the miR-106b～25 cluster or with EP300 downregulated.The MDR phenotype mainly shows as attenuating of the apoptotic effect of paclitaxel,which was expressed as the decreasing of the activity of apoptosis-related proteins caspase-3/7 and caspase-9,as well as ratio fo G2/M phase cells.In brief,MDR in cells overexpressing mi R-106b～25 cluster,or with downregulation of EP300,is due to apoptosis evasion.Meanwhile,downregulation of E-cadherin leads to the MDR phenotype which is transporter-independent,similar to overexpressing the miR-106b～25 cluster or downregulated EP300.