| OBJECTIVE: To explore the effects of miR-125b and miR-21gene expressionchanges on Taxol resistance in human breast cancer cells MCF-7/PR and SKBR3/PR.METHODS: Increasing the concentration of Taxol step by step to treat with theMCF-7and SKBR3cells, changes of cellular morphology duiring the process ofacquired drug resistance were observated and recorded under the binocular invertedmicroscope; the drug resistant ability to Taxel of sensitive or resistant cells wasmeasured by MTT assay. MDR1, BCRP, MRP1, Bax, Bcl-2, miR-125b and miR-21level of sensitive or resistant cells was determined by real time PCR, The expressionof P-gp,Bcl-2and Bax protein of sensitive or resistant cells were analyzed by westernblot,while the transwell assay and the wound-healing assay were applied in biologicalactivity of MCF-7/PR and SKBR-3/PR compared with their parental cells. SyuntheticmiR-125b mimics and miR-21inhibitors were transfected into MCF-7/PR andSKBR3/PR breast cancer cells with lipofectamine2000vector, miR-125b and miR-21levels were determined by real time PCR.Drug resistance of cells was detected byMTT assay. The expression of P-gp, Bcl-2and Bax protein were analyzed by westernblot.while biological activity of cells was detected by using Transwell andWound-healing assay,cell cycle and apoptpsis were assqyed by flow cytometry.RESULTS:1.We established Paclitaxel resistant breast cancer cell lines MCF-7/PRand SKBR3/PR successfully. In MCF-7/PR and SKBR3/PR, the mRNAs of MDR1,BCRP, MRP1, Bcl-2, miR-125b increased and miR-21decreased (P<0.05).Theprotein levels of P-gp, Bcl-2were up-regulated, and Bax was down-regulated.Transmembrane in MCF-7/PR and SKBR3/PR was significantly more than that inMCF-7and SKBR3cells. The scratch wound-healing assay confirmed thatMCF-7/PR and SKBR-3/PR cells have significantly increased motility activitycompared with their respective parental cells (P<0.05).2. Overexpression ofmiR-125b and downregulation of miR-21, the IC50of Taxol MCF-7/PR and SKBR3/PR decreased significantly. Furthermore, miR-125b mimics and miR-21inhibitor could enhance apoptosis mRNA and protein expression in MCF-7/PR andSKBR3/PR, decreased MDR1and Bcl-2and increased Bax (P<0.05).Transmembrane cells in miR-125b mimics and miR-21inhibitor transfected cellswere significantly less than that in blank control or negative cells. The scratchwound-healing assay confirmed that miR-125b mimics and miR-21inhibitortransfected cells have significantly increased motility activity compared with in blankcontrol or negative cells (P<0.05). In miR-125b mimics and miR-21inhibitortransfected cells,apoptosis rates were significantly increased and cell cycle analysisshowed that they have increased G0/G1phase(P<0.05).CONCLUSIONS:1.The established MCF-7/PR and SKBR3/PR breast cancer cellshave the typical multidrug resistant characteristics, which can be used as the modelsfor resistance mechanism study.2. The resistance on Taxol of human Taxol-resistantbreast cancer cells is reversed by miR-125b overexpression or miR-21down-regulating. |
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