The Effect Of Cytoplasmic Macrophage Colony-stimulating Factor On The Apoptosis And Expression Multidrug Resitance-associated Microrna In Human Breast Cancer MCF-7Cells

OBJECTIVES: To explore the effect of cytoplasmic macrophagecolony-stimulating factor (M-CSF) on the apoptosis and expression of multidrugresistance-associated microRNA in MCF-7cells.METHODS: The parent MCF-7cells (referred as to MCF-7cell),pCMV/cyto/myc-transfected MCF-7cells (referred as to MCF-7-C cell) and pCMV/cyto/myc-M-CSF-transfected MCF-7cells (referred as to MCF-7-M cell) werecultured in1640medium. The effect of cytoplasmic M-CSF on the sensitivity ofMCF-7cells to TAX and5-FU was evaluated by half maximal (50%) inhibitoryconcentration (IC50) based on the result from MTT. The intracellular drugconcentration was measured by high performance liquid chromatography.Differentially expressed microRNAs were detected by Gene chip and resulting geneswere verificated by quantitative real time PCR. The target genes of differentiallyexpressed miRNAs were predicted by bioinformatics method and resulting geneswere verificated by Western Blot. The apoprosis was observed by Hoechst-PIstaining and flow cytometry.RESULTS: The results from MTT showed that the sensitivity of MCF-7-M cellsto TAX and5-FU was markedly lower than that of either MCF-7-C cells or MCF-7cells(p<0.05), and that IC50of MCF-7-C cell，MCF-7cell and MCF-7-M cell to TAXwas10.37,9.58,77.35μg/mL and to5-FU was21.57,20.13,207.31μg/mL,respectively. The results from HPLC indicated that there was s significantly lower forthe intracellular TAX and5-FU in MCF-7-M cells than that of either MCF-7-C cellsor MCF-7cell（sp<0.05）. The results from Gene chip suggested that10miRNAs weremore than1.5-fold up-regulated (eg.hsa-miRPlus-F1159, hsa-miR-27a, hsa-let-7e,et al) and6miRNAs were less than0.5-fold down-regulated (eg. hsa-miR-34b,hsa-miR-143, hsa-miRPlus-F1042, et al). Of all miRNA, these resulting miRNAincluding hsa-miRPlus-F1159, hsa-miRPlus-E1045and hsa-miRPlus-F1042werepredicted by computer software. The results from RT-PCR showed that expression ofhas-miR-27a, has-let-7e and has-miR-143in MCF-7-M cells was markedly higherthan that in either MCF-7-C cells or MCF-7cells(p<0.05). The results from Westernblotting indicated that expression of ABL2protein in MCF-7-M cells wassignificantly higher than that in either MCF-7-C cells or MCF-7cells(p<0.05). Theresults from Hoechst-PI staining demonstrated that the feature of apoptosis waspresented in both MCF-7-C and MCF-7cells, but not in MCF-7-M cells. The resultsfrom flow cytometry showed that apoptosis rate of MCF-7-M cells, MCF-7-C cells andMCF-7cells was40.2%,51.4%and12.6%when cells treated by2.0μg/ml TAX for24h，whereas44.9%,42.1%and0.0%when cells treated by13.5μg/ml5-FU，respectively., which suggested that apoptosis rate of MCF-7-M cells was markedlylower than that of either MCF-7-C cells or MCF-7cells(p<0.05).CONCLUSIONS:1. Cytoplasmic M-CSF decreased the drug sensitivity ofMCF-7cells to TAX and5-FU.2. Cytoplasmic M-CSF up-regulated the expression of these multidrugresistance-associated microRNA including hsa-miR-27a, has-let-7e and has-miR-143.3. Cytoplasmic M-CSF enhanced the anti-apoptosis ability of MCF-7cells andinduced the expression of anti-apoptosis protein ABL2in MCF-7cells.