MiR-125Modulate Multidrug Resistance In A549/DDP Cells Through Targeting Bcl-2and ABCC4

AimsBoth of incidence and mortality rates of the lung cancer are the highest in the world.Multidrug resistance (MDR) was the leading cause of failure of chemotherapy and poorprognosis in lung cancer patients. Studies had shown that BCL2and ABCC4proteinswere highly expressed in a variety of malignant cells, and might mediate the occurrence oftumor MDR. This series of studies aims to observe the differential expression ofmiR-125b between the cell lines A549and its drug-resistant cell lines A549/DDP, andthereby see insight into the regulation of miR-125b on Bcl-2and ABCC4expression atboth protein level and mRNA level and its significance.Methods1. The miRNA expression profiles were detected and differentially expressed miRNAswere identified in lung adenocarcinoma cell line A549and its multidrug-resistant celllines A549/DDP using miRNA microarray.2. The accuracy of the miRNA microarray results was verified by quantitative real-timeRT-PCR.3. The target genes regulated by miR-125b were predicted by refering to elevant literatures and using bioinformatics web sites or software.4. Experimental validation of target genes were conducted using luciferase reporter assays.5. The miR-125b mimics were transfected into the cell line A549/DDP, and the targetgene’s protein expression were then detected.6. The effect of miR-125b on drug sensitivity of the multi-drug-resistant lung cancer cellline A549/DDP was determined by MTT assay.7. The impact of miR-125b on apoptosis in A549/DDP multidrug-resistant human lungadenocarcinoma cells was assessed by flow cytometry analysis.Results1. The miRNAs expression profile of A549and A549/DDP was analyzed, and22differentially expressed miRNAs were identified when the fold change determined forthe expression ratio of A549/CDDP vs A549was higher than1.19times (P <0.05).Among them,14miRNAs were significantly upregulated and8miRNAs weredown-regulated in A549/DDP.2. The two significantly differentially expressed miRNAs were measured usingquantitative RT-PCR assays, and the results demonstrated that miR-125b expressionwas significantly decreased and mir-24expression was somehow increased inA549/DDP cells when compared with A549cells, which was consistent with that of themiRNA microarray results suggesting the miRNA microarray results can accuratelyreflect the differences in miRNA expression between A549and A549/DDP cell lines.3. The predicted target genes of miR-125b including Bcl-2and ABCC4was identifiedbased on reference to relevant literatures and applying of bioinformatics web sites orsoftware, and it had been confirmed the two genes played a role in a variety ofmalignancies.4. It was revealed that miR-125b inhibited luciferase expression by binding to Bcl-23’UTR and ABCC4-23’UTR cloned downstream of the luciferase coding region,therefore, the destruction of the binding sites can abolish the inhibitory effect ofmiR-125b on the luciferase expression. 5. Comparing with the cells in the control group, the protein expression levels of Bcl-2and ABCC4were significantly reduced in the A549/DDP cells transfected withmiR-125b mimics.6. The drug sensitivity testing in vitro showed a significantly increased sensitivity tovarious chemotherapeutic agents in the miR-125b mimics-transfected A549/DDP cellswhen compared with the control cells.7. Flow cytometry analyses indicated that the sensitivity to drug-induced apoptosis in themiR-125b mimics-transfected A549/DDP cells was significantly increased as comparedto control cellsConclusions1. The miRNAs were differentially expressed between the human lung adenocarcinomacell line A549and the multidrug-resistant human lung cancer cell line A549/DDP, andthose differentially expressed miRNAs may be involved in the development of MDRin lung cancer.2. miR-125b may modulate the sensitivity of lung cancer cells to apoptosis induced bychemotherapy agents via suppressing Bcl-2and ABCC4gene expression...