The Expression And Mechanism Of MicroRNAs In Reversal Effect Of5-Aza-dc On Cisplatin-resistance In NSCLC Cells

Background:Cisplatin is the main chemotherapy drug for non-small cell lung cancer (NSCLC), and cisplatin-resistance is considered a major impediment in the treatment. How to reverse cisplatin-resistance in NSCLC is a research hotspot. Our previous research has demonstrated that5-aza-2’-deoxycytidine (5-Aza-dc) could reverse cisplatin-resistance in NSCLC cells. However, the mechanism is not clear. The latest research showed that miRNA is mediated drug resistance in various cancers, and miRNA expression profiles in cancer cells can be affected by5-Aza-dc. This study compared differential expression of miRNAs between A549/DDP cells and the same cells treated by5-Aza-dc, and then predicted and analyzed the target genes of miRNAs, in order to identify whether miRNA is associated with reversal effect of5-Aza-dc on cisplatin-resistance in NSCLC A549/DDP cell line.Objective:To explore the correlation between miRNA and reversal effect of5-Aza-dc on cisplatin-resistance in NSCLC A549/DDP cell line. To screen the miRNAs and target genes which probably regulate the reversal effect of5-Aza-dc on cisplatin-resistance in NSCLC A549/DDP cell lineMethods:MTT method for test of cisplatin resistance index in A549/DDP cells, and cisplatin-resistance reverse index of A549/DDP cells treated by5-Aza-dc. Microarray was employed to compare differential expression of miRNAs between A549/DDP cells and the same cells treated by 5-Aza-dc; the results of microarray were confirmed by RT-qPCR. TargeScanHuman, PicTar2005and miRanda v5.1were used to predict the target genes of miRNAs; GO Tree Machine software was used to explore GO annotation clustering of those target genes; then the function of those genes would be suggested from those enrichment of GO items. Finally, DAVID online platform was used to predict the biological pathways of those genes.Results:1. The IC50of cisplatin in A549and A549/DDP cells were6.79±1.58μM,39.39±2.86μM, the resistance index of A549/DDP was5.80, A549/DDP cells was identified as cisplatin-resistant NSCLC cells; Non-toxic dose of5-Aza-dc(20μM) reduced the IC50of cisplatin to17.21±3.13μM, cisplatin-resistance reverse index of A549/DDP was2.29.2. The results of miRNA microarray showed that131miRNAs expressed differentially between A549/DDP cells and the same cells treated by5-Aza-dc, the expressions of27miRNAs were up-regulated by more than two folds, while expressions of16miRNAs were down-regulated by more than two folds. MiR-320a, let-7b, miR-548j were further confirmed by RT-qPCR, the results of RT-qPCR were consistent with that of microarray.3. The result of TargeScanHuma, PicTar2005and miRanda v5.1prediction showed that target genes of miR-320a, let-7b, miR-548j; analysis of GO Tree Machine laid out that target genes may related to cancer; the enriched pathways of target genes were predicted by DAVID, including JAK/STAT signaling pathway, MAPK signaling pathway, PPAR signaling pathway, adherens junction and Wnt signaling pathway, all of which involved in the cancer pathway.Conclusion:The miRNA expression profiles between A549/DDP cells and the same cells treated by5-Aza-dc is conspicuously different, miRNAs may play an important role in reversal effect of5-Aza-dc on cisplatin-resistance in NSCLC cells by target gens.9figures,12tables and38references...