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MicroRNA expression profiles associated with acquired gefitinib-resistance in human lung adenocarcinoma cells.

Molecular medicine reports (2014-10-24)
Xiaojun Ge, Limei Zheng, Min Huang, Yonglun Wang, Feng Bi
ABSTRACT

The aim of the present study was to establish, characterize and elucidate the potential mechanisms of acquired gefitinb resistance, using the A549 human lung cancer cell line. A gefitinib-resistant A549 sub-clone was established by exposure to escalating gefitinib concentrations over a period of 16-24 months. Half maximal inhibitory concentration (IC50) values were quantified using a real time cytotoxicity assay. The expression profiles of the parent and resistant sub-clone A549 cells were detected using the µParaflo® Microfluidics Biochip microRNA (miRNA) Microarray. The ArrayPro software was used to analyze the differential expression levels of the miRNA, and bioinformatics software was used to predict the potential target genes of the differentially expressed miRNAs. Quantitative polymerase chain reaction (qPCR) was used to confirm the results of the miRNA microarray. A miRNA mimic was transfected into the gefitinib-resistant cells, in order to predict target gene interaction effects, following gefitinib treatment. Protein expression level differences were confirmed by western-blot analysis. Real time cytotoxicity assays revealed a 3-fold increase in the IC50 values of the gefitinib-resistant sub-clones, as compared with the parent cells. There were marked morphological differences between the parent and resistant cells. In the microarray analysis, the gefitinib-resistant sub-clones had 25 upregulated and 18 downregulated miRNAs, as compared with the parent cells. The qPCR revealed that miR-7 was significantly downregulated, which was concordant with the results of the microarray. The results of the present study suggest that miR-7 may significantly improve the sensitivity of cancer cells to gefitinib. The data presented in the present study provides an experimental basis and theory that miRNAs may be involved in acquired gefitinib-resistance of lung adenocarcinoma, and miR-7 may have potential clinical effects in the reversal of drug resistance.

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