Multiple clinical and experimental studies have suggested that epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) may be effective at treating advanced non-small cell lung cancer (NSCLC), however, the molecular basis of primary resistance to EGFR-TKIs in NSCLC remains unclear. In the current study, the insulin-like growth factor 1 receptor (IGF-1R) gene in the gefitinib-resistant human lung adenocarcinoma epithelial cell line A549 (A549/GR) was silenced using small interfering RNA (siRNA) in order to determine the role of microRNA (miRNA) in the development of resistance against epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in lung adenocarcinoma. The relative gefitinib-resistant capacity in A549 and A549/GR cells was determined using a cell counting kit 8. A549/GR cells were transfected with chemically synthesized siRNA to silence the IGF-1R gene. A total of 48 h after siRNA transfection, IGF-1R expression in A549/GR cells was evaluated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. miRNA expression in A549/GR cells and A549/GR cells with silenced IGF-1R was analyzed using a miRNA microarray. The microarray results of 10 miRNAs were then compared with the results of RT-qPCR. The results demonstrated that the gefitinib-resistance capacity of A549/GR cells was six times higher than that of A549 cells. Additionally, RT-qPCR and western blotting demonstrated that the IGF-1R gene in A549/GR cells was successfully silenced by siRNA. The highest silencing rate (72%) of the IGF-1R gene was obtained using siRNA-2. The microarray identified 72 miRNAs with significantly different expression in A549/GR cells with silenced IGF-1R compared with A549/GR cells. Of the 72 differentially expressed miRNAs, 13 miRNAs (including miR-497-3p and miR-1273c) were up-regulated and 59 miRNAs (including miR-361-3p and miR-345-3p) were down-regulated in A549/GR cells with silenced IGF-1R compared with A549/GR cells. The changes in the expression of 10 different miRNAs were confirmed by RT-qPCR. Thus, the present study successfully established an A549/GR cell line with silenced IGF-1R. The results suggest that a number of miRNAs associated with the IGF-1R signaling pathway, including miR-497-3p and miR-144-5p, were involved in the development of resistance against EGFR-TKIs in A549 cells. These miRNAs may provide novel targets to treat lung adenocarcinoma exhibiting resistance against EGFR-TKIs.
Keywords: drug resistance; gefitinib; insulin-like growth factor receptor 1; microRNA; non-small cell lung cancer.