| Objective Though epidermal growth factor receptor tyrosine kinase inhibitors(EGFR-TKI) have been used clinically and achieved remarkable efficacy in the treatment of advanced non-small cell lung cancer patients who had mutant EGFR gene, those patients will eventually experience acquired resistance to TKI after median 9?13 months.EGFR exon 20 T790M mutation is the most important mechanism of resistance. As the third generation TKI has been applied in the clinical, the survival of patients has been further extending. However, there is no better way to overcome the other mechanisms of drug resistance. New research methods are urgently needed. There are numourous evidences indicated that miRNAs are associated with anti-tumor durg resistance. The aim of this study was to screen the miRNA related to Gefitinib resistance, and to explore the mechanism of the interaction between miRNA and drug resistance related genes in human lung adenocarcinoma cell lines.Methods PC9/GR cells were developed from PC9 cells by repeated exposure to increasing concentrations of gefitinib, meanwhile A549 cells and H1975 cells were also involved in this study. Half inhibition rates (IC50) of gefitinib on 4 cells were calculated by MTT assay. EGFR gene 19?21 exon mutation spectrums were performed by direct DNA sequence. Differential expression of miRNAs between PC9 and PC9/GR was detected by microarray technology. The target genes of miRNA were predicted by using bioinformatics software and network database such as TargetScans, miRanda, PicTar and FindTar. After constrction of miRNA over-expression and low-expresision cell lines,MTT assays and flow cytometry analysis were performed to detect the alteration of cell viability and apoptosis rate. Quantitiative real-time PCR and western blot were used to detect the expression variation of target genes and protein. Target genes of PC9 cell line were silenced by siRNA transinfection and then, MTT and flow cytometry assay were perfomed to verify whether the target genes are related to acquired resistance to gefitinib. The relationship between miRNA and target gene was further investigated by plasmid construction and dual-luciferase reporter assay.Results PC9/GR was induced by gefitinib and final IC50 was up to 26.599?mol/L.The PC9 was 1.187?mol/L, A549 was 16.648?mol/L and H1975 was 9.002?mol/L. 78 miRNAs were overexpressed with >2 fold increase and 129 miRNAs were showed low-expression with >2 fold in PC9/GR cell line than in PC9. Among the increased miRNA group, miR-221-3p showed a 2.45-fold increase and this results from microarray was confirmed by quantitative RT-PCR, p=0.0002. Direct sequence assay showed that PC9 cell was EGFR 19 exon del E746-749, PC9/GR 19 exon del E746-749,A549 19?21 full wild type,H1975 20 exon T790M combined with 21 L858R. With the exception of A549 cell, MTT assay indicated that PC9 cell was dramatic increased drug-resistance by up-regulation of miR-221-3p and IC50 was up to 4 fold increasing,conversely, the resistance of PC9/GR and H1975 cell was dramatic decreased by down-regulation of miR-221-3p. Similar results were observed in flow cytometry assay.The apoptosis rate in miR-221-3p down-regulated PC9/GR and H1975 cells were increased and in miR-221-3p up-regulated PC9 cells were decreased when compared to their parent cells, respectively. CDKN1B / p27, DKK2 and PTEN genes were predicted as the potential target gene of miR-221 by Targetsan and microRNA database. Neither the gene nor the protein level, A549 cells did not show any relationship to the miR-221-3p overexpression. RT-PCR showd significant changes of target gene expression in PC9, PC9/GR and H1975 when regulated miR-221-3p expression. With the exception of CDKN1B/p27 protein in PC9 and PC9/GR, WB confirmed that DKK2 and PTEN protein were corresponding changed by regulating miR-221-3p. The content of 3 proteins in H1975 cells was significantly changed by down-regulating miR-221-3p(p=0.0183 for CDKN1B/p27, p=0.0074 for PTEN, p=0.0165 for DKK2). After silencing PTEN and DKK2 genes by transinfecting siRNAs to PC9 cell, MTT and flow cytometry showed that PC9 cell obtain drug-resistance, whereas after silenc CDKN1B/p27, tests didn't show corresponding changes in MTT and flow cytometry.Dual luciferase reporter gene assay showed that PTEN and DKK2 genes are direct target of miR-221-3p.Conclusion MiR-221-3p was highly expressed in gefitinib-resistance lung cancer cells. PTEN and DKK2 were target genes of miR-221-3p. Acquaired resistance to gefitinib in EGFR exon 19 and 21 mutant cells is induced by miR-221-3p via inhibition of PTEN and DKK2 gene expression. |
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