Abstract
BackgroundTo investigate the regulatory mechanism behind miR‐34a‐altered Axl levels in non‐small‐cell lung cancer (NSCLC) with gefitinib‐acquired resistance.MethodsThe expression of miR‐34a, Axl, Gas6 and related downstream signaling proteins in the EGFR mutant NSCLC cell lines were determined by qRT‐PCR and Western blot; PC9‐Gef‐miR‐34a and HCC827‐Gef‐miR‐34a cells were established by transfecting the parent cells with a miR‐34a overexpressing virus, then the expression of Axl, Gas6 and the downstream channel‐related proteins were also compared in PC9‐Gef‐miR‐34a and HCC827‐Gef‐miR‐34a and drug‐resistant strains. The survival rate of the cells were measured by CCK8 assay. A luciferase reporter detected whether Axl was the target of miR‐34a. Finally, a tumor‐bearing nude mouse model was established to verify the relationship between the expression of miR‐34a, Axl and Gas6 mRNA in vivo.ResultsThe expression levels of Axl mRNA and protein, Gas6 mRNA and protein, and related downstream proteins in PC9‐Gef and HCC827‐Gef cell lines were higher than those in PC9 and HCC827 parental cell lines, while the expression of miR‐34a was lower than it was in the parental cell lines (P < 0.05). The expression of Axl mRNA and protein, Gas6 mRNA and protein, and related downstream signaling proteins in PC9‐Gef and HCC827‐Gef cell lines was higher than the expression in PC9‐Gef‐miR‐34a and HCC827‐Gef‐miR‐34a cells, which overexpressed miR‐34a (P < 0.05).ConclusionThe miR‐34a regulation of Axl plays an important role in NSCLC‐acquired gefitinib resistance, and their expression is inversely correlated, which suggests that they can be used as prognostic markers or potential therapeutic targets for NSCLC.
Highlights
The discovery of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib, significantly improve the clinical efficacy of treatments for patients with EGFR-mutated advanced non-small-cell lung cancer (NSCLC), improving the patient quality of life and the prognosis.[1,2] acquired drug resistance will occur in most patients after a median of 9 to 13 months of treatment.[3,4,5] The acquired resistance of EGFR-TKI allows the disease to progress in patients and becomes the bottleneck restricting the continued use of EGFR-TKI
NSCLC can acquire drug resistance through a secondary mutation of exon 20 of EGFR gene (T790M) and the amplification of c-MET gene; Axl has been recently found to correlate with the acquired drug resistance of EGFR-TKI,[5,6] but the molecular mechanism of Axl leading to EGFR-TKI resistance in NSCLC lung cancer cells is not fully understood
The results showed that the expression levels of AKT, p-AKT, ERK, p-ERK, STAT3 and p-STAT3 in HCC827-Axl and PC9-Axl cells were significantly higher than those in HCC827-control and PC9-control cells; further, the sensitivity of HCC827-Axl and PC9-Axl cells to gefitinib was decreased after overexpressing Axl, and the differences were significantly different (P < 0.05)
Summary
The discovery of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib, significantly improve the clinical efficacy of treatments for patients with EGFR-mutated advanced NSCLC, improving the patient quality of life and the prognosis.[1,2] acquired drug resistance will occur in most patients after a median of 9 to 13 months of treatment.[3,4,5] The acquired resistance of EGFR-TKI allows the disease to progress in patients and becomes the bottleneck restricting the continued use of EGFR-TKI. Our previous studies found that miR-34a expression was significantly lower and Axl was more highly expressed in gefitinib-resistant cell lines than in controls.
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