Targeting Epidermal Growth Factor Receptor by MiRNA-145 Inhibits Cell Growth and Sensitizes NSCLC Cells to Erlotinib

Abstract

Background: Despite effective activity of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), such as erlotinib, all non-small cell lung cancer (NSCLC) patients eventually acquire resistance to these agents. Studies have demonstrated that down-regulation of miRNA-145 leads to enhancement of EGFR expression, cell proliferation and metastasis. The aim of this study was to investigate the effect of miRNA-145 on sensitivity of the A549 NSCLC cells to erlotinib. Methods: Quantitative real-time PCR was used to examine the effect of miRNA-145 on EGFR expression. The effect of miRNA-145 on cell growth and sensitivity the lung cancer cells to erlotinib was examined by trypan blue and MTT assays, respectively. The combination index was calculated using the non-constant method of Chou-Talalay. Apoptosis was determined by ELISA cell death assay. Results: We found that miRNA-145 was markedly suppressed the expression of EGFR and inhibited the cancer cell growth, relative to blank control and negative control miRNA (p<0.05). Pretreatment with miRNA-145 synergistically enhanced the sensitivity of the lung cancer cells to erlotinib. Results of apoptosis assay revealed that miRNA-145 can induce apoptosis and increase the erlotinib-mediated apoptosis. Conclusions: Our data demonstrate that miRNA-145 play a critical role in the lung cancer cell growth, survival and EGFR-TKIs resistance possibly by regulation of EGFR. Therefore, miRNA-145 replacement therapy can become a new therapeutic strategy in lung cancer.

Keywords: Apoptosis; Lung cancer; MiRNA-145; growth; tyrosine kinase inhibitor.

Figure 1

Expression of EGFR in A549 Lung Cancer Cells Transfected with miRNA-145. To measure the expression of EGFR in lung cancer cells, the cells were transfected with miRNA-145 and negative control (NC) miRNA for 24, 48 and 72 h. Then, the EGFR gene expression was measured by quantitative real-time PCR and 2 ^{- (∆∆Ct)} method. Data are expressed as mean±SD of three independent experiments; *p<0.05 significantly different from corresponding blank control and NC miRNA. #p<0.05

Figure 2

Effect of miRNA-145 on Sensitivity of the Lung Cancer Cells to Erlotinib. Cells were treated with miRNA-145 (50 nM) and various concentrations of erlotinib for 24 h (A and B) and 48 h (C and D). After treatment, cell survival was determined by the MTT assay as described in the method section. Cell survival curves were plotted using Prism 6.01 software. The data represent mean±SD (n=3). Data from three independent experiments were used to plot the combination index (CI) versus fractional effect (Fa) according the method of Chou and Talalay. Dashed lines indicate CI=1.

Figure 3

Growth Curve of A549 Cells Transfected with miRNA-145 and Negative Control (NC) miRNA. Cell growth was measured using trypan blue exclusion assay over a period of 5 days. The results are expressed as mean±SD (n=3). *p<0.05 versus blank control or NC miRNA

Figure 4

Combination Effects of miRNA-145 and Erlotinib on Lung Cancer Cell Apoptosis. Cells were transfected with miRNA-145 (50 nM) and negative control (NC) miRNA (50 nM) for 6 h. Then, erlotinib (IC₅₀ doses of 24 and 48 h) was added to the cells. After 24 and 48 h of transfection, the apoptosis was determined using ELISA cell death assay. The data presented are mean±SD (n=3) of independent experiments; *p<0.05 compared with blank control or NC miRNA; #p<0.05 versus miRNA-145 or erlotinib