Up-Regulation of MiRNA-125a-5p Inhibits Cell Proliferation and Increases EGFR-TKI Induced Apoptosis in Lung Cancer Cells

Abstract

Background: Despite the dramatic efficacy of erlotinib, an EGFR tyrosine kinase inhibitor (TKI), most of non-small cell lung cancer (NSCLC) patients ultimately acquire resistance to this agent. Different studies indicated that miRNA-125a-5p is down-regulated in human lung cancer cells and may function as a tumor suppressor by targeting EGFR. However, the biological function of miRNA-125a-5p in NSCLC resistance to EGFR-TKIs is not fully understood. In this study the effect of miRNA-125a-5p on cell proliferation, apoptosis and sensitivity of the A549 lung cancer cells to erlotinib was investigated.

Methods: After miRNA-125a-5p transfection, the expression levels of EGFR mRNA were measured by QRT-PCR. Trypan blue assays were performed to evaluate the proliferation of the A549 lung cancer cells. The cytotoxic effects of miRNA-125a-5p and erlotinib, alone and in combination, were determined using MTT assay. Combination index study was performed using the method of Chou-Talalay. Apoptosis was assessed using an ELISA cell death assay kit.

Results: MiRNA-125a-5p clearly reduced the expression of EGFR mRNA in a time dependent manner, causing marked cell proliferation inhibition and spontaneous apoptosis (p<0.05, relative to control). Pretreatment with miRNA-125a-5p synergistically increased the cytotoxic effect of erlotinib and decreased its IC50. Furthermore, miRNA-125a-5p significantly enhanced the apoptotic effect of erlotinib. Negative control miRNA had no significant effect on biological parameter of the tumor cells.

Conclusions: Our data suggest that suppression of EGFR by miRNA-125a-5p can effectively trigger apoptosis and overcome EGFR-TKs resistance of lung cancer cells. Therefore, miRNA-125a-5p may be a potential therapeutic adjuvant in patients with lung cancer.<br />.

Keywords: Apoptosis; EGFR; Lung cancer; MiRNA-125a-5p; erlotinib.

Figure 1

Effect of miRNA-125a-5p on the Expression of EGFR in A549 Cells. The EGFR expression determined by RT-qPCR at 24, 48 and 72 h after transfection of the cells with miRNA-125a-5p and negative control (NC) miRNA. Relative EGFR mRNA expression was measured using the 2^{- (∆∆Ct)} method. The EGFR mRNA decreased clearly at the three time points compared with corresponding blank control and NC miRNA groups (*p<0.05). The results presented are mean±SD of three independent experiments. #p <0.05

Figure 2

Effect of miRNA-125a-5p in Combination with Erlotinib on Cell Survival. Human A549 cells were treated with miRNA-125a-5p (50 nM) and different concentrations of erlotinib for 24 h (A and B) and 48 h (C and D). Cell survival was determined by the MTT assay as described in the method section. Dose-response curves were plotted using GraphPad Prism 6.01 software. Bars represent mean±SD (n=3). Data from three independent experiments were used to calculate the combination index (CI) according the method of Chou-Talalay. A horizontal dashed marks CI=1

Figure 3

Effect of Down-Regulation of EGFR by miRNA-125a-5p on Lung Cancer Cell Proliferation. The A549 cells were transfected with miRNA-125a-5p and negative control (NC) miRNA and then cell viability was tested by trypan blue assay over a period of 5 days. The results represent mean±SD of three independent experiments. *p<0.05 versus blank control or NC miRNA

Figure 4

Cell Apoptosis of A549 Cells Treated with miRNA-125a-5p and Erlotinib. The cells were treated with miRNA-125a-5p (50 nM), negative control (NC) miRNA (50 nM) and erlotinib (IC₅₀ doses of 24 and 48 h), alone and in combination, and then apoptosis was measured by cell death ELISA. The data are expressed as mean±SD (n=3); *p<0.05 versus blank control or NC miRNA; #p<0.05 versus miRNA-125a-5p or erlotinib