Effect and mechanism of miR-146a on malignant biological behaviors of lung adenocarcinoma cell line

Abstract

The aim of the present study was to assess the expression of microRNA-146a (miR-146a) in human lung adenocarcinoma cells, its effect on cellular behaviors, and the underlying molecular mechanisms. Reverse transcription-quantitative PCR (RT-qPCR) was used to measure miR-146a expression in the human normal lung epithelial cell line, BEAS-2B, and human lung adenocarcinoma cell lines, A549, PC-9 and H1299, to determine whether miR-146a acts as an oncogene or anti-oncogene. miR-146a mimics were transfected into target cells to observe the proliferation, apoptosis, invasion and migration of human lung adenocarcinoma cells. The target genes of miR-146a were predicted using bioinformatics analysis, and binding sites were validated by dual-luciferase reporter assay. Target gene expression at the mRNA and protein levels was measured by RT-qPCR and western blot analysis, respectively. The expression levels of miR-146a in human lung adenocarcinoma cell lines were lower than its expression in BEAS-2B (P<0.01). A549 cell line is a EGFR wild-type lung adenocarcinoma cell line, which is also the most widely studied in NSCLC, and therefore this was chosen as the target cell line for further investigation. Overexpression of miR-146a in A549 cells can inhibit cell proliferation (P<0.05), promote apoptosis (P<0.05), and reduce the cells' migratory ability (P<0.01). Bioinformatics prediction indicated that interleukin-1 receptor-associated kinase 1 (IRAK1) and TNF receptor associated factor 6 (TRAF6) are the target genes of miR-146a. Dual-luciferase reporter assay showed that miR-146a could specifically bind to 3'-untranslated regions of IRAK1 and TRAF6. The protein and mRNA levels of IRAK1 and TRAF6 were significantly downregulated after miR-146a overexpression in A549 cells (P<0.01). The results of this study demonstrated that the expression of miR-146a in human lung adenocarcinoma cells was significantly lower than in normal lung epithelial cells, indicating that miR-146a acts as an anti-oncogene. miR-146a suppresses the proliferation and migration of human lung adenocarcinoma cells by downregulating the expression of IRAK1 and TRAF6.

Keywords: lung adenocarcinoma cell; malignant biological behavior; mechanistic research; microRNA-146a.

Figure 1.

Relative mRNA expression of miR-146a in the four cell lines. Expression of miR-146a in three lung adenocarcinoma cell lines (A549, H1299, and PC9) and a human normal bronchial epithelial cell line (BEAS-2B). Expression of miR-146a was significantly lower in all three lung adenocarcinoma cell lines than in the normal epithelial cell line. **P<0.01. miR-146a, microRNA-146a.

Figure 2.

Transfection efficiency of miR-146a mimics in A549 cells. (A) A549 cells were photographed under a microscope 6 h after transfection. The overall transfection efficiency was 95.4%. Scale bar, 100 µm (×100 magnification). (B) At 48 h after transfection, the expression of miR-146a in A549 cells was measured and the expression of miR-146a was significantly higher in the mimic group than in the NC and mock groups. **P<0.01 vs. NC and mock group. miR-146a, microRNA-146a; NC, negative control.

Figure 3.

Effects of miR-146a overexpression on proliferation and apoptosis in A549 cells. (A) The OD values of A549 cells were measured at 12, 24, 36, 48, 60 and 72 h after transfection. The values of the mimics group were smaller than the nc group and the mock group at 24, 36, 48, 60 and 72 h. (B and C) At 48 h after transfection, miR-146a overexpression was found to promote apoptosis of A549 cells. *P<0.05 vs. mock and NC group. miR-146a, microRNA-146a; NC, negative control; OD, optical density; PI, propidium iodine.

Figure 4.

miR-146a overexpression inhibits migration of A549 cells. (A) Overexpression of miR-146a was found to inhibit the migratory ability of A549 cells in the Transwell experiment. (C and D) Images of wound-healing were taken at 0 h and 48 h, and the area of healing represented the migration ability. The assay showed that miR-146a inhibited the migration ability of A549 cells Scale bar, 200 µm (×100 magnification) **P<0.01 and *P<0.05 vs. mock and NC group. miR-146a, microRNA-146a; NC, negative control.

Figure 5.

Binding sites of miR-146a with IRAK1 and TRAF6. IRAK1, interleukin-1 receptor-associated kinase 1; TRAF6, TNF receptor associated factor 6; UTR, untranslated region; miR-146a, microRNA-146a.

Figure 6.

Detection of miR-146a targeted genes using a dual-luciferase reporter assay system. There was a profound decrease in the luciferase activity in A549 cells co-transfected with miR-146a mimics and (A) IRAK1-WT or (B) TRAF6-WT, *P<0.05 and **P<0.01 vs. mock and NC group. miR-146a, microRNA-146a; NC, negative control; WT, wild-type; MUT, mutant.

Figure 7.

Overexpression of miR-146a and inhibition of IRAK1 and TRAF6 in A549 cells. (A) mRNA expression levels, and (B) western blot analysis and (C) densitometry analysis of protein expression levels indicated that at 48 h after transfection, overexpression of miR-146a inhibited the mRNA and protein expression levels of IRAK1 and TRAF6. **P<0.01 vs. mock and NC group. miR-146a, microRNA-146a; NC, negative control; IRAK1, interleukin-1 receptor-associated kinase 1; TRAF6, TNF receptor associated factor 6.