microRNA-664 enhances proliferation, migration and invasion of lung cancer cells

Abstract

Altered microRNA (miR) expression serves an important role in the development and progression of lung cancer. In the present study, the effect of miR-664 on proliferation, migration and invasion of lung cancer cells was assessed. The proliferation of lung cancer cells with an overexpression of miR-664 was examined via MTT assay. The Caspase-Glo3/7 assay was used to examine the effect of miR-664 on cisplatin-induced apoptosis in lung cancer cells. The migration and invasion of lung cancer cells were assessed by Transwell migration and matrigel invasion assays. Western blot analysis was used to examine the protein expression levels. miR-664 improved the proliferation of lung cancer cells and inhibited cisplatin-induced apoptosis of A549 and A427 cells. Furthermore, altered expression of miR-664 affected migration and invasion of lung cancer cells. In addition, a miR-664 mimic decreased E-cadherin expression and increased vementin and Snail expression in lung cancer cells. Notably, the expression level of protein kinase B in A549 cells was changed following altered expression of miR-664. The results of the present study suggest that miR-664 serves an essential role in tumor development and progression in lung cancer.

Keywords: apoptosis; metastasis; microRNA; microRNA-664; protein kinase B.

Figure 1.

miR-664 decreases the chemo-sensitivity of lung cancer cells. (A) miR-664 expression level was examined using reverse transcription-quantitative polymerase chain reaction on lung cancer cells transfected with either miR-664 mimic or miR-664 inhibitor. ***P<0.05 vs. control. (B) Proliferation of A549 cells following transfection with a miR-664 mimic. (C) Proliferation of A427 cells following transfection with miR-664 mimic. miR, micro RNA; ctrl, control; OD, optical density.

Figure 2.

miR-664 decreases apoptosis in lung cancer cells. (A) Caspase 3/7 activity in A549 cells transfected with miR-664 following cisplatin treatment. (B) Caspase 3/7 activity in A427 cells transfected with miR-664 following cisplatin treatment. (C) The level of apoptotic proteins in A549 and A427 cells following cisplatin treatment. Bar graph indicates the relative quantity of proteins on A549 and A427 cells following transfection with miR-664 mimic by densitometric analysis. P<0.05 represents a statistically significant difference vs. control. miR, microRNA; ctrl, control; OD, optical density; RFU, relative fluorescence units; Bcl-2, B-cell lymphoma-2; Bax, Bcl-2 associated X protein.

Figure 3.

miR-664 increases migration and invasion of A549 cells, stained with Diff Quik stain. (A-D) miR-664 significantly increases migration and invasion of A549 cells. (E-H) Inhibitor of miR-664 significantly decreases the migration and invasion of A549 cells. All experiments were performed in triplicate, P<0.05 represents a statistically significant difference. miR, microRNA. Scale bar=100 µm.

Figure 4.

miR-664 increases migration and invasion of A427 cells, stained with Diff Quik stain. (A-D) Significantly increased migration and invasion of A427 cells with miR-664 treatment and (E-H) significantly decreased migration and invasion of A427 cells following treatment with miR-664 inhibitor. All experiments were performed in triplicate, P<0.05 represents a statistically significant difference. miR, microRNA. Scale bar=100 µm.

Figure 5.

miR-664 regulates expression of EMT markers by activating AKT in lung cancer cells. (A) The level of EMT markers in A549 cells following transfection with either miR-664 mimic or anti-miR-664 inhibitor. (B) Bar graph indicates the relative quantity of proteins in A549 cells following transfection with miR-664 mimic by densitometric analysis. (C) Expression of AKT and PTEN in A549 cells following transfection with either miR-664 mimic or anti-miR-664 inhibitor. (D) Bar graph indicates the relative quantity of proteins in A549 cells following transfection with miR-664 mimic by densitometric analysis. P<0.05 represents a statistically significant difference. miR, microRNA; EMT, epithelial-mesenchymal transition; AKT, protein kinase B; PTEN, phosphatase and tensin homolog; Ctrl, control; E-cad, E-cadherin; N-cad, N-cadherin; p-AKT, phosphorylated-AKT.