MicroRNA-215 suppresses the proliferation, migration and invasion of non-small cell lung carcinoma cells through the downregulation of matrix metalloproteinase-16 expression

Abstract

The present study investigated the expression of microRNA (miR)-215 in non-small cell lung carcinoma (NSCLC) at tissue and cellular levels, as well as its biological functions and mechanism of action. A total of 56 patients with NSCLC were included in the present study. NSCLC tissues and tumor-adjacent normal tissues were resected and collected. Reverse transcription-quantitative polymerase chain reaction was used to measure the expression of miR-215. Following transfection with miR-215 mimics, A549 cell proliferation, migration and invasion were determined using a Cell Counting Kit-8 and Transwell assay. Western blotting was employed to measure the expression of matrix metalloproteinase (MMP)-16 protein. A dual-luciferase reporter assay was conducted to determine the existence of a direct interaction between miR-215 and the MMP-16 gene. Reduced expression of miR-215 in NSCLC was closely associated with lymphatic metastasis and TNM staging. Overexpression of miR-215 inhibited the proliferation of A549 cells in vitro. Upregulated expression of miR-215 inhibited the migration and invasion of A549 cells in vitro. miR-215 exerted its biological functions possibly by regulating the expression of MMP-16. Elevated expression of MMP-16 promoted the proliferation, migration and invasion of A549 cells. miR-215 regulated the proliferation, migration and invasion of A549 cells by binding with the seed 3'-untranslated region of MMP-16 mRNA. The present study demonstrates that reduced expression of miR-215 in NSCLC is negatively associated with lymphatic metastasis and TNM staging. In addition, miR-215 acts as a tumor suppressor gene by inhibiting the proliferation, migration and invasion of NSCLC cells via the downregulation of MMP-16 expression.

Keywords: matrix metalloproteinase-16; microRNA-215; non-small cell lung carcinoma.

Figure 1.

Expression of miR-215 in NSCLC and its clinical significance. (A) Relative expression of miR-215 in NSCLC tissues and tumor-adjacent normal tissues. (B) Relative expression of miR-215 in patients in groups N1 and N0. (C) Relative expression of miR-215 in patients at TNM stages I, II and III/IV. The data in multiple groups were compared using one-way analysis of variance followed by a post hoc test. *P<0.05 vs. normal tissues; ^#P<0.05 vs. N0; ^$P<0.05 as indicated. miR, microRNA; NSCLC, non-small cell lung carcinoma; N1, lymphatic metastasis; N0, without lymphatic metastasis.

Figure 2.

Effect of miR-215 on the proliferation of A549 cells in vitro. (A) Expression level of miR-215 in A549 cells determined by reverse transcription-quantitative polymerase chain reaction. (B) Proliferation of A549 cells. A549 cells were transfected with miR-215 mimics or miR-NC, and a Cell Counting Kit-8 assay was performed to measure the absorbance (490 nm) of the cells at 0, 24 and 48 h. A cell proliferation curve was plotted. *P<0.05 vs. miR-NC group. miR, microRNA; NC, negative control.

Figure 3.

Effect of miR-215 on the migration and invasion of A549 cells in vitro. A549 cells were transfected with miR-215 mimics or miR-NC, and migratory and invasive abilities of the cells were determined using Transwell assays. Magnification, ×100. *P<0.05 vs. with corresponding miR-NC group. miR, microRNA; NC, negative control.

Figure 4.

Effect of miR-215 on the expression of MMP-16 protein in A549 cells. Western blotting was used to determine protein expression. *P<0.05 vs. miR-NC. miR, microRNA; MMP-16, matrix metalloproteinase-16; NC, negative control.

Figure 5.

Effect of MMP-16 on the biological functions of A549 cells. (A) Expression of MMP-16 protein in A549 cells transfected with NC or pcDNA3.1-MMP-16 plasmids. Western blotting was used to determine protein expression. (B) Proliferation of A549 cells transfected with NC or pcDNA3.1-MMP-16 plasmids. A Cell Counting Kit-8 assay was used to measure the absorbance (490 nm) of the cells at 0, 24 and 48 h. A cell proliferation curve was plotted. (C) Migration and invasion of A549 cells transfected with NC or pcDNA3.1-MMP-16 plasmids. Migratory and invasive abilities of the cells were determined using Transwell assays. Magnification, ×100. *P<0.05 vs. the NC group. MMP-16, matrix metalloproteinase-16; NC, negative control.

Figure 6.

Effect of miR-215 and MMP-16 on the biological functions of A549 cells. (A) Expression of MMP-16 protein in A549 cells transfected with NC, miR-215 or miR-215 and pcDNA3.1-MMP-16 plasmids. Western blotting was used to measure protein expression. (B) Proliferation of A549 cells transfected with NC, miR-215 or miR-215 and pcDNA3.1-MMP-16 plasmids. A Cell Counting Kit-8 assay was used to measure the absorbance (490 nm) of the cells at 0, 24 and 48 h. A cell proliferation curve was plotted. (C) Migration and invasion of A549 cells transfected with NC, miR-215 or miR-215 and pcDNA3.1-MMP-16 plasmids. Migratory and invasive abilities of the cells were determined using Transwell assays. Magnification, ×100. The data in multiple groups for migration or invasion assays were compared using one way analysis of variance followed by a post hoc test. *P<0.05 vs. the NC group; ^#P<0.05 vs. the miR-215 mimics group. miR, microRNA; MMP-16, matrix metalloproteinase-16; NC, negative control.

Figure 7.

Identification of interaction between miR-215 and MMP-16. Results of the dual-luciferase reporter assay. Plasmids (0.5 µg) with WT or mutant 3′-untranslated region DNA sequences were co-transfected with miR-215 mimics into A549 cells. Following cultivation for 24 h, the cells were lysed using a dual-luciferase reporter assay kit and fluorescence intensity was measured using GloMax 20/20 luminometer. Using Renilla fluorescence activity as an internal reference, the fluorescence values of each group of cells were measured. *P<0.05 as indicated. miR, microRNA; MMP-16, matrix metalloproteinase-16; WT, wild-type; NC, negative control.