miR-202-3p negatively regulates MMP-1 to inhibit the proliferation, migration and invasion of lung adenocarcinoma cells

Abstract

Lung adenocarcinoma (LUAD) is one of the common cancers. Studies show that MMP-1 is involved in tumor progression, yet relevant regulatory mechanism in LUAD remains to be further elucidated. Here, we demonstrated from bioinformatics analysis for GEO data that MMP-1 was differentially up-regulated in LUAD. miR-202-3p, identified as the upstream regulator of MMP-1 by both bioinformatics and dual-luciferase assays, was differentially down-regulated in LUAD and presented a negative correlation with MMP-1. Following cell biological experiments proved that knocking down the expression of MMP-1 inhibited the proliferation, migration and invasion of LUAD cells, while overexpressed miR-202-3p posed a similar suppressive effect on cancer progression. Additionally, rescue assay further identified that overexpression of MMP-1 attenuated the suppressive effect of up-regulated miR-202-3p on malignant progression of LUAD cells. In all, this research suggests a mechanism by which MMP-1 under the regulation of miR-202-3p modulates the proliferation, migration and invasion of LUAD cells, which may contribute to the development of new therapeutic strategies.

Keywords: Mir-202-3p; lung adenocarcinoma; mmp-1.

Figure 1.

Screening of LUAD-related genes and prediction of regulatory miRNA. A: Heat map of DEGs in LUAD dataset GSE19804; B: KEGG enrichment analysis of the DEGs in GSE19804; C: MMP-1 gene expression level in LUAD (red) and normal (gray) samples in TCGA and GTEx databases; D: Prediction of upstream regulatory miRNAs of MMP-1; E: Correlation analysis of MMP-1 and miR-202-3p; * indicates p< 0.05

Figure 2.

MMP-1 and miR-202-3p expression levels in LUAD cells. A, B: The relative mRNA and protein expression levels of MMP-1 were detected by qRT-PCR and western blot in LUAD cells A549, PC9 and normal cells BEAS-2B; C: The relative expression of miR-202-3p in BEAS-2B, A549 and PC9 cells was measured by qRT-PCR; * indicates p< 0.05

Figure 3.

MMP-1 regulates the proliferation, migration and invasion of LUAD cells. A: qRT-PCR was used to detect the mRNA expression of MMP-1 in si-MMP-1, si-NC and control groups; B, C: Western blot was used to detect the protein expression of MMP-1 in si-MMP-1, si-NC and control groups; D, E: Transwell assay was used to detect the invasion and migration of LUAD cells A549 and PC9 after knocking down MMP-1 (100×); F, G: MTT assay was used to detect LUAD A549 and PC9 cell viability after knockdown of MMP-1; H, I: Colony formation assay was used to detect cell colony formation ability after knockdown of MMP-1; * indicates p< 0.05

Figure 4.

miR-202-3p targets MMP-1 expression and inhibits LUAD cell proliferation, migration and invasion. A549 and PC9 cells were transfected with control, miR-202-3p mimic and mimic NC. A: Bioinformatics analysis (TargetScan) predicted the binding sites of miR-202-3p on MMP-1 3ʹUTR; B: qRT-PCR was used to detect the relative mRNA expression of MMP-1 after miR-202-3p was overexpressed in different LUAD cells; C: Western blot was used to detect the protein expression level of MMP-1; D: Dual luciferase assay was used to detect the luciferase activity in different treatment groups after miR-202-3p was overexpressed; E, F: Transwell experiment was used to detect cell invasion and migration of A549 and PC9 cells (100×); G, H: MTT assay was used to detect LUAD A549 and PC9 cell proliferation; I, J: Colony formation assay was used to detect cell colony formation ability of A549 and PC9 cells; * means px< 0.05

Figure 5.

miR-202-3p exerts its function in LUAD by targeting MMP-1. The A549 cells were transfected with mimic-NC+oe-NC, miR-mimic+oe-NC, and miR-mimic+oe-MMP-1, and then the cells were collected. A: Cell transfection efficiency was detected; B: MTT assay was used to measure cell proliferation; C, D: Cell apoptosis and cell cycle were detected by flow cytometry; E: Transwell was used to detect cell migration and invasion; * means p< 0.05