MiroRNA-31-3p Promotes the Invasion and Metastasis of Non-Small-Cell Lung Cancer Cells by Targeting Forkhead Box 1 (FOXO1)

Abstract

Objective: To explore the possibility of microRNA miR-31-3p as a biomarker for bone metastasis of non-small-cell lung cancer (NSCLC) and its molecular mechanism to the invasion and metastasis of NSCLC cells.

Methods: Real-time quantitative PCR (RT-qPCR) was used to detect the expression levels of miR-31-3p and forkhead box 1 (FOXO1) in NSCLC tissues, serum, and cells to analyze the correlation between the expression levels of miR-31-3p and the clinicopathology of NSCLC. After interference with or overexpressing miR-31-3p, NSCLC cell proliferation, apoptosis, invasion ability, and migration ability were detected by MTT, flow cytometry, Transwell, and scratch experiment, respectively. The interaction between miR-31-3p and FOXO1 was further verified by the dual-luciferase reporter experiment. Western blot was performed to detect the protein expression of FOXO1 in tissues and FOXO1, RhoA, p-RhoA, ROCK-2, and p-ROCK-2 in cells.

Results: In tissues, serum, and NSCLC cell line A549 of the NSCLC patients, the expression of FOXO1 was notably lower, and the miR-31-3p expression was significantly higher. Overexpression of miR-31-3p could distinctly improve the proliferation, invasion, and migration of A549 cells, meanwhile inhibit cell apoptosis, and activate the RhoA/ROCK-2 signaling pathway, while interfering with the expression of miR-31-3p has the opposite function. Besides, bioinformatics analysis and luciferase reporter assay confirmed that FOXO1 was a target gene of miR-31-3p. Overexpressing FOXO1 could inhibit the proliferation and metastasis of A549 cells, but overexpressing miR-31-3p reverses the results.

Conclusion: This study confirmed that miR-31-3p promotes the proliferation, invasion, and migration of NSCLC cells and inhibits apoptosis through targeted regulating FOXO1 and be a potential therapeutic targets for the treatment of NSCLC.

Figure 1

High expression of miR-31-3p in NSCLC patients. (a) Observation of the expression of miR-31-3p in the tissues of NSCLC patients and the normal control group using RT-qPCR. (b) Evaluation of the expression of miR-31-3p in the serum of normal people and NSCLC patients by RT-qPCR. (c) Determination of the expression of miR-31-3p in each group cells using RT-qPCR. ^∗P < 0.05, ^∗∗P < 0.01.

Figure 2

The effect of abnormal expression of miR-31-3p on the proliferation, apoptosis, and bone metastasis of NSCLC cells. (a) Determination of the expression level of miR-31-3p in each group of cells using RT-qPCR. (b) Evaluation of the proliferation of each group of cells by MTT; ^∗P < 0.05, ^∗∗P < 0.01 vs. NC group (48 h). (c) Assessment of the apoptosis rate of each group of cells by flow cytometry. (d) Measurement of the invasion ability of each group of cells using Transwell test. (e) Observation of the migration ability of each group of cells by scratch test. (f) Evaluation of the protein expression level and gray-scale statistics of RhoA, p-RhoA, ROCK-2, and p-ROCK-2 in each group of cells using Western blot. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001.

Figure 3

Targeted binding of miR-31-3p with FOXO1. (a) Verification of the targeting relationship between miR-31-3p and FOXO1 by dual-luciferase reporter gene experiment. (b) Determination of the expression level of FOXO1 in the tissues of NSCLC patients in each group using RT-qPCR. (c) Detection of 16HBE-T and FOXO1 expression in A549 cells using RT-qPCR. (d) RT-qPCR evaluation of FOXO1 expression in A549 cells after overexpression or interference with miR-31-3p. (e) Assessment of FOXO1 protein expression in each group of cells by Western blot. ^∗P < 0.05 and ^∗∗P < 0.01 vs. NC group.

Figure 4

MiR-31-3p regulates the proliferation, apoptosis, invasion, and migration of NSCLC cells through FOXO1. (a) Measurement of the proliferation rate of each group of cells using MTT; ^∗P < 0.05, ^∗∗P < 0.01 vs. NC group (48 h). (b) Verification of the apoptosis rate of each group of cells by flow cytometry. (c) Evaluation of the migration ability of each group of cells by scratch test. (d) Assessment of the cell invasion ability by Transwell assay. ^∗∗P < 0.01 and ^∗∗∗P < 0.001.