Upregulated microRNA‑671‑3p promotes tumor progression by suppressing forkhead box P2 expression in non‑small‑cell lung cancer

Abstract

In the present study, the expression of microRNA (miR)‑671‑3p in non‑small‑cell lung cancer (NSCLC) was detected via reverse transcription‑quantitative polymerase chain reaction analysis, and its role in cell proliferation, apoptosis, migration and invasion was investigated via Cell Counting Kit‑8, colony formation, flow cytometry, Transwell and scratch assays, respectively. It was observed that the expression of miR‑671‑3p was upregulated in NSCLC tissues and cell lines (A549 and H1975). Treatment with miR‑671‑3p inhibitors suppressed cell proliferation, migration and invasion, and increased apoptosis in vitro, suggesting that miR‑671‑3p functions as an oncogene in NSCLC. In addition, forkhead box P2 (FOXP2) has been reported to be a tumor suppressor that is downregulated in several types of cancer, and its low expression was confirmed in NSCLC tissues and cell lines in the current study via western blotting. The results of the luciferase reporter assay also demonstrated that miR‑671‑3p targeted directly the 3'‑untranslated region of FOXP2. Furthermore, overexpression of FOXP2 in A549 and H1975 cell lines suppressed the growth, migration and invasion, and promoted apoptosis, whereas these effects were reversed by transfection with miR‑671‑3p mimics, suggesting that miR‑671‑3p promoted tumor progression via regulating FOXP2. Taken together, the results reported in the present study implied that miR‑671‑3p may be a potential therapeutic target in NSCLC.

Figure 1.

High expression of miR-671-3p in NSCLC tissues and cell lines. (A) Expression levels of miR-671-3p in NSCLC tissues and adjacent normal tissues (**P<0.01). (B) Expression levels of miR-671-3p in NSCLC and normal cell lines (*P<0.05 vs. normal 16HBE cells). (C) Expression levels of miR-671-3p in NSCLC cell lines transfected with miR-671-3p inhibitors, miR-671-3p mimics or the corresponding NC (*P<0.05 vs. inhibitors NC; ^#P<0.05 vs. mimics NC). Data are presented as the mean ± standard deviation of three independent experiments. miR, microRNA; NSCLC, non-small-cell lung cancer; NC, negative control.

Figure 2.

Downregulation of miR-671-3p inhibits the proliferation and induces the apoptosis of non-small-cell lung cancer cells in vitro. A549 and H1975 cell lines were transfected with miR-671-3p inhibitors or NC, and then subjected to various assays. (A) Cell Counting Kit-8 and (B) EdU assays were performed to examine the cell proliferation (×100 magnification). (C) Colony formation assay. (D) Flow cytometry assay and (E) apoptosis rate. (F) Western blotting was used to determine the Bcl-2, Bax, caspase-3 and caspase-9 protein levels. All data are presented as the mean ± standard deviation, and each experiment was performed in triplicate. *P<0.05 vs. NC group. miR, microRNA; NC, negative control; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein.

Figure 3.

Downregulation of miR-671-3p inhibits non-small-cell lung cancer cell migration and invasion in vitro. A549 and H1975 cells were transfected with miR-671-3p inhibitors or NC. (A) Cell migration was examined by scratch assay, scale bar 100 μm. (B) Cell invasion was examined by transwell assay (×100 magnification). (C) Western blotting for MMP-2 and MMP-9 protein level detection. All data are presented as the mean ± standard deviation, and each experiment was performed in triplicate. *P<0.05 vs. NC group. miR, microRNA; NC, negative control; MMP, matrix metalloproteinase.

Figure 4.

miR-671-3p directly targets FOXP2 in non-small-cell lung cancer. (A) Predicted binding sites in the 3′UTR of FOXP2 mRNA and seed sequence of miR-671-3p. (B) Dual luciferase report assay, conducted to confirm whether miR-671-3p targets FOXP2. All data were presented as the mean ± standard deviation and each was performed in triplicate. **P<0.01 vs. pGL3-FOXP2-3′UTR. miR, microRNA; FOXP2, forkhead box P2; 3′UTR, 3′-untranslated region; mut, mutant; NC, negative control.

Figure 5.

miR-671-3p expression is negatively correlated with FOXP2 expression in NSCLC tissues and cell lines. (A) FOXP2 mRNA expression level in NSCLC tissues. (B) Western blotting was used to determine FOXP2 protein expression in NSCLC tissues. (C) Pearson correlation analysis between miR-671-3p level and FOXP2 mRNA expression. (D) FOXP2 mRNA expression in A549 and H1975 cell lines. (E) Western blotting was applied to detect FOXP2 protein levels in A549 and H1975 cell lines transfected with pcDNA3.1-FOXP2 or NC. All data are presented as the mean ± standard deviation, and each experiment was performed in triplicate. *P<0.05 vs. corresponding control group; ^#P<0.05 vs. pcDNA3.1-FOXP2 group. miR, microRNA; FOXP2, forkhead box P2; NSCLC, non-small-cell lung cancer; T, tumor tissue; N, normal tissue; NC, negative control.

Figure 6.

miR-671-3p promotes NSCLC cell proliferation and suppresses apoptosis by downregulating FOXP2 in vitro. A549 and H1975 cell lines transfected with NC or pcDNA3.1-FOXP2 or pcDNA3.1-FOXP2 and miR-671-3p mimics. (A) Cell Counting Kit-8 assay. (B) Flow cytometry apoptosis assay. (C) Western blotting for detection of Bcl-2, Bax, caspase-3 and caspase-9 protein levels. All data are presented as the mean ± standard deviation, and each experiment was performed in triplicate. *P<0.05 vs. NC group; ^#P<0.05 vs. pcDNA3.1-FOXP2 group. miR, microRNA; FOXP2, forkhead box P2; NSCLC, non-small-cell lung cancer; NC, negative control; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein.

Figure 7.

miR-671-3p promotes NSCLC cell proliferation, migration and invasion, and suppresses apoptosis by downregulating FOXP2 in vitro. A549 and H1975 cell lines transfected with NC or pcDNA3.1-FOXP2 or pcDNA3.1-FOXP2 and miR-671-3p mimics. (A) Scratch migration assay, scale bar 100 μm. (B) Transwell invasion assay (×100 magnification). (C) Western blotting for detection of MMP-2 and MMP-9 protein levels. All data are presented as the mean ± standard deviation, and each experiment was performed in triplicate. *P<0.05 vs. NC group; ^#P<0.05 vs. pcDNA3.1-FOXP2 group. miR, microRNA; FOXP2, forkhead box P2; NSCLC, non-small-cell lung cancer; NC, negative control; MMP, matrix metalloproteinase.