MicroRNA‑671‑5p inhibits cell proliferation, migration and invasion in non‑small cell lung cancer by targeting MFAP3L

Abstract

MicroRNA (miR)‑671‑5p serves as a tumor suppressor in several types of cancer, including gastric and breast cancer. However, the function of miR‑671‑5p in non‑small cell lung cancer (NSCLC) has not been described in detail. The present study aimed to investigate the role of miR‑671‑5p in NSCLC. The expression levels of miR‑671‑5p were determined in NSCLC tissue samples and cell lines using reverse transcription‑quantitative PCR. Prediction of miR‑671‑5p targets was performed using the TargetScan database and verified by luciferase reporter assay and western blot analysis. Functional experiments, including Cell Counting Kit‑8, wound healing and Transwell assays, were performed in NSCLC cells. The results of the present study demonstrated that lower expression levels of miR‑671‑5p were observed in NSCLC tissues and cell lines compared with those in the corresponding controls. Low miR‑671‑5p levels were significantly associated with an advanced Tumor‑Node‑Metastasis stage and lymph node metastasis in patients with NSCLC. Microfibril‑associated protein 3‑like (MFAP3L) was confirmed to be a direct target of miR‑671‑5p. The proliferative, migratory and invasive abilities of NSCLC cells were suppressed following transfection with miR‑671‑5p mimics and promoted by the miR‑671‑5p inhibitor compared with those in the respective control groups. In addition, the effects of miR‑671‑5p on cell proliferation, migration and invasion, as well as the expression levels of proliferating cell nuclear antigen, E‑cadherin, N‑cadherin and vimentin were reversed by MFAP3L overexpression. In conclusion, targeting the miR‑671‑5p/MFAP3L signaling pathway may be a promising therapeutic strategy for NSCLC treatment.

Keywords: metastasis; microRNA‑671‑5p; microfibril‑associated protein 3‑like; non‑small cell lung cancer; proliferation.

Figure 1.

miR-671-5p expression is downregulated in NSCLC tissues and cell lines compared with normal lung tissues and cells. (A) miR-671-5p levels in 56 NSCLC and paired adjacent tissues were determined by RT-qPCR. (B) miR-671-5p levels in the normal human bronchial epithelial cell line BEAS-2B and three NSCLC cell lines were analyzed by RT-qPCR; U6 was used as an internal control. *P<0.05 and ***P<0.001 vs. adjacent tissues or BEAS-2B. miR, microRNA; NSCLC, non-small cell lung cancer; RT-qPCR, reverse transcription-quantitative PCR.

Figure 2.

miR-671-5p inhibits the proliferation, migration and invasion in non-small cell lung cancer cells. (A) miR-671-5p expression levels in 95D or A549 cells transfected with miR-671-5p mimics or miR-NC were examined by RT-qPCR. (B) Cell proliferative ability was compared between miR-671-5p mimics- and miR-NC-transfected 95D or A549 cells by the CCK-8 assay. (C) Cell migration analysis was performed in 95D or A549 cells transfected with miR-671-5p mimics or miR-NC. (D and E) Transwell invasion assay was performed in 95D and A549 cells transfected with miR-671-5p mimics or miR-NC. (F) miR-671-5p expression levels in H1299 cells transfected with the miR-671-5p inhibitor or inhibitor NC were examined by RT-qPCR. (G) Cell proliferative ability was compared between miR-671-5p inhibitor- and inhibitor NC-transfected H1299 cells by the CCK-8 assay. (H) Cell migration and (I) Transwell invasion assays were performed in H1299 cells transfected with miR-671-5p inhibitor and inhibitor NC. All experiments were performed in triplicate. **P<0.01 and ***P<0.001 vs. miR-NC or inhibitor NC. miR, microRNA; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR; CCK-8, Cell Counting Kit-8; OD, optical density.

Figure 3.

miR-671-5p negatively regulates MFAP3L by directly binding to its 3′-UTR. (A) The sequence alignment of human miR-671-5p and the 3′-UTR of MFAP3L. (B) 95D and (C) A549 cells were co-transfected with the WT or MUT MFAP3L plasmids and miR-671-5p mimics or miR-NC, and the interaction was analyzed by the luciferase reporter assay. (D) 95D and (E) A549 cells were transfected with miR-671-5p mimics or miR-NC. The ATF2 protein levels were determined by western blot analysis. All experiments were performed in triplicate. (F) MFAP3L expression levels in 56 NSCLC and paired adjacent tissues were determined by reverse transcription-quantitative PCR. (G) The correlation between miR-671-5p and MFAP3L expression in NSCLC tissues was determined by Spearman's correlation analysis. **P<0.01 and ***P<0.001 vs. miR-NC. MFAP3L, microfibril-associated protein 3-like; miR, microRNA; 3′-UTR, 3′-untranslated region; WT, wild-type; MUT, mutant; NC, negative control.

Figure 4.

Restoration of MFAP3L expression reverses the miR-671-5p mimic-mediated effects on cancer cell viability, migration and invasion. (A) The protein levels of MFAP3L were determined in 95D and A549 cells following transfection with the MFAP3L overexpression plasmid or empty vector. (B) The protein expression levels of MFAP3L were detected in 95D and A549 cells co-transfected with either miR-NC or miR-671-5p mimics and MFAP3L or empty vector using western blot analysis. (C) The proliferative ability of co-transfected 95D and A549 cells was assessed by CCK-8 assay. (D) Wound healing assay was performed to assess the migratory ability of co-transfected 95D and A549 cells. (E and F) Transwell assay was performed to determine the invasive ability of co-transfected 95D and A549 cells. All experiments were performed in triplicate. **P<0.01 and ***P<0.001 vs. vector or miR-NC + vector; ^###P<0.001 vs. miR-671-5p mimics + vector. MFAP3L, microfibril-associated protein 3-like; miR, microRNA; NC, negative control; CCK-8, Cell Counting Kit-8; OD, optical density.

Figure 5.

Restoration of MFAP3L expression attenuates the effects of miR-671-5p on the epithelial-to-mesenchymal transition-associated protein expression. 95D cells were transfected with miR-NC or miR-671-5p mimics and MFAP3L or empty vector. Western blot analysis was performed to detect the protein expression levels of PCNA, E-cadherin, N-cadherin and vimentin in transfected 95D cells. **P<0.01 and ***P<0.001 vs. miR-NC + vector; ^#P<0.05 and ^###P<0.001 vs. miR-671-5p mimics + vector. MFAP3L, microfibril-associated protein 3-like; miR, microRNA; NC, negative control; PCNA, proliferating cell nuclear antigen.