miR‑491‑3p functions as a tumor suppressor in non‑small cell lung cancer by targeting fibroblast growth factor 5

Abstract

The present study aimed to identify the function of miR‑491‑3p in regulating non‑small cell lung cancer (NSCLC). Tumor tissues and adjacent normal tissues were collected from 43 patients with NSCLC. A549 and H1299 cells were transfected with microRNA (miR)‑491‑3p mimic, mimic negative control (NC), miR‑491‑3p inhibitor, inhibitor NC, pcDNA3.1‑FGF5 vector and control vector. Cell counting kit‑8 assay and Edu experiments were performed to assess cell viability and proliferation. Matrigel experiment, wound healing assay and flow cytometric analysis were performed to explore cell invasion, migration and apoptosis, respectively. A dual‑luciferase reporter experiment was performed to identify the relationship between miR‑491‑3p and fibroblast growth factor 5 (FGF5). In vivo study was conducted by using nude mice. The miR‑491‑3p and FGF5 protein expression levels were investigated using reverse transcription‑quantitative polymerase chain reaction and western blot analysis. In NSCLC tumor tissues, miR‑491‑3p was downregulated and FGF5 was upregulated (P<0.01). Low miR‑491‑3p expression and high FGF5 mRNA expression was associated with poor outcomes in patients, including advanced TNM stage and lymph node metastasis (P<0.05). upregulation of miR‑491‑3p suppressed viability, proliferation, invasion and migration of NSCLC cells; however, it promoted apoptosis (P<0.01). FGF5 was a target gene for miR‑491‑3p. miR‑491‑3p directly inhibited FGF5 expression. upregulation of FGF5 significantly reversed the inhibitory effects of miR‑491‑3p on malignant phenotypes of NSCLC cells (P<0.01). miR‑491‑3p overexpression suppressed the in vivo growth of NSCLC. Thus, it was identified that miR‑491‑3p functions as a tumor suppressor in NSCLC by directly targeting FGF5.

Keywords: apoptosis; invasion; microRNA‑491‑3p/fibroblast growth factor 5; non‑small cell lung cancer; proliferation.

Figure 1.

miR-491-3p is upregulated and FGF5 is downregulated in NSCLC tissues. (A) RT-qPCR revealed that miR-491-3p was upregulated in the tumor tissues of patients with NSCLC. (B) Western blot analysis indicated that FGF5 protein was downregulated in tumor tissues of patients with NSCLC. (C) miR-491-3p and FGF5 mRNA expression in NSCLC cells was monitored by RT-qPCR. (D) FGF5 protein expression detection in NSCLC cells was implemented by western blot analysis. **P<0.01. miR, microRNA; FGF5, fibroblast growth factor 5; NSCLC, non-small cell lung cancer; RT-qPCR, reverse transcription-quantitative PCR.

Figure 2.

miR-491-3p overexpression inhibits viability, proliferation, migration and invasion, and promotes apoptosis in NSCLC cells. (A) Cell Counting Kit-8 assay illustrated that miR-491-3p overexpression inhibited NSCLC cell viability. (B) Edu experiment indicated that miR-491-3p overexpression inhibited NSCLC cell proliferation. 200× magnification. Scale bar: 50 µm. (C) Matrigel experiment indicated that miR-491-3p overexpression suppressed NSCLC cell invasion. 200× magnification. Scale bar: 50 µm. (D) Wound healing assay revealed that miR-491-3p overexpression suppressed NSCLC cell migration. (E) Flow cytometry was used to measure cell apoptosis. miR-491-3p overexpression promoted NSCLC cell apoptosis. *P<0.05 and **P<0.01. miR, microRNA; NSCLC, non-small cell lung cancer; NC, negative control.

Figure 3.

miR-491-3p directly inhibits the expression of FGF5. (A) TargetScan 7.1 online prediction software revealed that FGF5 possessed a binding site for miR-491-3p in the 3′-UTR region. (B) Dual luciferase reporter gene assay verified that FGF5 is a target gene of miR-491-3p. (C) FGF5 mRNA expression in clinical samples of patients with NSCLC was detected by RT-qPCR. Pearson's correlation coefficient analysis was employed to identify the correlation between miR-491-3p and FGF5 mRNA in tumor tissues of patients NSCLC. (D) RT-qPCR and western blotting indicated that miR-491-3p suppressed the expression of FGF5 mRNA and protein. **P<0.01. miR, microRNA; FGF5, fibroblast growth factor 5; UTR, untranslated; RT-qPCR, reverse transcription-quantitative PCR; WT, wild-type; MUT, mutant; NC, negative control.

Figure 4.

FGF5 upregulation reverses the inhibitory effects of miR-491-3p on NSCLC cell malignant phenotype. (A) A549 cells were successfully transfected using pcDNA3.1-FGF5 plasmid and control vector. (B) FGF5 upregulation reversed the inhibitory effects of miR-491-3p on NSCLC cell viability. (C) FGF5 upregulation reversed the inhibitory effects of miR-491-3p on NSCLC cell proliferation. 200× magnification. Scale bar: 50 µm. (D) FGF5 upregulation reversed the inhibitory effects of miR-491-3p on NSCLC cell invasion. 200× magnification. Scale bar: 50 µm. (E) FGF5 upregulation reversed the inhibitory effects of miR-491-3p on NSCLC cell migration. (F) FGF5 upregulation reversed the effects of miR-491-3p on NSCLC cell apoptosis. **P<0.01. FGF5, fibroblast growth factor 5; miR, microRNA; NSCLC, non-small cell lung cancer; NC, negative control.

Figure 5.

miR-491-3p overexpression suppresses the in vivo growth of NSCLC. (A and B) The volume and weight of xenograft tumor tissues was measured. (C) Reverse transcription-quantitative PCR was implemented to detect the expression of miR-491-3p and FGF5 mRNA in xenograft tumor tissues. (D) Immunohistochemical analysis was carried out to detect the expression of Ki67 and FGF5 protein in xenograft tumor tissues. 200× magnification. Scale bar: 100 µm. **P<0.01. miR, microRNA; NSCLC, non-small cell lung cancer; FGF5, fibroblast growth factor 5; NC, negative control.

Figure 6.

Mechanism diagram of the present study.