A total of 43 NSCLC cancer and corresponding adjacent tissue specimens were obtained from patients with NSCLC who underwent curative resection at Ningbo No. 2 Hospital (Ningbo, China). No patients had undergone chemotherapy or radiotherapy prior to surgery. Fresh samples were collected at the time of surgery, and were rapidly frozen in liquid nitrogen and stored at −80°C until use. The clinicopathological characteristics of the patients with NSCLC are summarized in Table I. Informed consent was signed by each patient. The present study was approved by the Ethics Committee of Ningbo No. 2 Hospital.

Human NSCLC A549, H1299, H1650 and H1975 cell lines and the non-tumorigenic human bronchial epithelial NL20 cell line were purchased from the American Type Culture Collection (Manassas, VA, USA). All the cells were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with penicillin (100 U/ml), streptomycin (100 U/ml) (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), and 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in 5% CO₂.

miR-671-3p mimics, miR-671-3p negative control (miR-NC), miR-671-3p inhibitors and miR-671-3p inhibitor negative control (anti-miR-NC) were purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). These were individually and transiently transfected with A549 cells at a final concentration of 100 nM using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The miR-671-3p inhibitors were modified antisense oligonucleotides designed specifically to bind to and inhibit endogenous miR-671-3p with a rare off-target effect: miR-NC: 5′-ACAUCUGCGUAAGAUUCGAGUCUA-3′; miR-671-3p mimics: 5′-UCCGGUUCUCAGGGCUCCACC-3′; anti-miR-NC: 5′-GCGTAACTAATACATCGGATTCGT-3′; miR-671-3p inhibitors: 5′-GGUGGAGCCCUGAGAACCGGA-3′. The efficiency was measured 48 h after transfection, by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) as described below.

Total RNA was isolated from tissues or cultured cells using TRIzol^® reagent (Thermo Fisher Scientific, Inc.). RNA quality and concentration was measured using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc., Wilmington, DE, USA). A total of 1 µg RNA was used for cDNA synthesis using a PrimeScript 1st Strand cDNA Synthesis kit (Takara Bio, Inc., Otsu, Japan). qPCR was performed using the miRNA Q-PCR Detection kit (GeneCopoeia, Inc., Rockville, MD, USA). The PCR thermocycling conditions were: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 34 sec. The RT-qPCR data were analyzed using the 2^−∆∆Cq method (12) and relative to the small nuclear RNA U6 or GAPDH levels. RT primers were as follows: GAPDH forward (F), 5′-ACAACTTTGGTATCGTGGAAGG-3′; GAPDH reverse (R), 5′-GCCATCACGCCACAGTTTC-3′; U6 F, 5′-CTCGCTTCGGCAGCACA-3′; U6 R, 5′-AACGCTTCACGAATTTGCGT-3′; miR-671-3p F, 5′-CTGGCTGGACAGAGTTGTCAT-3′; miR-671-3p R, 5′-TCCGGTTCTCAGGGCTCCACC-3′; CCND2 F, 5′-TACCTGGACCGTTTCTTGGC-3′; CCND2 R, 5′-AGGCTTGATGGAGTTGTCGG-3′.

Western blot was performed as previously described (13). In brief, tumor cells were lysed with lysis buffer (0.5 mol/l Tris-HCl, pH 7.4, 1.5 mol/l NaCl, 2.5% deoxycholic acid, 10% NP-40 and 10 mmol/l EDTA) in the presence of cocktail protease inhibitors (Thermo Fisher Scientific, Inc.). The lysates were collected by centrifugation at 16,000 × g for 20 min at 4°C. Protein concentration was determined by Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). A total of 50 mg protein/lane was separated by 12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Thermo Fisher Scientific, Inc.) The membrane was blocked with 5% non-fat dry milk (Yili Group, Beijing, China) for 1 h at room temperature, followed by incubation with primary antibodies for 2 h at room temperature: Anti-Cyclin D2 (1:1,000; cat. no. ab207604; Abcam, Cambridge, MA, USA) and anti-GAPDH (1:1,000; cat. no. ab9485; Abcam). Following washing, the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5,000; cat. no. ab7090; Abcam) at room temperature for 1 h. An enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc.) was used to perform chemiluminescence detection.

Each group of A549 cells was collected at 24, 48, 72 and 96 h following transfection. Then, cells were incubated with 10 µl CCK-8 reagent (Beyotime Institute of Biotechnology, Haimen, China) for 2 h at 37°C. Next, absorbance at 450 nm was measured at each time point using an enzyme immunoassay analyzer. The experiment was conducted in three separate wells for each sample, and performed in triplicate.

To measure cell invasion, a Transwell invasion chamber coated with Matrigel^® (Corning Incorporated, Corning, NY, USA) at 37°C for 30 min was used to determine the cell invasion ability. Following fixation with 4% paraformaldehyde for 1 h at room temperature, the cells that had invaded the membrane were stained with 0.1% crystal violet for 30 min at room temperature and counted. The number of cells that had invaded through the Matrigel was counted in 5 fields of triplicate membranes at magnification, ×100, using an inverted light microscope (Olympus Corporation, Tokyo, Japan).

The potential targets and binding sites of miR-671-3p were analyzed using TargetScan7 tool (http://www.targetscan.org/vert_71/). The 3′-UTR of the CCND2 was obtained by gene synthesis, and inserted downstream of the luciferase reporter gene in a pmirGLO vector (Promega Corporation, Madison, WI, USA). For the luciferase reporter assay, A549 cells (2×10⁴/well) were seeded in a 24-well plate and incubated for 24 h prior to transfection. Next, firefly luciferase constructs containing the 3′-UTR and miR-671-3p mimics or the corresponding negative controls were co-transfected into A549 cells using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Cells were collected at 48 h after transfection, and measured using the Dual-Luciferase Reporter System (Promega Corporation), according to manufacturer's protocols. The pRL-TK Renilla luciferase activity was used for normalization.

All statistical analyses were performed using SPSS 20.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism v. 6 (GraphPad Software, Inc., La Jolla, CA, USA). A Student's t-test and one-way analysis of variance followed by Tukey's post-hoc test were used to analyze two or multiple groups, respectively. Kaplan-Meier curves were used to analyze survival rate and log-rank tests was used to calculate the corresponding P-values. Associations between miR-671-3p expression and clinicopathological characteristics of patients with NSCLC were analyzed using a χ² test. P<0.05 was considered to indicate a statistically significant difference.

In order to explore the function of miR-671-3p in NSCLC progression, the expression of miR-671-3p in 43 pairs of NSCLC tissues and adjacent normal tissues was first analyzed by RT-qPCR. The results demonstrated that miR-671-3p expression was significantly downregulated in NSCLC tissues compared with the adjacent normal tissues (Fig. 1A). Consistently, it was identified that the expression of miR-671-3p was also markedly downregulated in the NSCLC A549, H1975, H1299 and H1650 cell lines compared with the non-tumorigenic human bronchial epithelial NL20 cell line (Fig. 1B). Furthermore, the NSCLC tissues were divided into miR-671-3p high and miR-671-3p low groups using the median value of miR-671-3p level as the cut-off value. pTNM staging designations were made according to the postsurgical pathological staging system according to the 7th edition of the TNM classification of malignant tumors (14). It was observed that the level of miR-671-3p was negatively associated with tumor size, Tumor Node Metastasis stage and metastasis (Table I). Taken together, these results suggested that miR-671-3p may be involved in NSCLC progression.

To additionally investigate the biological functions of miR-671-3p, miR-671-3p was overexpressed in A549 cells, which exhibited the lowest level of miR-671-3p among all measured cell lines. RT-qPCR analysis indicated that miR-671-3p levels were significantly upregulated following transfection with miR-671-3p mimics in A549 cells (Fig. 2A). Then, CCK-8 and Transwell invasion assays were performed. The results indicated that ectopic expression of miR-671-3p significantly inhibited the proliferation and decreased the invading cell number (Fig. 2B and C), suggesting that miR-671-3p exerts a tumor-suppressive role in NSCLC.

To additionally confirm the role of miR-671-3p in NSCLC, experiments using miR-671-3p inhibitors were performed. As demonstrated by the RT-qPCR assay results, miR-671-3p expression was significantly downregulated in A549 cells following transfection with miR-671-3p inhibitors compared with the negative control (Fig. 3A). The CCK-8 assay revealed that miR-671-3p inhibition led to a decreased proliferation ability in A549 cells (Fig. 3B). Furthermore, knockdown of miR-671-3p significantly inhibited the invasion of A549 cells (Fig. 3C).

The present study then aimed to determine the mechanism of miR-671-3p in NSCLC. The potential target of miR-671-3p was identified using TargetScan7. The results implied that CCND2 may be a target of miR-671-3p, as a potential binding site of miR-671-3p in the CCND2 3′-UTR was identified (Fig. 4A). To validate this prediction, a luciferase reporter assay was then performed. It was identified that the overexpression of miR-671-3p significantly repressed the luciferase intensity of CCND2-3′-UTR-wild type in A549 cells (Fig. 4B). Notably, mutation of the predicted site in CCND2 3′-UTR abrogated the effect of miR-671-3p (Fig. 4B), indicating that miR-671-3p interacts with CCND2 mRNA directly. In addition, RT-qPCR and western blot analyses suggested that the overexpression of miR-671-3p markedly decreased the mRNA and protein levels of CCND2 in A549 cells (Fig. 4C and D). The expression levels of CCND2 were also upregulated in NSCLC tissues and cell lines (Fig. 4E and F). Taken together, these results suggested that CCND2 was directly targeted by miR-671-3p in NSCLC cells.

To confirm the role of CCND2 in the process of miR-671-3p-mediated NSCLC progression, rescue experiments were performed. The efficiency of CCND2 overexpression was first validated by RT-qPCR. The results revealed that the CCND2 mRNA level was significantly upregulated in A549 cells following transfection with the pCDNA3-CCND2 vector (Fig. 5A). Then, CCK-8 and Transwell invasion assays were conducted. The results demonstrated that miR-671-3p overexpression significantly inhibited the proliferation and invasion of NSCLC cells (Fig. 5B and C). However, restoration of CCND2 markedly rescued the inhibitory effects of miR-671-3p overexpression in A549 cells (Fig. 5B and C). In conclusion, these results demonstrated that miR-671-3p exerts its roles via directly targeting CCND2 in NSCLC cells.