The present study was approved by the Ethics Committee of Hunan Provincial People's Hospital (Changsha, China). The use of these tissue samples was approved by all of the patients prior to participation in the present study, and written informed consent was obtained from all patients with NSCLC. Paired NSCLC tissues and adjacent non-tumor lung tissues were collected from 47 patients (26 males and 21 females; age range, 39–72 years old) with NSCLC who underwent surgical resection at Hunan Provincial People's Hospital between June 2014 and October 2016. None of the patients underwent chemotherapy or radiotherapy prior to surgery. On the basis of the miR-433 median level, all patients with NSCLC were assigned into either miR-433 low-expression group (n=24) and miR-433 high-expression group (n=23). All tissues were quickly snap-frozen in liquid nitrogen following excision and then stored at −80°C until further experimentation.

A non-tumorigenic bronchial epithelium BEAS-2B cell line and four NSCLC cell lines (SK-MES-1, A549, H522 and H460) were obtained from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). BEAS-2B cells were cultured in LHC-9 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.). All NSCLC cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS, 100 U/ml penicillin G and 100 µg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.). All cells were cultured at 37°C in a humidified atmosphere with 5% CO₂. A549 and H460 cells were revealed to express relatively low levels of miR-432 compared with SK-MES-1 and H522 cells; therefore, A549 and H460 cell lines were chosen for subsequent functional experiments.

MiR-433 mimics and miRNA mimics negative control (miR-NC) were purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). The miR-433 mimic sequence was 5′-AUCAUGAUGGGCUCCUCGGUGU-3′ and the miR-NC sequence was 5′-UUCUCCGAACGUGUCACGUTT-3′. Small interfering RNA (siRNA) against the expression of E2F transcription factor 3 (E2F3; E2F3 siRNA), negative control siRNA (NC siRNA), E2F3 overexpression plasmid pcDNA3.1-E2F3 and blank pcDNA3.1 plasmid were chemically synthesized by GeneCopoeia, Inc. (Rockville, MD, USA). The E2F3 siRNA sequence was 5′-GCACTACGAAGTCCAGATA-3′ and the NC siRNA sequence was 5′-UUUTGATCAUTGATGAAA-3′. Cells were plated into 6-well cell culture plates and cultured to 60–70% confluence. miRNA mimics (100 pmol), siRNAs (100 pmol) or blank plasmids (4 µg) were transfected into A549 and H460 cells using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. A total of 8 h post-transfection, the culture medium in each well was replaced with fresh DMEM medium with 10% FBS.

Total RNA was prepared from tissues and all five cell lines using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocols. To analyze miR-433 expression, total RNA was reversed transcribed to complementary DNA (cDNA) using a TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. qPCR was performed with a TaqMan MicroRNA PCR kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) on a Bio-Rad CFX96 Real-Time PCR machine (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The thermocycling conditions used for qPCR were as follows: 50°C for 2 min, 95°C for 10 min; followed by 40 cycles of denaturation at 95°C for 15 sec; and annealing/extension at 60°C for 60 sec. For the detection of E2F3 mRNA expression levels, cDNA was synthesized from total RNA using a M-MLV cDNA Reverse Transcription kit (Invitrogen; Thermo Fisher Scientific, Inc.). Subsequently, qPCR was performed using a SYBR^® Premix Ex Taq™ kit (Takara Biotechnology Co., Ltd., Dalian, China). The thermocycling conditions used for qPCR were as follows: 5 min at 95°C, followed by 40 cycles of 95°C for 30 sec and 65°C for 45 sec. Relative miR-433 and E2F3 mRNA expression was normalized to U6 small nuclear RNA (U6) and GAPDH, respectively. Each sample was performed in triplicate and analyzed with the 2^−ΔΔCq method (25). The primers were designed as follows: miR-433 forward, 5′-GGATCATGATGGGCTCCT-3′ and reverse, 5′-CAGTGCGTGTCGTGGAGT-3′; U6 forward, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ and reverse, 5′-CGCTTCACGAATTTGCGTGTCAT-3′; E2F3 forward, 5′-GATGGGGTCAGATGGAGAGA-3′ and reverse, 5′-GAGACACCCTGGCATTGTTT-3′; and GAPDH forward, 5′-CAAGGTCATCCATGACAACTTTG-3′ and reverse, 5′-GTCCACCACCCTGTTGCTGTAG-3′.

The effect of miR-433 on the proliferative ability of A549 and H460 cells was determined using a CCK-8 assay. After 24 h post-transfection, 3,000 transfected cells were plated onto 96-well cell culture plates and cultured for 0, 24, 48 or 72 h. At each time point, a total of 10 µl CCK-8 solution (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added into each well, and then the cells were incubated at 37°C with 5% CO₂ for another 2 h. Finally, the optical density value was detected at a wavelength of 450 nm using a microplate reader (Bio-Rad, Laboratories, Inc.).

Matrigel (BD Biosciences, San Jose, CA, USA) coated Transwell plates with 8 µm pore polycarbonate membranes (BD Biosciences) were employed to assess the invasive capacity of A549 and H460 cells. A total of 1×10⁵ transfected cells in FBS-free DMEM were seeded into the upper chambers, and 600 µl DMEM supplemented with 20% FBS was added to the lower chambers as a chemoattractant. Following incubation at 37°C with 5% CO₂ for 24 h, the cells remaining on the upper side of the polycarbonate membranes were wiped with cotton swabs. The invasive cells were fixed with 100% methanol at room temperature for 20 min and stained with 0.5% crystal violet solution (Beyotime Institute of Biotechnology, Shanghai, China). Finally, the stained cells were photographed and counted under a light microscope (magnification, ×200) using five randomly selected fields per membrane.

TargetScan 7.1 (http://www.targetscan.org/) and miRanda (http://www.microrna.org/) were applied to predict the potential targets of miR-433.

The wild type and mutant sequences containing the predicted target sites of miR-433 in the 3′-UTR of E2F3 mRNA were synthesised by Shanghai GenePharma Co., Ltd., (Shanghai, China), cloned into the pMIR-REPORT luciferase reporter plasmids (Promega Corporation, Madison, WI, USA) and named as pMIR-Wt-E2F3-3′-UTR and pMIR-Mut-E2F3-3′-UTR, respectively. Cells were plated into 24-well cell culture plates at a density of 60–70% confluence. MiR-433 mimics or miR-NC with or without pMIR-Wt-E2F3-3′-UTR or pMIR-Mut-E2F3-3′-UTR, were transfected into A549 and H460 cells using Lipofectamine^® 2000 according to the manufacturer's protocols. A pRL-TK plasmid with a constitutive expression of Renilla luciferase (Promega Corporation) was also transfected into A549 and H460 cells to serve as a negative control. Following transfection for 48 h, the transfected cells were collected and the relative luciferase activity was measured using a Dual-Luciferase^® Reporter Assay System (Promega Corporation) according to the manufacturer's protocols. The activity of firefly luciferase was normalised to that of Renilla luciferase.

The primary antibodies used in the present study were acquired from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and included mouse anti-human monoclonal E2F3 (1:1,000; cat. no. sc-28308) and mouse anti-human monoclonal GAPDH (1:1,000; cat. no. sc-365062) antibodies. A total of 72 h post-transfection, A549 and H460 cells were harvested. Total protein isolated from tissues and A549 and H460 cells following transfection was isolated using a radioimmunoprecipitation assay lysis buffer (Nanjing KeyGen Biotech Co., Ltd. Nanjing, China). The concentration of total protein was detected using a Bicinchoninic Acid Protein Assay kit (Nanjing KeyGen Biotech Co., Ltd.). Equal amounts of protein (30 µg) was separated via 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were then blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) at room temperature for 1 h, washed with TBST three times and incubated with primary antibodies overnight at 4°C. Subsequent to washing with TBST three times, the membranes were further probed with goat anti-mouse IgG horseradish peroxidase-conjugated secondary antibodies (1:5,000; cat. no. sc-2005; Santa Cruz Biotechnology, Inc.) at room temperature for 1 h. Finally, the protein signals were visualized using an ECL™ Western Blotting Detection Reagents kit (GE Healthcare, Chicago, IL, USA), and analyzed with Quantity One software (version 4.62; Bio-Rad Laboratories, Inc.). GAPDH served as a loading control.

Data were expressed as the mean ± standard deviation of at least 3 independent experiments and analysed using a statistical software package (SPSS 19.0, IBM Corp., Armonk, NY, USA). Differences between groups were compared with Student's t-test or one-way analysis of variance for multiple comparisons, combined with post hoc analysis (Student-Newman-Keuls test). The association between miR-433 and clinicopathological features in NSCLC was evaluated by chi-square test. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the expression pattern of miR-433 in NSCLC, total RNA from 47 paired NSCLC tissues and adjacent non-tumor lung tissues was obtained. The data of RT-qPCR revealed that miR-433 was significantly underexpressed in the NSCLC tissues relative to that in the adjacent non-tumor lung tissues (Fig. 1A; P<0.05). After confirming the downregulation of miR-433 in NSCLC, the association between the miR-433 expression levels and clinicopathological data in the patients with NSCLC was investigated. On the basis of the miR-433 median level, patients with NSCLC were separated into two groups as follows: MiR-433 low-expression group (n=24) and miR-433 high-expression group (n=23). Decreased miR-433 expression levels were significantly associated with the tumor-node-metastasis (TNM) stage (P=0.006) and lymph node metastasis (P=0.028) but not associated with the other clinicopathological factors in NSCLC (Table I). Lastly, the miR-433 expression levels in four NSCLC cell lines and a non-tumorigenic bronchial epithelium BEAS-2B cell line were analysed. Compared with in BEAS-2B cells, the expression levels of miR-433 were lower in all the examined NSCLC cell lines (Fig. 1B; P<0.05). These results suggested that the downregulation of miR-433 may be associated with the progression of NSCLC.

To determine the effects of miR-433 on the progression of NSCLC, miR-433 mimics were transfected into A549 and H460 cells, which were revealed to express relatively low miR-433 levels among the four NSCLC cell lines. RT-qPCR analysis revealed that miR-433 was significantly overexpressed in A549 and H460 cells following transfection with miR-433 mimics compared with corresponding miR-NC-transfected cells (Fig. 2A; P<0.05). Subsequently, CCK-8 and cell invasion assays were employed to examine the effects of miR-433 overexpression on NSCLC cell proliferation and invasion, respectively. CCK-8 assay indicated that ectopic miR-433 expression significantly decreased A549 and H460 cell proliferation at both 48 and 72 h (Fig. 2B; P<0.05). As presented in Fig. 2C, the invasive capacities of A549 and H460 cells transfected with miR-433 mimics were significantly lower than cells transfected with miR-NC (P<0.05). These results suggested that miR-433 may exhibit a tumor suppressive role in NSCLC progression.

To elucidate the mechanisms underlying the inhibitory effects of miR-433 in NSCLC cells, bioinformatics analysis was performed to predict the potential targets of miR-433. E2F3 (Fig. 3A) was predicted as a primary target of miR-433 and selected for investigation, to further verify its previously reported contribution to NSCLC formation and progression (26–30). To confirm this hypothesis, luciferase reporter assays were performed using A549 and H460 cells cotransfected with miR-433 mimics or miR-NC and pMIR-Wt-E2F3-3′-UTR or pMIR-Mut-E2F3-3′-UTR. The results indicated that the ectopic expression of miR-433 significantly decreased the luciferase activities of the wild-type 3′-UTR of E2F3 compared with cells transfected with miR-NC (P<0.05), but did not affect the luciferase activities of the mutant 3′-UTR of E2F3 in the A549 and H460 cells (Fig. 3B). RT-qPCR and western blot analyses were performed to investigate whether miR-433 may exert regulatory effects on E2F3 expression in NSCLC cells. MiR-433 upregulation significantly suppressed E2F3 mRNA and protein expression levels in the A549 and H460 cells compared with miR-NC groups (Fig. 3C and D; P<0.05). The results of the present study indicated that E2F3 may be a direct target of miR-433 in NSCLC.

E2F3 was confirmed as a direct target of miR-433 in NSCLC in the present study. Hence, the tumor suppressive role of miR-433 in NSCLC cells may be induced by the downregulation of E2F3. To test this hypothesis, A549 and H460 cells were transfected with E2F3 siRNA to significantly knockdown the endogenous E2F3 expression levels compared with in cells transfected NC siRNA. The results were further confirmed by western blot analysis (Fig. 4A; P<0.05). Functional experiments demonstrated that E2F3 knockdown significantly reduced the proliferation (Fig. 4B; P<0.05) and invasion (Fig. 4C; P<0.05) of the A549 and H460 cells. These effects were similar to those observed with miR-433 overexpression. Hence, miR-433 may have prohibited the proliferation and invasion in NSCLC, at least partly, by E2F3 downregulation.

Rescue experiments were performed to determine whether the tumor-suppressing roles of miR-433 in NSCLC cells were mediated by E2F3. A549 and H460 cells were cotransfected with miR-433 mimics and E2F3 overexpression plasmid pcDNA3.1-E2F3 or blank pcDNA3.1 plasmid. Western blot analysis indicated that the cotransfection of pcDNA3.1-E2F3 significantly increased the level of E2F3 protein expression in A549 and H460 cells compared with cells transfected with miR-433 mimics and blank pcDNA3.1 plasmids (Fig. 5A; P<0.05). Subsequent functional assays revealed that the cotransfection of pcDNA3.1-E2F3 significantly decreased the proliferation (Fig. 5B; P<0.05) and invasion (Fig. 5C; P<0.05) of A549 and H460 cells compared with cells transfected with miR-433 mimics and blank pcDNA3.1 plasmids. Thus, the results of the present study suggested that the tumor suppressive roles of miR-433 on NSCLC cells may depend, at least in part, on the inhibition of E2F3 expression.