NSCLC tissues and adjacent non-cancerous tissues were collected from 42 patients at The Second Affiliated Hospital of Harbin Medical University (Harbin, China) between May 2014 and September 2016. Tissue samples were frozen and stored at −80°C. The samples were divided into LIMD2 low expression group and LIMD2 high expression group (the median value of LIMD2 expression was used as the cutoff value). The associations between the expression of LIMD2 and the clinicopathological features of patients with non-small-cell lung cancer is given in Table I. The present study was approved by the Research Ethics Committee of the Second Affiliated Hospital of Harbin Medical University. Informed consent was obtained from patients for the collection of samples in accordance with the Declaration of Helsinki. All patients had not undergone radical prostatectomy and/or any other treatment prior to the present study.

Human NSCLC cell lines H1299, A549, H358 and H460 were purchased from the American Type Culture Collection (Manassas, VA, USA). Human bronchial epithelial cells (HBEC) were obtained from the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI-1640 medium (Invitrogen; Thermo Fischer Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fischer Scientific, Inc.) at 37°C in a humidified 5% CO₂ incubator.

Small interfering (si)RNA against LIMD2 (5′-GACCCACCAACTTACCATA-3′) or negative control (si-NC; 5′-AATTCTCCGAACGTGTCAC-3′) and miR-34a (5′-UGGCAGUGUCUUAGCUGGUUGU-3′) mimic or negative control (NC mimic; 5′-ACAUCUGCGUAAGAUUCGAGUCUA-3′) were synthetized by Invitrogen (Thermo Fischer Scientific, Inc.) and 50 nM per plasmid was transfected into H1299 and A549 cells, in which LIMD2 expression levels were higher compared with the other cell lines, using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). The LIMD2 coding sequence was cloned into the pcDNA3.1 vector (Invitrogen) between Not I and EcoR I, and 1 mg plasmid was transfected into H1299 and A549 cells as well as 50 nM miR-34a or NC mimics using Lipofectamine^® 3000. Then, 48 h after transfection, the transfection efficiency was determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) as described below.

Each well of a 96-well plate was seeded with ~5,000 transfected cells. Cell proliferation was assessed using a Cell Counting Kit-8 assay (CCK-8, Shanghai U-Sea biotech, Shanghai, China). Following incubation for 72 h at 37°C, 10 µl CCK-8 was added to each well and incubated for 2 h in an incubator at 37°C. The absorbance was determined using a multimode microplate reader (Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany) at 450 nm.

Cell invasion assays were performed using Transwell chambers (Corning Incorporated, Corning, NY, USA). Transfected cells (2×10⁴) were seeded in the upper chamber (pre-coated with Matrigel) and incubated in serum-free RPMI-1640 media, and the lower chamber was coated with complete RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). After 24 h at 37°C, migrated cells on the bottom surface of the filters were fixed with 4% paraformaldehyde for 1 h at 25°C, and stained with 0.1% crystal violet solution (MedChem Express, Shanghai, China) for 30 min at 25°C, and counted under an inverted microscope at ×100 magnifications; 3 fields per view were analyzed. The experiments were performed in triplicate.

Total RNA was extracted from tumor tissues or cultured cells with TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. Then, cDNA was synthesized with the M-MLV reverse transcriptase (Promega Corporation, Madison, WI, USA) according to the manufacturer's protocol. Subsequently, transcripts were analyzed via qPCR using a TaqMan MicroRNA Assay kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) on an ABI 7300 qPCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). Each experiment was repeated 3 times. The thermocycling conditions were: Denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 sec and elongation at 60°C for 1 min. The relative expression levels or LIMD2 and miR-34a were calculated and normalized to endogenous that of GAPDH and U6, respectively. The primer sequences were: miR-34a forward, 5′-AACGAGACGACGACAGAC-3′ and reverse, 5′-UGGCAGUGUCUUAGCUGGUUGU-3′; U6 forward, 5′-AACGAGACGACGACAGAC-3′ and reverse, 5′-GCAAATTCGTGAAGCGTTCCATA-3′; LIMD2 forward, 5′-GCCCATCCTGTTGTGACCAA-3′ and reverse, 5′-ATGGTGAGTCTGCACCCTTC-3′; GAPDH forward, 5′-ATGTTGCAACCGGGAAGGAA-3′ and reverse, 5′-AGGAAAAGCATCACCCGGAG-3′. Expression fold was calculated based on the 2^−ΔΔCq method (13).

Following transfection, total protein was extracted from tumor tissues or cultured cells by radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc.). All the protein lysates were separated using 10% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (Thermo Fisher Scientific, Inc.). The membrane was blocked using 5% non-fat milk in PBS (Thermo Fisher Scientific, Inc.) containing 0.1% Tween-20 (Sigma-Aldrich; Merck KGaA) at room temperature for 3 h. Then, the membrane was incubated for 2 h at 25°C with specific primary anti-human antibodies: GAPDH (1:5,000; cat. no. SAB2701826; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and LIMD2 (1:2,000; cat. no. SAB1307182; Sigma-Aldrich; Merck KGaA), followed by incubation for 1 h at 25°C with a goat horseradish peroxidase-conjugated secondary antibody (1:2,000; sc-2005; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Membranes were then washed with PBS for 10 min, and the protein bands were visualized using an Enhanced Chemiluminescence Western Blotting kit (Pierce; Thermo Fisher Scientific, Inc.), in accordance with the manufacturer's protocol. Protein densitometry was performed using ImageJ software (version 1.41; National Institutes of Health, Bethesda, MD, USA). GAPDH was used as a control. The experiment was repeated 3 times.

The target gene LIMD2 and potential binding site were predicted using TargetScan7 (http://www.targetscan.org/vert_71/). A luciferase reporter assay was performed to determine the direct binding of miR-34a to the target gene LIMD2. The wild type (WT) and mutant (Mut) 3′-UTR sequence of LIMD2 was directly synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China) and then inserted into pGL3 plasmid (Ambion; Thermo Fisher Scientific, Inc.) between Mlu I and Bgl II. Cells (2×10⁴) were cultured in 24-well plates, and each well was transfected with 0.2 µg firefly luciferase reporter plasmid, and equal amounts of miR-34a (50 nM) and NC mimics (50 nM) using Lipofectamine^® 2000. After 24 h, Firefly and Renilla luciferase activities were measured using the Luc-Pair miRNA Luciferase Assay kit (GeneCopoeia, Inc., Rockville, MD, USA) according to the manufacturer's protocols. Luciferase activities were normalized to that of Renilla luciferase.

All experiments were performed 3 times and results expressed as mean ± standard deviation. All statistical analyses were performed using SPSS software version 20.0 (IBM Corp., Armonk, NY, USA). For comparisons between multiple groups, one-way analysis of variance followed by a Tukey's post-hoc test was applied and a two-tailed Student's t-test was performed for comparisons between two groups. Pearson's correlation coefficient analysis was used to determine the correlation between LIMD2 and miR-34a expression. The association between the expression of LIMD2 with the clinicopathological features of patients with non-small-cell lung cancer was analysis using a χ² test. P<0.05 was considered to indicate a statistically significant difference.

To investigate the function of LIMD2 in NSCLC, the expression levels of LIMD2 were determined by RT-qPCR. The present study reported that LIMD2 expression levels were significantly upregulated in NSCLC tissues (n=41) compared with in adjacent normal tissues (n=41) (Fig. 1A). Additionally, the expression levels of LIMD2 in NSCLC cell lines were determined. The results indicated that the expression levels of LIMD2 were significantly upregulated in NSCLC cell lines, including H1299, A549, H358 and H522 cells, compared with human bronchial epithelial cells (HBECs) (Fig. 1B). Consistently, the protein expression levels of LIMD2 in NSCLC tissues and cell lines were notably upregulated compared with in normal tissue and HBECs, respectively (Fig. 1C and D). In addition, the present study reported that LIMD2 expression levels were significantly higher in NSCLC tissues with metastasis than tissues without (Fig. 1E). These data suggest LIMD2 may be involved in NSCLC progression.

To determine the effects of LIMD2 on NSCLC cells, LIMD2 expression was downregulated in H1299 and A549 cells via transfection with specific siRNAs. RT-qPCR analysis revealed that the expression levels of LIMD2 were significantly downregulated in H1299 and A549 cells transfected with siLIMD2 compared with in the control group (Fig. 2A). The present study also performed CCK-8 and Transwell assays to determine the effects of LIMD2 on cell proliferation and invasion, respectively. The CCK-8 assay indicated that knockdown of LIMD2 significantly suppressed the proliferation of NSCLC cells at 72 h compared with in the control group at 72 h (Fig. 2B). Furthermore, the present study reported that LIMD2 silencing significantly reduced the number of migrated cells compared with in the control group (Fig. 2C). The above data indicated that LIMD2 contributes to NSCLC progression.

The present study investigated the mechanism underlying the regulation of LIMD2 expression in NSCLC cells. The results demonstrated that overexpression of miR-34a significantly suppressed the mRNA expression levels of LIMD2 in H1299 and A549 cells compared with in the control (Fig. 3A). Additionally, western blot analysis revealed that ectopic expression of miR-34a markedly decreased the protein expression levels of LIMD2 in H1299 and A549 cells compared with in the control (Fig. 3B). Furthermore, an inverse correlation between the expression of LIMD2 and miR-34a was observed in NSCLC tissues (Fig. 3C).

miRNAs have been demonstrated to regulate the degradation of target mRNAs (12). To investigate whether LIMD2 is a direct target of miR-34a, bioinformatics analysis was conducted using TargetScan. The results indicated that LIMD2 is a potential target of miR-34a. To verify this prediction, luciferase reporter assays with the WT and Mut 3′-UTR sequence of LIMD2 mRNA were performed. The results demonstrated that overexpression of miR-34a significantly suppressed the luciferase activity in H1299 and A549 cells transfected with WT-LIMD2 3′-UTR reporter plasmid compared with in the control group; no notable differences were observed within cells overexpressing miR-34a containing the Mut-3′UTR compared with in the controls (Fig. 3D). These results demonstrated that miR-34a directly targets LIMD2.

LIMD2 was proposed to act as an oncogene in NSCLC in the present study; however, miR-34a inhibited the expression of LIMD2 in NSCLC cells. To determine the role of miR-34a in NSCLC, the expression levels of miR-34a were determined by RT-qPCR. The results demonstrated that miR-34a expression levels were significantly downregulated in NSCLC tissues compared with in adjacent normal tissues (Fig. 4A). Additionally, lower expression levels of miR-34a were observed in NSCLC cell lines compared with in HBEC cells (Fig. 4B). Furthermore, the expression levels of miR-34a were significantly lower in NSCLC tissues with metastasis compared with in those without metastasis (Fig. 4C). These data implied that miR-34a may be a tumor suppressor in NSCLC.

The present study investigated whether the effects of miR-34a on NSCLC cells are associated with LIMD2. RT-qPCR analysis indicated that the overexpression of LIMD2 significantly increased the mRNA expression levels of LIMD2 in H1299 and A549 cells compared with in the vector-transfected group (Fig. 5A). CCK-8 and Transwell assays revealed that the overexpression of miR-34a significantly suppressed the proliferation and invasion, of NSCLC cells respectively, compared with in the control group (Fig. 5B). Of note, restoration of LIMD2 expression significantly reversed the inhibitory effects of miR-34a on H1299 and A549 cell proliferation and invasion compared with in the miR-34a overexpression group (Fig. 5B and C). Taken together, the data from the present study demonstrated that miR-34a suppressed NSLCL progression through targeting LIMD2.