A total of 30 tumor tissues and paired adjacent non-tumor lung tissues were obtained from patients with NSCLC (sex: Male, n=25; female, n=5; age: <60 years, n=4; ≥60 years, n=26) who had undergone primary surgical NSCLC resection at the Affiliated Hospital of Nantong University (Nantong, China) between April 2012 and December 2015. After collection, the tissues were frozen at −80°C. The present study was approved by the Ethics Committee of the Affiliated Hospital of Nantong University, and all of the experiments were performed in accordance with the approved guidelines of the Affiliated Hospital of Nantong University. Written informed consent was obtained from each patient prior to study enrollment.

The human NSCLC cell lines A549, H1299, H1975 and H1650, the human lung cell line BEAS-2B, and 293 cells (all from Cell Center of Central South University, Changsha, China) were maintained in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.) at 37°C in a humidified incubator containing 5% CO₂. Human miR-145 mimics and small interfering (si)RNAs were transfected into A549 and H1299 cells (5×10³ cells) using Lipofectamine^® 3000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol at a concentration of 50 nmol/l. A miR-145 sequence-scrambled RNA oligonucleotide was used as a miRNA negative control (miNC). miR-145 mimics, siRNAs and negative controls were designed and obtained from Biomics Biotechnologies Co., Ltd. (Nantong, China). The miRNA and siRNA sequences were as follows: miR-145 mimics sense, 5′-GUCCAGUUUUCCCAGGAAUCCCU-3′ and antisense, 5′-AGGGAUUCCUGGGAAAACUGGAC-3′; miNC sense, 5′-GUACGGCUCUCACCUUCCAUAGU-3′ and antisense, 5′-ACUAUGGAAGGUGAGAGCCGUAC-3′; RIOK2-targeted siRNA (siRIOK2) sense, 5′-CAAUCAAGCUUUAGAAGAAdTdT-3′ and antisense, 5′-UUCU UCUAAAGCUUGAUUGdTdT-3′; and NOB1-targeted siRNA (siNOB1) sense, 5′-GGAACAAGACCCUGAAGAAdTdT-3′ and antisense, 5′-UUCUUCAGGGUCUUGUUCCdTdT-3′. In addition, a siRNA with no homology with human genes was used as a siRNA negative control (siNC), the sequences of which were as follows: Sense 5′-UUCUCCGAACGUGUCACGUdTdT-3′ and antisense 5′-ACGUGACACGUUCGGAGAAdTdT-3′.

Total RNA was extracted from tissues and cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), whereas small RNA enriched with miRNAs was isolated using the mirPremier^® microRNA Isolation kit (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany); these kits were used according to the manufacturers' protocols. Stem-loop RT-qPCR was performed to detect miR-145 expression according to a method previously described by Chen et al (21) using EzQuick™ miR-145 qPCR Detection Primer Set (Biomics Biotechnologies Co., Ltd.) and SYBR Green I (Thermo Fisher Scientific, Inc.); U6 small RNA was used as an internal control. RT-qPCR was conducted using the SuperScript^® III Platinum^® SYBR^® Green One-Step RT-qPCR kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. β-actin was used as an internal control. The thermocycling conditions were as follows: RT for 30 min at 42°C; initial denaturation at 95°C for 5 min, followed by 45 cycles of denaturation at 95°C for 20 sec, annealing at 55°C for 30 sec, and extension at 72°C for 30 sec. The relative expression levels were evaluated using the 2^−ΔΔCq method (22). The RT-qPCR primer sequences were designed as follows: RIOK2 forward, 5′-ACAACAGGCAAGATGGTCA-3′ and reverse, 5′-GACGACAAGGCAATTAGATGAG-3′; NOB1 forward, 5′-ACATACCAGTTGGAAGCAGAG-3′ and reverse, 5′-CAGGTTCTCAGGCTCACAAG-3′; β-actin forward, 5′-CCACACCTTCTACAATGAG-3′ and reverse, 5′-ATAGC ACAGCCTGGATAG-3′; miR-145 forward, 5′-ACACTCCAG CTGGGGTCCAGTTTTCCCAGGA-3′ and reverse, 5′-GTG CAGGGTCCGAGGT-3′; and U6 forward, 5′-AACGCTTCACGAATTTGCGT-3′ and reverse, 5′-CTCGCTTCGGCAGCACA-3′.

TargetScan online software (http://www.targetscan.org/) was used to predict the miR-145-binding sites of RIOK2 and NOB1. Subsequently, RIOK2 (accession no. NM_018343, 111-1,769 sites) and NOB1 (accession no. NM_014062, 59-1,297 sites) human cDNA sequences (obtained from National Center for Biotechnology Information GenBank; https://www.ncbi.nlm.nih.gov/genbank/) of the predicted miR-145-binding regions in RIOK2 and NOB1 mRNA were inserted into pGL3 vectors (Promega Corporation, Madison, WI, USA) to obtain the following DLR vectors: Wild-type (wt)-plasmid (p) RIOK2- or wt-pNOB1-3′ untranslated region (UTR), which were used to evaluate miR-145 activity. In addition, mutant (mt)-pRIOK2-or mt-pNOB1-3′UTR vectors were constructed as negative controls, in which the miR-145-binding sites were scrambled. Briefly, 1×10⁵ 293 cells/well were plated in a 24-well plate and cultured for 24 h at 37°C in a humidified incubator containing 5% CO₂. Subsequently, to determine the effects of miR-145 on RIOK2 expression, 80 ng/well wt- or mt-pRIOK2-3′UTR and 50 nmol/l miR-145 mimics or miNC were cotransfected into 293 cells using Lipofectamine^® 3000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 48 h. To observe the effects of miR-145 on NOB1 expression, wt- or mt-pNOB1-3′UTR and miR-145 mimics or miNC were cotransfected into 293 cells. pRL-TK (Promega Corporation) was cotransfected into cells of each group as an internal control. Firefly and Renilla luciferase activities were measured 48 h post-transfection using the DLR assay system (Promega Corporation) according to the manufacturer's protocol.

A549 and H1299 cells were seeded into 6-well plates and cultured for 24 h. Following cell transfection for 48 h, cells were collected and lysed in protein lysis and extraction buffer (Thermo Fisher Scientific, Inc.) on ice. The lysates were then centrifuged at 13,800 × g for 20 min at 4°C and the protein concentration was determined by bicinchoninic acid assay (Promega Corporation). Subsequently, proteins (50 µg) were separated by 10% SDS-PAGE and electroblotted onto polyvinylidene difluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). Subsequently, the membranes were blocked with 1% bovine serum albumin (Sangon Biotech Co., Ltd., Shanghai, China), and incubated with anti-RIOK2 (1:1,000; cat. no. ab172717), anti-NOB1 (1:1,000; cat. no. ab228985) and anti-β-actin antibodies (1:5,000; cat. no. ab8226) (all from Abcam, Cambridge, MA, USA). β-actin was used as an internal control. After washing with Tris-buffered saline with 0.1% Tween-20 (TBST), the PVDF membranes were incubated with the following horseradish peroxidase-conjugated secondary antibodies: Goat anti-mouse immunoglobulin (Ig)G (1:2,000; cat. no. ab205719) for RIOK2 and NOB1; goat anti-rabbit IgG (1:2,000; cat. no. ab205718) for β-actin (all from Abcam) for 1.5 h at room temperature. Finally, membranes were washed with TBST and the specific proteins were detected using an enhanced chemiluminescence substrate (Thermo Fisher Scientific, Inc.). The blots were semi-quantified using ImageJ (v1.45) software (National Institutes of Health, Bethesda, MD, USA).

An MTT Cell Viability Assay kit (Thermo Fisher Scientific, Inc.) was used to measure cell viability. Briefly, the cultured A549 and H1299 cells were plated into a 96-well plate at 5×10³ cells/well and were grown to ~75% confluence over 24 h at 37°C. Following transfection for 0, 24, 48, 72 and 96 h, the medium was removed and replaced with 100 µl fresh DMEM, and 10 µl 12 mmol/l MTT solution was added to each well. A negative control well containing 100 µl medium only was treated with 10 µl MTT solution. Following incubation at 37°C for 4 h, 100 µl SDS-HCl (0.01 mol/l) solution was added to each well, mixed thoroughly and incubated at 37°C for 4 h. Finally, each sample was mixed again and absorbance was measured at 570 nm using a microplate reader.

The Transwell assay was used to measure cell migration. Briefly, 1.5×10⁵ A549 or H1299 cells/well were seeded into a 24-well plate and grown for 24 h. After transfection for 48 h, the cells were suspended in DMEM at a density of 1×10⁶ cells/ml. The Transwell chambers (Corning Incorporated, Corning, NY, USA) were initially incubated with DMEM at 37°C for 1 h; an 8-µm pore polycarbonate membrane separated the upper and lower chambers. Subsequently, 100 µl cell suspensions were added to the upper chamber, whereas 600 µl DMEM containing 10% FBS and conditioned medium (1:1 dilution) was added to the lower chamber. Conditioned media were obtained from the cultures of A549 or H1299 cells, which were transfected with miR-145 or miNC for 48 h. Post-incubation for 24 h at 37°C, the cells on the top surface of the membrane were carefully removed using a cotton swab, whereas cells on the bottom surface were fixed in 10% formaldehyde for 30 sec and stained with 0.5% crystal violet solution. After washing with PBS, the cells on the top surface of the membrane were carefully removed again. Cells on the bottom surface of the membrane were observed and counted under a microscope (Nikon 90i; Nikon Corporation, Tokyo, Japan).

Cell invasion was detected using Transwell chambers coated with Matrigel. The assay was performed using the same protocol as for the cell migration assay, with the exception that the 8-µm pore polycarbonate membrane was coated with 50 µl 0.5 mg/ml Matrigel (BD Biosciences, Franklin Lakes, NJ, USA).

All experiments were performed independently at least three times. Data are presented as the means ± standard deviation. Statistical analyses were performed using SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA); Spearman's correlation analysis was used to conduct correlations analyses. Significance between multiple groups was evaluated by one-way analysis of variance followed by a Dunnett post hoc test. Comparisons between two groups was performed by Student's t-test. All P-values are based on two-sided statistical analyses; P<0.05 was considered to indicate a statistically significant difference.

The mRNA expression levels of RIOK2 and NOB1 were detected in 30 NSCLC tumor tissues and paired adjacent non-tumor lung tissues using RT-qPCR. The results demonstrated that the mRNA expression levels of RIOK2 and NOB1 were increased in tumor tissues compared with in non-tumor lung tissues (P<0.05; Fig. 1A and B). In addition, miR-145 expression was detected in tumor tissues by stem-loop RT-qPCR; the results indicated that the expression levels of miR-145 were decreased in tumor tissues compared with in non-tumor lung tissues (P<0.05; Fig. 1C). Spearman's correlation analysis revealed that miR-145 was negatively correlated with RIOK2 and NOB1 expression (RIOK2, r=−0.886, P<0.05; NOB1, r=−0.893, P<0.05) (Fig. 2).

As predicted by TargetScan online software, RIOK2 and NOB1 each possess a miR-145-binding site, at position 423 in RIOK2 mRNA and position 161 in NOB1 mRNA. The following DLR vectors: wt-RIOK2 or NOB1-3′UTR, and mt-RIOK2 or NOB1-3′UTR, were constructed in order to verify the inhibitory effects of miR-145 (Fig. 3A). Following cotransfection of 293 cells with miR-145 mimics and wt-RIOK2 or NOB1-3′UTR, a marked decrease was observed in luciferase activity, as compared with in cells cotransfected with miNC and mt-RIOK2 or NOB1-3′UTR (P<0.05, Fig. 3B and C).

The expression levels of RIOK2, NOB1 and miR-145 were detected in various NSCLC cell lines by RT-qPCR. The results demonstrated that the mRNA expression levels of RIOK2 and NOB1 were increased in NSCLC cells (A549, H1299, H1975 and H1650) compared with in the normal human lung cell line BEAS-2B (P<0.05; Fig. 4A and B). In addition, the expression levels of miR-145 were decreased in NSCLC cells (A549, H1299, H1975 and H1650) compared with in the normal human lung cell line BEAS-2B (P<0.05; Fig. 4C). A549 and H1299 cells were selected as an NSCLC cell model for subsequent functional assays. Post-transfection of cells with miR-145 mimics, miR-145 expression was markedly increased compared with in the miNC-transfected cells (P<0.05; Fig. 4D).

The expression levels of RIOK2 and NOB1 were detected following transfection of NSCLC cells with miR-145 mimics, siRIOK2 and siNOB1 using western blotting. The results demonstrated that the protein expression levels of RIOK2 and NOB1 were inhibited by miR-145 overexpression in A549 and H1299 NSCLC cells compared with in miNC-transfected cells (P<0.05; Fig. 5A). Furthermore, RIOK2 protein expression was significantly inhibited by siRIOK2, whereas NOB1 protein expression was significantly inhibited by siNOB1, as compared with in the siNC-transfected groups (P<0.05; Fig. 5B).

An MTT assay was used to investigate the inhibitory effects of miR-145 or siRNAs on NSCLC cell growth. miR-145 markedly inhibited the growth of A549 and H1299 cells at 72 and 96 h compared with the miNC-transfected cells (P<0.05; Fig. 6A and B). In addition, cell growth was reduced in cells transfected with siRNAs that target RIOK2 or NOB1 compared with in siNC-transfected cells (P<0.05; Fig. 6C and D).

Transwell assays were used to evaluate the effects of miR-145-mediated RIOK2 and NOB1 inhibition on NSCLC cell migration and invasion. miR-145 transfection significantly decreased A549 and H1299 cell migration and invasion compared with in miNC-transfected cells (P<0.05; Figs. 7 and 8).