The procedures of our present study were approved by the Ethics Committee of Weifang People's Hospital (Weifang, China) and written informed consent was also provided by all patients. Samples of primary NSCLC tissues and adjacent normal tissues were collected from 53 patients (30 males, 23 females; age range, 47–71 years) who had received surgical resection at Weifang People's Hospital between June 2016 and August 2017. None of these patients had been treated preoperatively with either radiotherapy or chemotherapy. All tissues were immediately snap-frozen in liquid nitrogen and then stored at −80°C for further RNA extraction.

A non-tumorigenic bronchial epithelium cell line, BEAS-2B, and four human NSCLC cell lines (A549, SK-MES-1, H522, and H460) were purchased from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). All cells were routinely cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS; both from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1% penicillin/streptomycin mixture (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The cultures were maintained at 37°C in a humidified atmosphere containing 5% CO₂.

miR-889 mimics and corresponding miRNA mimics negative control (miR-NC), siRNA targeting the expression of TAB1 (si-TAB1) and negative control siRNA (si-NC) were constructed by GenePharma (Shanghai, China). The si-TAB1 sequence was 5′-GGAUGAGCUCUUCCGUCUUTT-3′ and the si-NC sequence was 5′-UUCUCCGAACGUGUCACGUTT-3′. TAB1 expression vector pcDNA3.1-TAB1 (pcTAB1) and empty vector (pcDNA3.1) was obtained from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). Before transfection, cells in logarithmic phase were plated into 6-well plates with a density of 4×10⁵ cells/well. When the culture confluency reached 60–70%, the cells were transfected with the miR-889 mimics (100 pmol), miR-NC (100 pmol), si-TAB1 (100 pmol), si-NC (100 pmol), pcTAB1 (4 µg) or pcDNA3 (4 µg) using Lipofectamine™ 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). All of the transfection procedures were based on the product specifications. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Transwell Matrigel invasion assays were performed 48 h after transfection. The MTT assay and western blotting were performed at 24 and 72 h post-transfection, respectively.

Total RNA was extracted from tissue samples or cultured cell lines using a TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). All-in-One™ miRNA qRT-PCR Detection kit (GeneCopoeia, Inc., Rockville, MD, USA) was used to detect miR-889 expression. The thermocycling conditions were as follows: 95°C for 10 min, and 45 cycles of denaturation at 95°C for 15 sec and annealing/elongation at 60°C for 15 sec. To quantify TAB1 mRNA expression, total RNA was reverse-transcribed into cDNA using a PrimeScript RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China). The temperature protocol for TAB1 mRNA reverse transcription was as follows: 37°C for 15 min and 85°C for 5 second. The quantitative PCR was carried out using an ABI 7500 Sequence Detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.) and a SYBR Premix Ex Taq™ (Takara Biotechnology Co., Ltd). The thermocycling conditions for TAB1 mRNA qPCR was as follows: 5 min at 95°C, followed by 40 cycles of 95°C for 30 sec and 65°C for 45 sec. U6 small nuclear RNA and GAPDH were used as the internal controls for miR-889 and TAB1 mRNA, respectively. All reactions were run in triplicate and relative gene expression was analyzed using the 2^−∆∆Cq method (19). The primers were designed as follows: miR-889 forward, 5′-ACACTCCAGCTGGGTTAATATCGGACAAC-3′, and reverse, 5′-TGGTGTCGTGGAGTCG-3′; U6 forward, 5′-GCTTCGGCAGCACATATACTAAAAT-3′, and reverse, 5′-CGCTTCACGAATTTGCGTGTCAT-3′; TAB1 forward, 5′-ATGAGCTCTTCCGTCTTTCG-3′, and reverse, 5′-ATCCCCACCTGCTTGATCT-3′; and GAPDH forward, 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse, 5′-GGCTGTTGTCATACTTCTCATGG-3′.

An MTT assay was performed to determine cellular proliferation. In brief, transfected cells were inoculated in 96-well plates with a density of 3,000 cells/well. Then the cells were incubated at 37°C supplied with 5% CO_2. for 0–3 days. The MTT assay was carried out every day by adding 20 µl of MTT reagent (5 mg/ml; Beyotime Institute of Biotechnology, Haimen, China) into each well. Following incubation at 37°C for an additional 4 h, the culture medium containing MTT solution was replaced with 150 µl of dimethyl sulfoxide (DMSO). A total of 15 min after incubation, the absorbance value of each well was detected at a 450 nm wavelength using an enzyme-linked immunosorbent assay reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Transwell insert chambers (8 µm pore size) covered with Matrigel (both from BD Biosciences, San Jose, CA, USA) were utilized to evaluate the invasive ability of NSCLC cells. Transfected cells were collected and suspended in FBS-free culture medium. A total of 5×10⁴ cells were added into the upper chambers. In the lower chambers, 500 µl DMEM containing 20% FBS was added to serve as a chemoattractant. Subsequent to incubation at 37°C with 5% CO_2. for 24 h, the non-invasive cells were gently wiped away with a cotton swab. The invasive cells that invaded through the pores were fixed with 100% methanol at room temperature for 30 min, stained with 0.5% crystal violet at room temperature for 30 min, and washed with phosphate buffer solution. Images of five different fields were captured from each insert, and the number of invasive cells was counted under an inverted light microscope (×200, magnification; Olympus Corporation, Tokyo, Japan).

A total of eight 6-week-old BALB/c male nude mice (20 g) were purchased from the Shanghai Laboratory Animal Center (Chinese Academy of Sciences, Shanghai, China). All animals were maintained under specific pathogen-free conditions at 25°C, 50% relative humidity, a 10 h light/14 h dark cycle and had ad libitum access to food and water. The animals were divided into two groups (n=4 mice/group), one injected with miR-NC-transfected cells and the other injected with miR-889 mimic-transfected cells. The width and length of the xenograft formed was detected with a vernier caliper. Tumor volume was calculated by the following formula: Volume (mm³) = width² (mm²) × length (mm)/2. At the end of the assay, all nude mice were sacrificed and the excited xenograft was weighted. All animal experiments were carried out in accordance with the Guide for Care and Use of Laboratory Animal and all experimental protocols were approved by the Animal Ethics Committee of the Weifang People's Hospital.

Three online miRNA target prediction software, including miRDB (http://www.mirdb.org/), miRanda (http://www.microrna.org), and TargetScan (http://www.targetscan.org/), were used to predict the potential target genes of miR-889.

The 3′-UTR region of TAB1 containing the predicted wild-type and mutant miR-889 binding sites was amplified by GenePharma and individually cloned into the pmiR-RB-REPORT™ vector (Promega Corporation, Madison, WI, USA). For the reporter assays, cells were plated into 24-well plates, and co-transfected with wild type or mutant reporter plasmid and miR-889 mimics or miR-NC using Lipofectamine™ 2000, according to the manufacturer's instructions. Forty-eight hours later, the luciferase activity of transfected cells was determined using a Dual-Luciferase Reporter Assay System (Promega Corporation). Firefly luciferase activity was normalized against Renilla luciferase activity.

Total protein was isolated from tissues or cells using cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA). The concentration of total protein was determined using a bicinchoninic acid protein assay (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts of protein (30 µg) were separated by 10% sodium dodecyl sulfate polyacrylamide gels electrophoresis and transferred onto polyvinylidene fluoride membranes (Beyotime Institute of Biotechnology), followed by blocking with 5% dried skimmed milk in Tris-buffered saline and 0.1% Tween-20 (TBS-T) at room temperature for 1 h. The membranes were subsequently incubated with the following primary antibodies at 4°C overnight: Rabbit anti-human TAB1 antibody (cat. no. ab76412; 1:1,000 dilution) and rabbit anti-human GAPDH antibody (cat. no. ab128915; 1:1,000 dilution; both from Abcam, Cambridge, UK). The membranes were then probed with horseradish peroxidase-conjugated secondary antibody (cat. no. ab205718; 1:5,000 dilution; Abcam,) for 2 h at room temperature. Finally, the protein bands were visualized using an enhanced chemiluminescence reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA). GAPDH was used as the internal control. Quantity One software version 4.62 (Bio-Rad Laboratories, Inc.) was used for densitometry analysis.

All assays were repeated at least three times. All data are presented as the mean ± standard deviation, and analyzed using SPSS 19.0 software (IBM Corp., Armonk, NY, USA). All functional experiments were repeated three times. Differences between two groups were examined using an unpaired Student's t-test, or a paired Student's t-test to compare expression data from NSCLC and adjacent normal tissues. One-way ANOVA with Tukey's post-hoc test was used for the comparison between multiple groups. A χ² test was performed to evaluate the association between miR-889 and the clinicopathological characteristics of NSCLC patients. A correlation between miR-889 and TAB1 mRNA expression was determined through Spearman's correlation analysis. P<0.05 was considered to indicate a statistically significant difference.

In total, 53 pairs of NSCLC tissue samples and adjacent normal tissue samples were collected. Total RNA was isolated from these tissues, and RT-qPCR was performed to assess miR-889 expression. The data revealed that the expression of miR-889 was notably lower in NSCLC tissue samples than in adjacent normal tissues (P<0.05; Fig. 1A). In addition, miR-889 expression in four human NSCLC cell lines (A549, SK-MES-1, H522, and H460) was determined by RT-qPCR. When compared with non-tumorigenic bronchial epithelium BEAS-2B cells, miR-889 expression was lower in all four NSCLC cell lines to varying degrees (P<0.05; Fig. 1B). These data indicated that miR-889 was underexpressed in both NSCLC tissue samples and cell lines.

To clarify the possible clinical meaning of low miR-889 expression in NSCLC, all enrolled NSCLC patients were categorized into two subgroups, ‘low miR-889 expression’ and ‘high miR-889 expression’, based on the median value of miR-889 expression in NSCLC tissue samples. Statistical analysis revealed that underexpression of miR-889 was significantly correlated with TNM stage (P=0.019) and distant metastasis (P=0.020); however, no significant associations with other pathological characteristics were observed, including sex (P=0.477), age (P=0.685), tumor size (P=0.407), histological tumor type (P=0.449), or tumor differentiation status (P=0.317; Table I). These results indicated that underexpression of miR-889 may be implicated in the pathogenesis of NSCLC.

To assess whether miR-889 affects NSCLC progression, cell lines H522 and H460 (possessing relatively lower miR-889 expression among the four assessed NSCLC cell lines) were selected to conduct the following experiments. miR-889 in H522 and H460 cells was overexpressed by transfection with miR-889 mimics (P<0.05; Fig. 2A). Then, an MTT assay was carried out to quantify cell proliferation; it was revealed that miR-889 upregulation significantly inhibited H522 and H460 cell proliferation when compared with the miR-NC group (P<0.05; Fig. 2B). Furthermore, a Transwell Matrigel invasion assay was employed to investigate the regulation of cell invasion by miR-889 in NSCLC. Ectopic miR-889 expression decreased the capacity for invasion in H522 and H460 cells relative to the cells treated with miR-NC (P<0.05; Fig. 2C). Collectively, these findings indicated that miR-889 may perform tumor-suppressive functions in NSCLC carcinogenesis.

To illustrate the potential molecular mechanisms by which miR-889 exerts its influence on NSCLC progression, bioinformatics tools, including miRDB, miRanda, and TargetScan, were applied to find a potential target gene (i.e., target mRNA) of miR-889. This analysis indicated a possible binding site of miR-889 in the 3′-UTR of TAB1 (Fig. 3A). TAB1 was selected for further analyses since this gene may participate in the initiation and progression of NSCLC (20). The luciferase reporter assay was conducted to determine whether miR-889 targets the 3′-UTR of TAB1 mRNA directly. To this end, luciferase reporter vectors were chemically synthesized and co-transfected into H522 and H460 cells along with miR-889 mimics or miR-NC. The exogenous miR-889 expression reduced the luciferase activity of the vector that carried the wild-type 3′-UTR of TAB1 (P<0.05). By contrast, mutation of the miR-889 binding site in the 3′-UTR of TAB1 abrogated the inhibitory effect of miR-889 overexpression on the luciferase activity (Fig. 3B).

To confirm that TAB1 contributes to NSCLC pathogenesis, RT-qPCR analysis was carried out to assess TAB1 mRNA expression in NSCLC tissue samples. The data indicated that the mRNA expression of TAB1 was notably higher in NSCLC tissue samples than in the adjacent normal tissues (P<0.05; Fig. 3C). Furthermore, miR-889 expression was revealed to be negatively correlated with TAB1 mRNA levels among NSCLC tissue samples (r=−0.5077, P=0.0001; Fig. 3D). Moreover, TAB1 mRNA and protein levels in H522 and H460 cells transfected with miR-889 mimics or miR-NC were examined by RT-qPCR and western blot analysis, respectively. Transfection of the miR-889 mimics significantly decreased TAB1 expression in H522 and H460 cells at both the mRNA (P<0.05; Fig. 3E) and protein levels (P<0.05; Fig. 3F). Thus, it was demonstrated that TAB1 mRNA is a direct target of miR-889 in NSCLC cells.

To precisely determine the involvement of TAB1 in NSCLC, endogenous TAB1 expression was knocked down in H522 and H460 cells by transfection with siRNA against TAB1 (si-TAB1). This transfection efficiently decreased TAB1 protein expression in H522 and H460 cells, as evidenced by western blot analysis (P<0.05; Fig. 4A). Then, MTT and Transwell Matrigel invasion assays were performed to evaluate the changes in proliferative and invasive abilities of H522 and H460 cells after transfection with si-TAB1 or si-NC. The knockdown of TAB1 inhibited the proliferation (P<0.05; Fig. 4B) and invasiveness (P<0.05; Fig. 4C) of H522 and H460 cells. These observations confirmed that TAB1 inhibition may imitate the tumor-suppressive roles of miR-889 mimics in NSCLC cells, further supporting the notion that TAB1 mRNA is a target of miR-889 in NSCLC cells.

To confirm that TAB1 is involved in the miR-889-induced tumor-suppressive effects in NSCLC cells, rescue experiments were conducted next by restoring TAB1 expression in miR-889 mimic-transfected H522 and H460 cells. Firstly, pcDNA3.1 or pc-TAB1 was introduced into H522 and H460 cells. RT-qPCR analysis confirmed that the expression level of TAB1 mRNA was increased in pc-TAB1-transfected H522 and H460 cells (P<0.05; Fig. 5A). Western blotting indicated that co-transfection with TAB1 expression vector pcDNA3.1-TAB1 (pc-TAB1) notably recovered the TAB1 protein amount that was significantly decreased by miR-889 mimics (P<0.05; Fig. 5B). Functional experiments revealed that the reintroduction of TAB1 reversed the anti-proliferative (P<0.05; Fig. 5C) and anti-invasive (P<0.05; Fig. 5D) effects of miR-889 upregulation in H522 and H460 cells. Overall, these results indicated that miR-889 exerts its anticancer actions on NSCLC at least partly by directly suppressing TAB1 expression.

A xenograft experiment was next conducted to examine the influence of miR-889 overexpression on in vivo tumor growth of NSCLC cells. H460 cells transfected with miR-889 mimics or miR-NC were subcutaneously injected into the flanks of nude mice. The tumor volume was measured for 1 month. Upregulation of miR-889 significantly decreased the tumor growth in vivo (P<0.05; Fig. 6A and B). At the end of the experiment, all the nude mice were euthanized, and the excised xenografts were weighed. The results indicated that the xenograft tumors derived from miR-889-overexpressing H460 cells had lower weight than did the xenograft tumors in the miR-NC group (P<0.05; Fig. 6C). Then, miR-889 expression was quantitated in the excised xenograft tumors and it was revealed that miR-889 was still overexpressed in the xenograft tumors of the ‘miR-889 mimics’ group (P<0.05; Fig. 6D). Furthermore, the protein amount of TAB1 in the xenograft tumors was significantly decreased by miR-889 overexpression, as revealed by western blot analysis (Fig. 6E). These observations indicated that miR-889 upregulation inhibited the tumor growth of NSCLC cells in vivo by suppressing TAB1 expression.