A cohort of 39 NSCLC tissues (29 male and 10 female; aged 31–63 years; median age, 51 years) and adjacent non-tumor tissues were collected at Yan'an Hospital Affiliated to Kunming Medical University from January 2015 to December 2016. The study was approved by the clinical research ethics committee of Yan'an Hospital Affiliated to Kunming Medical University. Written informed consent was obtained from all the patients. Diagnoses of NSCLC were given by two pathologists. All patients recruited to this study did not receive any preoperative treatment. NSCLC patients were staged according to the tumor node metastasis (TNM) staging system of the American Joint Committee on Cancer.

The human NSCLC cell lines (H1299, H1650, A549 and PC9) and human bronchial epithelial cell line (16HBE) were purchased from the Chinese Academy of Sciences (Shanghai, China) and cultivated in Dulbecco's modified Eagle's medium (DMEM; Hyclone; Ge Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and streptomycin (100 µg/ml), penicillin (100 U/ml). All the cells were cultured at 37°C in a humidified incubator with 5% CO₂.

According to the manufacturer's protocol, 1×10⁶ A549 or H1299 cells were transfected using Lipofectamine 2000 Reagent (Thermo Fisher Scientific, Inc.) and 48 h after transfection, subsequent experiments were performed. Scrambled small interfering (si)RNAs (siNC; 5′-UUCUCCGAACGUGUCACGU-3′) or LINC00978 siRNAs (siLINC00978; 5′-GGUGGCUAUGAGUCAGCAU-3′) were purchased from Invitrogen (Thermo Fisher Scientific, Inc.) and 100 nM was used for transfection. miR-6754-5p inhibitors (5′-TCCTCCAAACCAGCCTCCCTGG-3′), mimics (5′-CCAGGGAGGCUGGUUUGGAGGA-3′) and controls (5′-GCGUAACUAAUACAUCGGAUUCGU-3′) were purchased from Genecopoeia Inc., (Rockville, MD, USA) and 100 nM was used for transfection.

For the Cell Counting Kit-8 (CCK-8) assay, A549 or H1299 cells were seeded in 96-well plates and then cells were cultured for 24, 48, 72 and 96 h prior to performing the CCK-8 assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Following incubation with CCK-8 at 37°C, absorbance (optical density value) at a wavelength of 450 nm was detected and used for calculating cell viability.

Cell invasion assay was performed using 24-well Transwell chambers with polycarbonate membranes containing 8-mm diameter pores (Corning Inc., Corning, NY, USA). A total of 1×10⁵ A549 or H1299 cells were seeded on the top side of the membrane precoated with Matrigel in DMEM without serum (BD Biosciences, Franklin Lakes, NJ, USA). The lower chambers were filled with DMEM with 10% FBS. Following incubation at 37°C for 24 h, the non-invasive cells on the top side of the membrane were removed by scraping. Invasive cells on the lower membrane were fixed with 20% methanol for 30 min at 25°C and stained with 0.1% crystal violet for 15 min at 25°C. Invasion was quantified by counting cells in 6 randomized fields of view in each well under a light microscope (Olympus Corporation, Tokyo, Japan) at the level of ×100 magnification.

Total RNA was isolated from cultured cells (H1299, H1650, A549, PC9 and 16HBE) or NSCLC tissues utilizing the TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). First strand complementary DNA was generated with a Reverse Transcription System kit (Takara Biotechnology Co., Ltd., Dalian, China), according to the manufacturer's protocol. The expression levels of LINC00978 and miR-6754-5p were detected using SYBR^® Premix Ex Taq™ (Takara Biotechnology Co., Ltd.) on ABI 7500 system (Applied Biosystems; Thermo Fisher Scientific, Inc.), respectively. The thermocycling conditions were as follows: 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. U6 small nuclear RNA (snRNA) was used as the internal controls for LINC00978 and miR-6754-5p, respectively. The RT-qPCR results were calculated using the 2^−ΔΔCq method (16). The primer sequences were as follows: LINC00978 (forward, 5′-TATGCCCAGGTTCTTCTGAGC-3′; reverse, 5′-GCATGGTGTGGTTCATTCTGG-3′), U6 (forward, 5′-AACGAGACGACGACAGAC-3′; reverse, 5′-GCAAATTCGTGAAGCGTTCCATA-3′) and miR-6754-5p (forward, 5′-AACGAGACGACGACAGAC-3′; reverse, 5′-CCAGGGAGGCTGGTTTGGAGGA-3′).

The RIP experiment was performed using the Magna RIP™ RNA-Binding Protein Immunoprecipitation kit (EMD Millipore, Billerica, MA, USA) following the manufacturer's protocol. A total of 2×10⁶ A549 cells were lysed using complete RIP lysis buffer and 100 µl of the whole A549 cell extract was incubated with RIPA buffer (Thermo Fisher Scientific Inc.) containing magnetic beads conjugated with human anti-Argonaute2 (Ago2) antibody (cat. no. 07-590; EMD Millipore) for 6–8 h at 4°C. A total of 4 µg normal mouse IgG (cat. no. PP542-K; EMD Millipore) conjugated to magnetic beads were used as a negative control. Samples were washed with washing buffer and incubated with 1 µg/ml proteinase K (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 55°C for 30 min to isolate the RNA-protein complexes from beads. Then immunoprecipitated RNA was extracted and subjected to RT-qPCR analysis.

The miRBD online tool (http://mirdb.org/miRDB/custom.html) was used to predict the potential miR targets of LINC00978.

Apoptosis was determined by staining with Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. Apoptosis was analyzed using a flow cytometer and BD Accuri C6 Software version 1.0.264.21 (BD Biosciences). The apoptosis rate was the sum of % FITC⁺/PI⁻ and FITC⁺/PI⁺ cells.

The mutant segments of LINC00978 were constructed. The wild type (Wt) and mutated type (Mut) of LINC00978 were inserted into luciferase reporter assay vector pmirGLO (Promega Corporation, Madison, WI, USA) was co-transfected with miR-NC or miR-6754-5p mimic into 2×10⁴ A549 cells using Lipofectamine 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The luciferase activity of each sample was measured using the Dual-Luciferase^® Reporter Assay system (Promega Corporation) and normalized to the Renilla luciferase activity.

Statistical analyses were performed using SPSS 20.0 (IBM, Corp., Armonk, NY, USA) and GraphPad Prism version 6 (GraphPad, Inc., La Jolla, CA, USA. The results were from three independent experiments and are presented as the mean ± standard deviation. A student's t-test or one-way analysis of variance followed by Tukey's post hoc test was used to analyze 2 or multiple groups, respectively, for statistical significance. Pearson's correlation coefficient analysis was used to determine the correlations. P<0.05 was considered to indicate a statistically significant difference.

To investigate the function of LINC00978 in NSCLC progression, RT-qPCR was used to measure its expression patterns. Results demonstrated that LINC00978 expression was significantly upregulated in NSCLC tissues compared with the adjacent normal tissues (P<0.05; Fig. 1A). Additionally, it was demonstrated that LINC00978 expression was significantly positively associated with grade III/IV tumor TNM stage and lymph node metastasis (P<0.05; Fig. 1B and C). Consistently, it was demonstrated that the expression of LINC00978 was also significantly upregulated in NSCLC cell lines, including H1299, H1650, A549 and PC9 cells compared with the 16HBE cell line (P<0.05; Fig. 1D). The data from the present study indicated that LINC00978 may exert an oncogenic role in NSCLC progression.

To investigate the influence of LINC00978 on NSCLC, LINC00978 was knocked down in A549 and H1299 cells by transfection with specific siRNA against LINC00978. RT-qPCR analysis indicated that LINC00978 expression was significantly downregulated following transfection (P<0.05; Fig. 2A). CCK-8 assays demonstrated that the proliferation of A549 and H1299 cells in the siLINC00978 group was decreased compared with the control group (Fig. 2B). Additionally, it was demonstrated that LINC00978 knockdown significantly promoted cell apoptosis by flow cytometry (P<0.05; Fig. 2C). Furthermore, the effects of LINC00978 on NSCLC cell migration and invasion were analyzed by transwell assays. It was demonstrated that LINC00978 silencing significantly inhibited the cell numbers of migration and invasion (P<0.05; Fig. 2D and E). These results suggested that LINC00978 contributed to the malignancy of NSCLC.

It has been acknowledged that lncRNA could function as a competing endogenous RNA to regulate gene expression by binding miR (17). Therefore, it was hypothesized that LINC00978 possesses a similar function in NSCLC. Through bioinformatics analysis, it was demonstrated that LINC00978 may directly interact with miR-6754-5p (Fig. 3A). To determine whether LINC00978 and miR-6754-5p were in the same RNA-induced silencing complex, RIP assays were performed with A549 cell lysates using anti-Ago2 or IgG. It was demonstrated that LINC00978 and miR-6754-5p were both significantly enriched in Ago2-containing miRs relative to control group (P<0.05; Fig. 3B). Furthermore, luciferase reporter assays were performed with reporter plasmids containing the wild-type (WT) or mutated miR-6754-5p binding sites in LINC00978. As presented, it was demonstrated that the overexpression of miR-6754-5p significantly inhibited the luciferase activity in A549 cells transfected with WT-LINC00978 (P<0.05; Fig. 3C). In addition, RT-qPCR analysis demonstrated that knockdown of LINC00978 promoted the expression of miR-6754-5p in A549 and H1299 cells (Fig. 3D). Consistently, the ectopic expression of miR-6754-5p significantly repressed the expression of LINC00978 in A549 and H1299 cells (P<0.05; Fig. 3E). Taken together, these data indicated that LINC00978 could directly act as a sponge for miR-6754-5p.

The function of miR-6754-5p remains largely unknown. To determine the function of miR-6754-5p in NSCLC, its expression patterns were examined using RT-qPCR. It was demonstrated that miR-6754-5p was significantly downregulated in NSCLC tissues compared with the adjacent normal tissues (P<0.05; Fig. 4A). Furthermore, it was demonstrated that the expression of miR-6754-5p was significantly inversely associated with TNM stage and tumor metastasis in NSCLC (P<0.05; Fig. 4B and C). In addition, it was demonstrated that there was a negative correlation between miR-6754-5p and LINC00978 expressions in NSCLC tissues (Fig. 4D). The above data suggested that miR-6754-5p may possess an opposite role to LINC00978 in NSCLC progression.

To further investigate whether LINC00978 regulates NSCLC progression by inhibiting miR-6754-5p expression, A549 cells were transduced with siLINC00978 and siLINC00978+miR-6754-5p inhibitor. RT-qPCR analysis indicated that miR-6754-5p expression was downregulated in siLINC00978+miR-6754-5p inhibitor group compared with the siLINC00978 group (Fig. 5A). Then CCK-8 assays, flow cytometry and transwell assays were performed to determine cell viability, apoptosis, migration and invasion. The results demonstrated that LINC00978 knockdown repressed the proliferation, migration and invasion while promoting cell apoptosis in A549 cells (Fig. 5B-E). However, inhibition of miR-6754-5p at the same time reversed the effects of LINC00978 knockdown on A549 cells at least in part (Fig. 5B-E). In conclusion, the results of the present study indicated that LINC00978 promotes NSCLC cell proliferation, migration an invasion by inhibiting miR-6754-5p expression.