A total of 22 freshly frozen LSCC specimens and 22 adjacent non-tumor tissues (used as the normal control) (at least 5 cm away from the carcinoma) were obtained from patients who underwent pneumonectomy at the Department of Oncology, The First Affiliated Hospital of Xinxiang Medical College (Weihui, China) from January 2019 to July 2020. The inclusion criteria in present study were as follows: i) LSCC; ii) no radiotherapy or chemotherapy prior to surgery; iii) a patient age of ≥18 years; iv) weight loss of ≤10% in the 3 months before diagnosis; and v) Karnofsky performance status ≥70% (18). The exclusion criteria were as follows: i) Histology other than SCC; ii) metastatic lung cancer; iii) patients with serious medical or psychiatric illness, or a history of serious cardiac disease; iv) prior radiation therapy to the thorax or total surgical resection; v) prior systemic chemotherapy; and vi) an age of >80 years. The clinicopathological information of the patients is shown in Table I. The present study was approved by the Ethics Committee of The First Affiliated Hospital of Xinxiang Medical College (Weihui, China), and written informed consent was obtained from each patient.

The miRNA dataset (GSE74190) was searched and downloaded from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). The GSE74190 dataset was based on the Agilent-019118 Human miRNA Microarray V1 G4470A platform (19). GEO2R (www.ncbi.nlm.nih.gov/geo/geo2r/), an interactive web tool, was applied to compare the samples in two different groups under the same experimental condition. Fold change ≥2 and P<0.05 served as basic screening parameters. Hierarchical clustering of differentially expressed miRNA was performed using the Multiple Experiment Viewer 4.7.1 software program (The Institute for Genomic Research, USA).

Total RNA of the cultured cells and tissues was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) as described previously (20). The TaqMan Reverse Transcription kit (Takara Biotechnology Co., Ltd.) was used to obtain cDNA for mRNA detection (42˚C for 1 h), while the TaqMan MicroRNA Reverse Transcription kit (Takara Biotechnology Co., Ltd.) was used for miRNA detection (42˚C for 1 h). qPCR was performed using SYBR Premix Ex Taq (Takara Bio, Inc.) on an ABI PRISM 7500 Sequence Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.). Primers for cDNA amplification were as follows: miR-494 forward, 5'-TGACCTGAAACATACACGGGA-3', and universal reverse primer, 5'-TATCGTTGTACTCCACTCCTTGAC-3'; U6 forward, 5'-GCTTCGGCAGCACATATACTAAAAT-3', and reverse, 5'-CGCTTCACGAATTTGCGTGTCAT-3'; PUMA-α forward, 5'-CGGCGGAGACAAGAGGAGC-3', and reverse, 5'-CAGGGTGTCAGGAGGTGGGAG-3'; and GAPDH forward, 5'-TCAACGACCCCTTCATTGACC-3', and reverse, 5'-CTTCCCGTTGATGACAAGCTTC-3'. The reaction mixtures were denatured at 95˚C for 3 min, followed by 40 cycles of 95˚C for 10 sec and 60˚C for 30 sec. The relative expression levels of miRNA and mRNA were normalized to that of U6 and GAPDH, respectively. The relative expression of each gene was calculated and normalized using the 2^-ΔΔCq method (21). All reactions were conducted in triplicate.

A total of four LSCC cell lines (NCI-H520, SW900, EBC-1 and SK-MES-1) were purchased from the American Type Culture Collection (ATCC) and cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Sigma-Aldrich; Merck KGaA), 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.) at 37˚C in a 5% CO₂ atmosphere. A human bronchial epithelial cell line (16HBE; ATCC) was used as the control. 16HBE cells were cultured in medium provided by Procell Life Science & Technology (cat. no. CM-0249) at 37˚C with 5% CO₂.

miR-494 mimics (5'-UGAAACAUACACGGGAAACCUC-3'), negative control (NC) mimics (5'-CACGAUAAACAAUACGGGUACC-3'), inhibitor (5'-GAGGUUUCCCGUGUAUGUUUCA-3') and inhibitor NC (5'-UGUGUCGUUAACUAUGGGCUCU-3') were obtained from Shanghai GenePharma Co., Ltd. SW900 and EBC-1 cells were transfected with 20 nM miR-494 mimic/inhibitor and mimic NC/inhibitor NC using Lipofectamine™ RNAiMAX (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol at 37˚C in a 5% CO₂ incubator. Following 48 h of transfection, cells were collected for cell proliferation, RT-qPCR and western blot analysis. PUMA-α overexpression plasmid (pcDNA3.1-PUMA-α) and pcDNA vector, siRNAs against PUMA-α (si-PUMA-α) and the corresponding scrambled NC (si-NC) were designed and synthesized by Guangzhou RiboBio Co., Ltd. The sequences of si-PUMA-α and si-NC are as follows: si-PUMA-α sense, 5'-GGGUCCUGUACAAUCUCAUCAUGGG-3' and antisense, 5'-CCCAUGAUGAGAUUGUACAGGACCC-3'; and si-NC sense, 5'-GGGUGUCAACACUCUACUAUCUGGG-3' and antisense, 5'-CCCAGAUAGUAGAGUGUUGACACCC-3'. A total of 50 nM si-PUMA-α and si-NC were co-transfected with 50 nM miR-494 inhibitor or inhibitor NC into SW900 and EBC-1 cells at 37˚C using Lipofectamine™ RNAiMAX according to the manufacturer's instructions. SW900 and EBC-1 cells were cultured at 37˚C for 24 h prior to transfection. After 48 h of transfection, cells were harvested and used for analysis. All the transfections were repeated >3 times independently.

Cell viability was measured using the Cell Counting Kit (CCK)-8 (Dojindo Laboratories, Inc.) assay. Briefly, SW900 and EBC-1 cells were seeded into a 96-well plate at a density of 3,000 cells/well and cultured at 37˚C for 24 h prior to transfection. Then, cells were transfected with corresponding oligonucleotides. After 24, 36 or 48 h, 10 µl CCK-8 solution was added into each well of the plate. The plates were incubated at 37˚C for 1 h, and the absorbance at 450 nm was measured. The experiment was repeated three times.

SW900 and EBC-1 cells were harvested 48 h after cell transfection, and then washed twice with phosphate-buffered saline. Next, the cells were stained with Annexin V (FITC) and propidium iodide reagent (Thermo Fisher Scientific, Inc.) at 37˚C for 10 min in the dark at room temperature according to the manufacturer's instructions. Cell apoptosis rates were detected via FACSAria flow cytometry (BD Biosciences) and analyzed using FlowJo 7.6 software (FlowJo, LLC).

For western blotting, SW900 and EBC-1 cells were lysed using RIPA lysis buffer (Beyotime Institute of Biotechnology). Protein concentrations were determined with the bicinchoninic acid assay (Beyotime Institute of Biotehnology). Cell lysate (40 µg total protein per lane) were separated on 15% gels using SDS-PAGE and transferred to polyvinylidene difluoride membranes (Roche Applied Science). The membranes were first blocked with 5% skimmed milk at room temperature for 30 min, after which the membranes were incubated with primary antibodies overnight at 4˚C. Membranes were incubated with primary antibodies against PUMA-α (1:1,000; cat. no. sc-377015; Santa Cruz Biotechnology, Inc.), cleaved caspase-3 (1:1,000; cat. no. ab2302; Abcam) and Bcl-2 (1:1,000; cat. no. ab32124; Abcam) overnight at 4˚C, followed by incubation with HRP-conjugated anti-rabbit secondary antibody (1:10,000; cat. no. ab205718; Abcam) for 1 h at room temperature. β-actin antibody (1:1,000; cat. no. ab8227; Abcam) was used as an internal control. The protein bands were developed using an ECL kit (GE Healthcare) and quantified using ImageJ Software (version 1.46; National Institutes of Health).

The potential binding site between PUMA-α and miR-494 was searched in TargetScan (http://www.targetscan.org) and PicTar (https://pictar.mdc-berlin.de/). A whole fragment of 3'UTR PUMA-α mRNA and a mutant form were cloned into pGL-3-Luc (Promega Coporation). The 293T cells were seeded in 24-well plates at 5x10⁴ cells per well and co-transfected with the pGL-3-PUMA-α wild-type or mutant portion and TK100 Renilla combined with the aforementioned miR-494 mimic, miR-494 inhibitor, mimic NC or inhibitor NC using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Cells were harvested 48 h after transfection, and luciferase activity was measured using a Dual-Luciferase^® Reporter System (Promega Corporation) according to the manufacturer's protocol. Relative firefly luciferase activity was normalized to Renilla luciferase activity. All the dual-luciferase reporter assays were performed in triplicate within each experiment.

Data are reported as the mean ± SD (unless otherwise presented). Statistical significance among different groups was determined using one-way ANOVA followed by Tukey's post-hoc test and the significance of the difference in means between two groups was evaluated using unpaired Student's t-test. The paired Student's t-test was used to compare data from cancerous vs. non-cancerous tissues. The correlation between PUMA-α and miR-494 expression was assessed using a two-tailed Pearson's correlation analysis. Statistical significance was analyzed using GraphPad Prism software (version 5.0; GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

To explore the role of miRNAs in LSCC, miRNA expression patterns were initially profiled using GSE74190 microarray data. Cluster analysis based on the miRNA expression pattern indicated a significant difference between LSCC tissues and adjacent non-cancerous tissues (Fig. 1A). Among the aberrantly expressed miRNAs, miR-494 was chosen for further study, which has been reported as an oncogenic miRNA in several types of human cancer (16,22-24). Moreover, several studies have revealed that miR-494 acts as an oncomir and is involved in tumor development, progression and metastasis, and confers resistance to chemotherapeutic drugs by targeting a number of molecules in several types of human cancer, such as endometrial cancer and hepatocellular carcinoma (25,26). However, the function and underlying molecular mechanism of miR-494 in LSCC has not been fully elucidated. To validate the expression trend of miR-494 in the LSCC tissues, RT-qPCR was performed to detect miR-494 expression in 22 pairs of LSCC tissues and adjacent non-cancerous tissues. As presented in Fig. 1B, the expression level of miR-494 in LSCC tissues was significantly higher compared with that in adjacent non-cancerous tissues. In addition, miR-494 levels were assessed in four LSCC cell lines (NCI-H520, SW900, EBC-1 and SK-MES-1) and in 16HBE, which acted as a normal control. miR-494 expression in all LSCC cells was upregulated compared with 16HBE, especially in SW900 and EBC-1 cells (Fig. 1C). These findings suggest that upregulation of miR-494 could be involved in the progression of LSCC.

Given the upregulation of miR-494 in LSCC tissues, it was predicted that miR-494 may function as an oncogene. To verify this hypothesis, miR-494 inhibitor was transfected into two LSCC cell lines, namely SW900 and EBC-1. The miR-494 level was significantly downregulated in both SW900 and EBC-1 cells after miR-494 inhibitor transfection (Fig. 2A). To evaluate whether miR-494 could affect cell proliferation, a CCK-8 assay was performed, and it was revealed that knockdown of miR-494 significantly suppressed the viability of SW900 and EBC-1 cells compared with inhibitor NC transfected cells (Fig. 2B and C). The effects of miR-494 knockdown on cell apoptosis were further examined. As shown in Fig. 2D-F, knockdown of miR-494 promoted apoptosis in both SW900 and EBC-1 cells compared with the inhibitor NC. Taken together, these data suggest that miR-494 may function as an oncogene in LSCC.

To uncover the potential molecular mechanisms involved in the suppressive role of miR-494 inhibition in LSCC cells, the potential downstream targets of miR-494 were searched for using publicly available databases, including TargetScan and PicTar. According to bioinformatics analysis, PUMA-α was selected for further study due to its promotive role in apoptosis (27). The binding sites between miR-494 and PUMA-α are illustrated in Fig. 3A. To examine whether PUMA-α is a direct target of miR-494, a luciferase activity assay was conducted. As shown in Fig. 3B, miR-494 overexpression reduced the luciferase reporter activity of the PUMA-α 3'UTR, while inhibition of miR-494 had the opposite effect. Additionally, PUMA-α 3'UTR luciferase reporter activity was unaffected by point mutations in the miR-494-binding seed region. Moreover, it was observed that miR-494 overexpression inhibited the mRNA and protein expression levels of PUMA-α, whereas miR-494 knockdown enhanced the expression levels of PUMA-α (Fig. 3C and D). These results suggested that miR-494 suppressed the expression of PUMA-α at the transcriptional level.

In addition, RT-qPCR was used to determine the PUMA-α expression levels in 22 pairs of LSCC tissues and adjacent non-tumor tissues. It was demonstrated that the PUMA-α expression levels were significantly decreased in LSCC tissues compared with the adjacent non-tumor tissues (Fig. 3E). Correlation analysis revealed a strong negative correlation between the expression levels of miR-494 and PUMA-α mRNA levels in LSCC tissues (Fig. 3F). These data indicated that PUMA-α is a functional target of miR-494.

To investigate the function of PUMA-α in SW900 and EBC-1 cells, SW900 and EBC-1 cells were transiently transfected with pcDNA-PUMA-α for 24 h. As shown in Fig. 4A, PUMA-α expression levels were increased in SW900 and EBC-1 cells after pcDNA-PUMA-α transfection compared with the pcDNA-vector group. CCK-8 assay demonstrated that overexpression of PUMA-α significantly inhibited the proliferation of SW900 and EBC-1 cells compared with the pcDNA-vector group (Fig. 4B and C). Additionally, a significant increase in the percentage of apoptotic cells was also observed in the pcDNA-PUMA-α-transfected cells compared with the pcDNA-vector transfected cells (Fig. 4D-F). These findings suggest that overexpression of PUMA-α has a similar role to miR-494 inhibition in SW900 and EBC-1 cells.

Since PUMA-α was identified as the target gene of miR-494 in LSCC cells, whether PUMA-α is involved in miR-494-mediated roles in LSCC cells was determined. As shown in Fig. 5A, PUMA-α knockdown could rescue the suppression effect of miR-494 inhibitor on SW900 and EBC-1 cell viability. Furthermore, the data showed that miR-494 inhibition significantly increased the expression levels of pro-apoptotic protein PUMA-α and cleaved caspase-3, and also decreased the expression levels of anti-apoptotic protein Bcl-2 in the SW900 and EBC-1 cells compared with the Blank group. However, the effects of the miR-494 inhibitor were partially attenuated by the knockdown of PUMA-α (Fig. 5B-E). Subsequently, it was confirmed that the promotion of apoptosis mediated by miR-494 inhibition was also attenuated by the knockdown of PUMA-α (Fig. 5F). These results indicated that the knockdown of miR-494 induced apoptosis in LSCC cells by targeting PUMA-α.