A total of 31 NSCLC tissues and 31 adjacent non-tumor tissues (5 cm from tumor tissue) were collected from 31 patients with NSCLC (16 men and 15 women; age range, 40–57 years; mean age: 47.21±8.33 years) who underwent tumor resection at the People's Hospital of Xinjiang Uygur Autonomous Region between February 2015 and January 2017. Patients who had received radiotherapy or chemotherapy prior to the surgery were excluded from the study. The tumors were staged according to the 8^th edition of American Joint Committee on Cancer Tumor-Node-Metastasis (TNM) system (16). This study was approved by the Ethics Committee of People's Hospital of Xinjiang Uygur Autonomous Region. Patients provided written informed consent.

The interaction between miR-21-5p and MAPK10 was predicted by TargetScan/miRanda. Starbase V2.0 project (http://starbase.sysu.edu.cn/) was used for the analysis of miR-21-5p and MAPK10 expression levels in NSCLC tumor and normal tissues, and the relation between miR-21-5p and MAPK10 in NSCLC tumor tissues from The Cancer Genome Atlas (TCGA) datasets (TCGA-LUAD and TCGA-LUSC) (17).

GEPIA software version 1.0 developed by Zhang Laboratory at the Peking University was used for the survival analysis based on TCGA data (960 patients) (18). Long-rank test was used to perform the analysis. For group cutoff, MAPK10 median expression threshold was chosen to split the cohort between high expression and low expression. P<0.05 was used for significance threshold.

The human NSCLC cell lines A549 and H1299 and the human lung epithelial cell line BEAS-2B were purchased from the American Type Culture Collection. Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (HyClone; GE Healthcare Life Sciences) and 1% penicillin/streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.) and placed at 37°C in a humidified incubator containing 5% CO₂.

miR-21-5p mimic and negative control (NC) were synthesized by Shanghai GenePharma Co., Ltd. pcDNA3-MAPK10 and pcDNA3 were purchased from Addgene (cat. no. 13758). A549 (1×10³/well) and H1299 (1×10³/well) cells were seeded in 96-well plates, cultured for 24 h and transfected with 50 nM miR-21-5p mimic, 50 nM miR-NC mimic, 2 µg pcDNA3 or 2 µg pcDNA3-MAPK10 using Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. After 48 h, A549 and H1299 cells were collected for subsequent experiments.

A549 and H1299 cells were treated with 8 µg/ml propofol (Sigma-Aldrich; Merck KGaA) for 48 h as previously described (13). Following propofol treatment for 48 h, and/or transfection with miR-21-5p mimic and/or pcDNA3-MAPK10 for 48 h, the A549 and H1299 cells were harvested for use in subsequent experiments.

Cell viability was determined using a Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc.). Briefly, A549 and H1299 cells (5×10³ cells/well) were seeded into 96-well plates. Following propofol treatment and/or transfection with miR-21-5p mimic and/or pcDNA3-MAPK10 for 48 h, 10 µl CCK-8 solution was added to each well. Plates were then incubated for 1 at 37°C in a humidified incubator containing 5% CO₂. Absorbance was read at 450 nm using a microplate reader (Bio-Rad Laboratories, Inc.).

Cell apoptosis was determined using an Annexin-V/Dead Cell Apoptosis kit (cat. no. V13242; Invitrogen; Thermo Fisher Scientific, Inc.). Briefly, A549 (1×10⁶ cells/ml) and H1299 cells (1×10⁶ cells/ml) were diluted in 100 µl binding buffer. Subsequently, Annexin V (5 µl) and propidium iodide (1 µl) were added to the cell suspensions. A549 and H1299 cells were then incubated in the dark at room temperature for 15 min. Subsequently, the cell apoptosis rate was determined by flow cytometric analysis using a FACSCalibur flow cytometer (BD Biosciences). Results were analyzed using FlowJo software (version 10.2; FlowJo LLC).

Total RNA was extracted from NSCLC and adjacent tissues, and from the BEAS-2B, A549 and H1299 cells using TRIzol reagent (Thermo Fisher Scientific, Inc.). miRNA was extracted from NSCLC and adjacent tissues, and from the BEAS-2B, A549 and H1299 cells using a miRNA Extraction kit (cat. no. 217004; Qiagen GmbH). RNA integrity was determined using a Nano Drop ND-1000 spectrophotometer. RT was carried out using a miRNA cDNA Synthesis kit (Qiagen GmbH) and TransScript First-Strand cDNA Synthesis SuperMix (Beijing TransGen Biotech co., ltd.). qPCR was carried out using SYBR-Green Premix Ex Taq II (Qiagen GmbH) with the ABI Prism 7500 Sequence Detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.). qPCR reactions were performed as follows: Initial denaturation at 95°C (10 min), then 95°C (5 sec), 63°C (30 sec) and 72°C (30 sec) for 40 cycles, followed by an extension step at 72°C (5 min). The sequences of the primers were as followed: MAPK10, forward 5′-CTTCCCAGATTCCCTCTTCC-3′, reverse 3′-GCTGGGTCATACCAGACGTT-5′; GAPDH, forward 5′-GAGTCAACGGATTTGGTCGT-3′, reverse 3′-TTGATTTTGGAGGGATCTCG-5′; miR-193-3p, forward 5′-ACACTCCAGCTGGGAACTGGCCTACAAAGT-3′, reverse 3′-TGGTGTCGTGGAGTCG-5′; miR-221-3p, forward 5′-CUUUGGGUCGUCUGUUACAUCGA-3′, reverse 3′-AUUGCUUCUCUUCAUGCAAGUCA-5′; miR-203a-3p, forward 5′-GAUCACCAGGAUUUGUAAAGU-3′, reverse 3′-GCAAAUUAAAUUUGGGCUUGUUCU-5′; miR-22-3p, forward 5′-UGUCAAGAAGUUGACCGUCGAA-3′ and reverse, 3′-AUGGUUGUUUCAAAUGCAUUGGG-5′; miR-125-5p, forward 5′-AGUGUUCAAUCCCAGAGUCCCU-3′, reverse 3′-CAGGCUUCAUGAUGUCUCCAUAUGAA-5′; miR-21-5p, forward 5′-AGUUGUAGUCAGACUAUUCGAU-3′, reverse 3′-CUUUACGAUGAAUUCAUUUCC-5′; U6, forward 5′-CGCTTCGGCAGCACATATACTAA-3′ and reverse 3′-TATGGAACGCTTCACGAATTTGC-5′. GAPDH was used as the endogenous control for MAPK10. U6 was used as the endogenous control for miR-21-5p. The expression levels of genes were calculated using the 2^−ΔΔCq method (19).

Proteins were extracted from NSCLC and adjacent tissues, and from the BEAS-2B, A549 and H1299 cells using RIPA lysis buffer (Beyotime Institute of Biotechnology) that contains protease inhibitors (Roche Diagnostics) on ice for 30 min. Following the quantification of protein concentration using BCA (Beyotime Institute of Biotechnology), proteins (20 µg/lane) were separated by 8% SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked with 5% non-fat milk for 2 h at room temperature and were incubated with primary antibodies against BAX (cat. no. 2772; 1:1,000), Bcl-2 (cat. no. 4223; 1:1,000), MAPK10 (cat. no. 2305; 1:1,000) and GAPDH (cat. no. 5174; 1:1,000) at 4°C overnight. Membranes were then incubated with goat-anti rabbit secondary antibody (cat. no. 7074; 1:2,000; Cell Signaling Technology, Inc.) at room temperature for 2 h. All antibodies were purchased from Cell Signaling Technology, Inc. Bands were detected using enhanced chemiluminescence substrate (EMD Millipore). The band intensities were analyzed using Image Lab™ (version 4.0; Bio-Rad Laboratories, Inc.).

The binding between miR-21-5p and the 3′-UTR of MAPK10 was predicted by TargetScan version 7.1 (http://www.targetscan.org/vert_71/) and validated by dual-luciferase reporter assay. The 3′-UTR of MAPK10 mRNA was amplified from the cDNA, which was obtained from 293T cells, and inserted into the pGL3-basic vector (Promega Corporation) between the KpnI and Xhol restriction enzyme sites. Two site mutations were introduced into the pGL3-MAPK10 3′-UTR-wild-type (WT) sequence to obtain the pGL3-MAPK10 3′-UTR-Mutant (MT) sequence by using a Quick Site-directed mutation kit (cat. no. 200518; Agilent Technologies, Inc.). 293T cells (1×10⁵/well) were plated in 24-well plates, co-transfected with pGL3-MAPK10 3′-UTR-WT (0.4 mg) or pGL3-MAPK10 3′UTR-MT (0.4 mg) together with miR-NC mimic (20 nM) or miR-21-5p mimic (20 nM) using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. After 48 h, the relative luciferase activity in each group was determined using the dual luciferase reporter system kit (Promega Corporation). Renilla luciferase activity (Promega Corporation) acted as the reference control.

Each experiment was repeated at least three times in the present study. Statistical analysis was carried out using GraphPad Prism 7.0 software (GraphPad Software, Inc.). Differences between two groups were analyzed by Student's t-test, and differences among three or more groups were analyzed by one-way analysis of variance followed by Newman-Keuls test. The association between MAPK10 expression and the clinicopathological characteristics of patients was analyzed using χ² test. Pearson's correlation test was performed to analyze the correlation between the miR-21-5p and MAPK10 mRNA levels. P<0.05 was considered to indicate a statistically significant difference.

The results from CCK-8 assay demonstrated that propofol significantly decreased A549 and H1299 cell viability compared with control group (Fig. 1A and B). Furthermore, results from flow cytometry indicated that propofol significantly increased NSCLC cell apoptosis compared with control group (Fig. 1C and D). In addition, western blotting analysis revealed that BAX/Bcl-2 ratio was significantly increased in NSCLC cells following propofol treatment, which was consistent with induction of cell apoptosis (Fig. 1E and F).

The levels of apoptotic-related molecules were examined following propofol treatment. As an important pro-apoptotic gene (20), a decrease in MAPK10 expression has been reported in breast, gastric, hepatocellular carcinoma and nasopharyngeal carcinomas (21,22). However, to the best of our knowledge, the effects of propofol on MAPK10 expression in NSCLC remain unknown, which was investigated in the present study. In the present study, propofol significantly increased MAPK10 mRNA and protein expression in A549 and H1299 cells compared with control group (Fig. 2A-C).

To the best of our knowledge, expression profile of MAPK10 in NSCLC remains unknown. In the present study, results from RT-qPCR and western blotting demonstrated that MAPK10 mRNA (Fig. 3A) and protein expression (Fig. 3B and C) was significantly lower in the human NSCLC cell lines A549 and H1299 compared with the human lung epithelial cell line BEAS-2B.

The expression profile of MAPK10 in NSCLC was subsequently identified via Starbase V2.0 project. The database showed that compared with the 56 normal samples, MAPK10 expression was significantly lower in the 526 tumor samples with lung adenocarcinoma (TCGA-LUAD; Fig. 3D). Compared with the 49 normal samples, MAPK10 expression was significantly lower in the 501 tumor samples with lung squamous cell carcinoma (TCGA-LUSC; Fig. 3E). In addition, MAPK10 mRNA and protein levels were significantly lower in tumor tissues from patients with NSCLC compared with adjacent normal tissues (Fig. 3F-H). These findings suggested that MAPK10 may serve a tumor-suppressive role in NSCLC.

The present study aimed to determine the oncogenes that target MAPK10. By using the online softwares miRanda and TargetScan, it was found that the 3′UTR of MAPK10 included the complementary sites for 21 miRNAs in miRanda and 15 miRNAs in TargetScan, and that six miRNAs overlapped, including miR-193-3p, miR-203a-3p, miR-22-3p, miR-125-5p, miR-221-3p and miR-21-5p (Fig. 4A).

Compared with the control group, propofol had no effect on miR-193-3p or miR-203a-3p expression level in A549 and H1299 cells; however, propofol treatment significantly increased miR-221-3p expression level in A549 cells and significantly decreased miR-221-3p expression level in H1299 cells. In addition, propofol significantly increased miR-22-3p expression level in A549 cells; however, it had no effect on miR-22-3p expression level in H1299 cells. Furthermore, propofol treatment significantly decreased and increased miR-125-5p level in A549 and H1299 cells, respectively. However, treatment with propofol downregulated miR-21-5p expression level in both A549 and H1299 cells (Fig. 4B and C). miR-21-5p was therefore selected in the present study and its role in NSCLC was further investigated.

The binding site between MAPK10 and miR-21-5p is illustrated in Fig. 5A. Before exploring the interaction between MAPK10 and miR-21-5p by dual-luciferase reporter assay, the present study aimed to verify the successful transfection of miR-21-5p mimic in A549 and H1299 cells. The results demonstrated a significantly higher miR-21-5p expression level in the miR-21-5p mimic group compared with the miR-NC group, confirming the successful transfection (Fig. 5B). Furthermore, results from dual-luciferase reported assay demonstrated that miR-21-5p mimic inhibited the relative luciferase activity in cells transfected with WT-MAPK10 but not with MT-MAPK10 in A549 and H1299 cells (Fig. 5C and D).

In addition, compared with the miR-NC mimic group, miR-21-5p mimic significantly inhibited the mRNA and protein levels of MAPK10 in A549 and H1299 cells (Fig. 5E-G).

The expression profile of miR-21-5p in NSCLC was identified via Starbase V3.0 project. As presented in Fig. 6A, miR-21-5p expression level was significantly increased in the 521 tumor tissues compared with the 20 normal samples in LUAD. Furthermore, as presented in Fig. 6B, miR-21-5p expression level was also significantly increased in the 475 tumor tissues compared with the 38 normal samples in LUSC.

Consistently, miR-21-5p expression level was higher in tumor tissues compared with adjacent normal tissues from the patients with NSCLC included in the present study (Fig. 6C). Furthermore, results from RT-qPCR demonstrated that miR-21-5p expression level was significantly increased in the human NSCLC cell lines A549 and H1299 compared with the human lung epithelial cell line BEAS-2B (Fig. 6D). Taken together, these results further indicated that miR-21-5p may act as an oncogene in NSCLC.

According to the results from Starbase V3.0 project, there was a negative correlation between miR-21-5p and MAPK10 expression in LUAD (n=512) and LUSC (n=475) (Fig. 7A and B). Furthermore, miR-21-5p is known to be associated with the DFS of patients with NSCLC (23). Subsequently, results from GEPIA software analysis demonstrated that a higher MAPK10 expression (n=480) was significantly associated with the prolonged DFS of patients with NSCLC in LUAD and LUSC, which was not the case for patients with lower MAPK10 expression (n=480; Fig. 7C).

Furthermore, miR-21-5p expression was negatively correlated with MAPK10 expression in the tumor tissues of patients with NSCLC included in the present study (Fig. 7D). In addition, the characteristics of patients with NSCLC are presented in Table I, and MAPK10 expression was demonstrated to be significantly associated with lymph node metastasis and Tumor-Node-Metastasis stage; however, MAPK10 expression was not associated with sex, age, histological type, EGFR mutation or KRAS mutation.

Subsequently, the present study investigated whether the effects of propofol on NSCLC cell viability and apoptosis were associated with miR-21-5p and MAPK10 expression. A549 and H1299 cells were therefore randomly divided into four different groups, including the control group, propofol group, propofol + miR-21-5p mimic group and the propofol + miR-21-5p mimic + pcDNA3-MAPK10 group.

In A549 and H1299 cells, increased expression of MAPK10 mRNA and protein was confirmed following transfection with pcDNA3-MAPK10 (Fig. 8A-C). Furthermore, results from CCK-8 assay demonstrated that transfection with miR-21-5p mimic attenuated the propofol-induced decrease in cell viability, which was reversed by pcDNA3-MAPK10 (Fig. 8D and E).

The results from flow cytometric assay demonstrated that transfection with miR-21-5p mimic attenuated the propofol-induced increase in A549 and H1299 cell apoptosis, which was reversed by pcDNA3-MAPK10 (Fig. 9A and B). In addition, results from western blotting revealed that the ratio BAX/Bcl-2 was consistent with an increase in cell apoptosis (Fig. 9C and D).