Tumor tissues were collected from 45 patients with NSCLC between August 2016 and August 2017 at Linyi Central Hospital (Linyi, China). The clinicopathological features (including gender, age, tumour stage, and tumor, node and metastasis staging) of patients with NSCLC enrolled in our study were presented in Table I. The tumor and the paracancerous tissues were fixed in 10% formalin for 48 h at room temperature. Tumor and non-tumor samples were confirmed by pathological examination. No patients received chemotherapy or radiotherapy prior to surgery. In addition, the present study was approved by the ethics committee of Linyi Central Hospital, and written informed consent was obtained from all patients prior to enrolment.

A549 cells were purchased from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China) and cultured in RPMI-1640 medium (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in 5% CO₂ in a humidified incubator.

The cells were divided into three groups: Control, negative control (NC) and miR-654-3p mimics. The cells in the control group were incubated in medium without treatment; cells in the NC group were transfected with miR-NC mimics, and cells in miR-654-3p mimics group were transfected with miR-654-3p mimics. All cells were incubated at 37°C in a humidified 5% CO₂ atmosphere for 48 h. The miR-654 mimics and control were obtained from New England Biolabs, Inc. (Ipswich, MA, USA). The cells were seeded in triplicate in 24-well plates and transfected with 500 ng miR-654-3p mimics or miR-NC mimics using 2.5 µl Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc.). After 6 h post-transfection, the medium was replaced with fresh medium containing 10% FBS. The sequences of the transfected mimics were the following: miR-654 mimics forward, 5′-UGGUGGGCCGCAGAACAUGUGC-3′; miR-654 mimics reverse, 5′-ACAUGUUCUGCGGCCCACGAAU-3′; NC mimics forward, 5′-UUCUCCGAACGUGUCACGUUU-3′; NC mimics reverse, 5′-ACGUGACACGUUCGGAGAAUU-3′.

RT-qPCR was employed to determine miR-654-3p and mRNA expression levels. An RNeasy Mini kit (Qiagen GmbH, Hilden, Germany) was used to isolate RNA from cells or tissues according to the manufacturer's protocol, and theconcentration of RNA was determined using a NanoDrop 2000 spectrophotometer (NanoDrop; Thermo Fisher Scientific, Inc., Wilmington, DE, USA). An M-MLV reverse transcriptase cDNA synthesis kit (Thermo Fisher Scientific) was applied to synthesize cDNA via RT; samples were incubated at 43°C for 30 min, 97°C for 5 min and 5°C for 5 min. The PrimeScript™ RT-PCR kit (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was used for qPCR. The thermocycling conditions were as follows: 95°C for 6 min (denaturation), 94°C for 30 sec (initiation), 60°C for 30 sec (annealing), and 75°C for 1.5 min (elongation) for 36 cycles. U6 was used to normalize the expression of miR-654-3p and GAPDH was used as an internal reference for mRNA expression. This experiment was performed in triplicate. Expression was quantified using the 2^−ΔΔCq method (16). The sequences of the primers used are as follows: miR-654-3p, forward: 5′-GGGATGTCTGCTGACCA-3′; reverse: 5′-CAGTGCGTGTCGTGGA-3′; U6, forward: 5′-CTCGCTTCGGCAGCACA-3′, reverse: 5′-AACGCTTCACGAATTTGCGT-3′; Bcl-2-associated × protein (Bax), forward: 5′-CACCAGCTCTGAACAGATCATGA-3′, reverse: 5′-TCAGCCCATCTTCTTCCAGATGT-3′; B cell lymphoma-2 (Bcl-2), forward: 5′-CACCCCTGGCATCTTCTCCTT-3′, reverse: 5′-AGCGTCTTCAGAGACAGCCAG-3′; RASAL2, forward: 5′-TGTTCTGTCCTTGAGCCAGT-3′, reverse: 5′-TCCACCTCAGACATCACCAA-3′; and GAPDH, forward: 5′-GCACCACCAACTGCTTAGC-3′, reverse: 5′-GGCATGGACTGTGGTCATGAG-3′.

Protein expression in tissues and cells was investigated by western blotting. Cells and tissue samples were lysed in radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc.) and the total protein concentration was determined using a Bicinchoninic Acid assay (Thermo Fisher Scientific, Inc.). GAPDH was used as a loading control; 2 µg protein was loaded for 15% SDS-PAGE, and then transferred to a polyvinylidene fluoride membrane (Thermo Fisher Scientific, Inc.). The membrane was blocked with 5% skimmed milk for 2 h at 37°C, then incubated with the following primary antibodies: Anti-RASAL-2 (cat. no. ab216127; 1:5,000; Abcam, Cambridge, UK), anti-Bcl-2 (cat. no. ab209039; 1:5,000; Abcam), anti-Bax (cat. no. ab32503; 1:5,000; Abcam) and anti-GAPDH (cat. no. ab181602; 1:10,000; Abcam) for 1 h at room temperature. The membrane was then incubated with a horseradish peroxidase-conjugated anti-rabbit antibody (ab6721; 1:5,000; Abcam) for 45 min at room temperature. Subsequently, an enhanced chemiluminescence western blotting kit (Pierce; Thermo Fisher Scientific, Inc.) was used to visualise the proteins. The gray values were obtained using ImageJ image analysis software (National Institutes of Health, Bethesda, MD, USA). This experiment was performed in triplicate.

Transfected cells were seededinto 24-well plates (2×10⁶ cells/ml) and incubated at 37°C with 5% CO₂. Subsequently, an MTT Cell Viability Assay kit (Sigma-Aldrich; Merck KGaA) was used, according to the manufacturer's protocols. The optical density was measured at a wavelength of 570 nm using a microplate reader at 0, 12, 24 and 48 h. The experiment was conducted in triplicate.

Transfected cells were plated into 24-well plates (2×10⁶ cells/ml) and incubated at 37°C with 5% CO₂ for 72 h. Then, the cells were treated with propidium iodide (10 µg/ml; Sigma-Aldrich; Merck KGaA) and Annexin V-fluorescein isothiocyanate (50 µg/ml; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) in the dark for 15 min at room temperature. Subsequently, the stained cells were analyzed using a FACScanflow cytometer (Becton, Dickinson and Company) and Diva software (version 8.0; Becton, Dickinson and Company)to determine the apoptotic rate.

Bioinformatics analysis using TargetScan 7.1 (http://www.targetscan.org/vert_71/) revealed that RASAL2 was a target of miR-654-3p. A luciferase reporter assay was performed to verify whether RASAL2 was a direct target of miR-654-3p. The cells were seeded in 96-well plates (2×10³ cells/well) and incubated at 37°C with 5% CO₂ for 24 h. Subsequently, RASAL2-3′UTR-wild type (WT) and RASAL2-3′UTR-mutant (MUT) plasmids (2.5 µg, Addgene, Inc., Cambridge, MA, USA), miR-654-3p mimics and miR-NC were transfected to cells using 2.5 µl Lipofectamine 2000. A BioLux^®Gaussia Luciferase Reporter Assay kit (New England Biolabs, Inc.) was utilized for the analysis of luciferase activity, according to the manufacturer's protocols. Luciferase activity was normalized to that of Renilla luciferase.

All data are presented as the mean ± standard deviation and were analyzed using GraphPad Prism version 5.01 (GraphPad Software, Inc., La Jolla, CA, USA). A paired Student's t-test was used to examine differences between the two groups. An unpaired Student's t-testwas used to compare the luciferase activity between NC and miR-654-3p groups. One-way analysis of variance followed by a Newman-Keuls post-hoc test was used to examine differences among three groups. The correlation between miR-654-3p and RASAL2 was analysed using Pearson's correlation coefficient. A χ² test was performed to analyze the data presented in Table I. P<0.05 was considered to indicate a statistically significant difference.

The expression levels of miR-654-3p and RASAL2 were determined by RT-qPCR and western blotting. As presented in Fig. 1A and B, significantly downregulated expression of miR-654-3p and upregulated expression of RASAL2 were observed in the tumor tissues compared with control. A negative correlation between miR-654-3p and RASAL2 expression was identified (Fig. 1C). Furthermore, the expression of RASAL2 protein was notably upregulated in tumor tissues compared with in the control (Fig. 1D and E). In addition, the expression levels of miR-654-3p and RASAL2 were significantly associated with the clinicopathological features of NSCLC, including tumor size and lymph node metastasis (Table I).

After 6 h following transfection with miR-654-3p mimics and a 48-h incubation at 37°C, miR-654-3p expression was analyzed by RT-qPCR. As presented in Fig. 2, miR-654-3p expression was significantly upregulated in the mimics group compared with the control groups.

After 48 h incubation at 37°C with 5% CO₂, the expression levels of RASAL2, Bax and Bcl-2 were determined by RT-qPCR and western blotting. As presented in Fig. 3A and B, the mRNA expression levels of RASAL2 and Bcl-2 were significantly decreased in the miR-654-3p mimics group compared with the control groups, respectively. Furthermore, the mRNA expression levels of Bax were significantly upregulated in the mimics group compared with the control groups (Fig. 3C). Additionally, compared with the NC group, significantly decreased expression of RASAL2 and Bcl-2, and increased expression of Bax protein were observed in response to miR-654-3p transfection (Fig. 3D and E). Therefore, miR-654-3p may inhibit the expression of RASAL2 and Bcl-2 expression, while inducing that of Bax.

Cell viability was determined at 0, 12, 24 and 48 h. As presented in Fig. 4, cell viability was significantly inhibited in the miR-654-3p mimics group compared with the control groups at 24 and 48 h. These results suggest that cell viability is suppressed by miR-654-3p upregulation.

As presented in Fig. 5, a significantly increased rate of apoptosis was observed in the miR-654-3p mimics group compared with the control groups (Fig. 5A and B). Thus, overexpression of miR-654-3p may induce apoptosis by targeting RASAL2.

Bioinformatics analysis using TargetScan suggested that RASAL2 was a target of miR-654-3p (Fig. 6A). Additionally, the luciferase activity was significantly decreased in the RASAL2-3′UTR-WT group treated with miR-654-3p mimics compared with the control (Fig. 6B), which indicated that RASAL2 was a target of miR-654-3p.