Tissue samples were taken from a total of 60 patients (60–75 years, 69.5±5.2 years, 39 males and 21 females) with NSCLC between July 2016 and July 2017 from Weifang Yidu Central Hospital (Weifang, China). The inclusion criteria included: i) Patients diagnosed with NSCLC via biopsy; ii) >18 years of age; iii) histopathological stage confirmed as early or metastatic, adenocarcinoma or squamous cell carcinoma. Exclusion criteria included: i) Currently suffering from any other types of cancer; and ii) currently taking medications or any adjuvant treatments.

The tumor and paracarcinoma tissues (5 cm away from the tumor tissues) were removed by surgical section, and immediately placed in liquid nitrogen or 10% formalin. All samples were confirmed by pathological examination, and no radiotherapy or chemotherapy was performed prior to surgery. In addition, the present study was approved by the Ethics Committee of Weifang Yidu Central Hospital, and written informed consent was obtained from each participant prior to the study.

The A549 lung adenocarcinoma cell line was purchased from Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China) and cultured in Hyclone™ Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 80 U/ml penicillin and 0.08 mg/ml streptomycin (all from Gibco, Thermo Fisher Scientific, Inc.). Cells were cultured in an incubator at 37°C with an atmosphere of 5% CO₂. The medium was replaced every other day. Cells in the exponential phase of growth were used for subsequent experiments.

Cells were seeded in 6-well plates at a density of 2×10⁴ cells/well, and subsequently divided into three groups: i) Control group (Control); ii) negative control group (NC); and iii) miR-139-5p mimics group (mimics). The miR-139-5p mimics and control plasmids were obtained from New England Biolabs, Inc. The sequences used were as follows: miR-NC, 5′-GUGUAUUCUACAGUGCACGUGUCUCCAGUGU-3′; miR-139-5p mimics, 5′-GGCUCGGAGGCUGGAGACGCGGCCCUGUUGGAGUAAC-3′. Cells were transfected with 500 ng plasmids or miRusing 2.5 µl Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Following cell incubation for 6 h, the medium was replaced with fresh medium containing 10% FBS. Experiments were performed in triplicate 48 h post-transfection.

In a separate experiment, 1×10⁵ cells/well cells were seeded into 24-well plates and divided into four groups: i) Control group; ii) DDP group; iii) DDP + miR-NC mimics; and iv) mimics group (DDP + miR-139-5p mimics). Prior to transfection, all cells were treated with 1.0 µM DDP (cat. no. R00313; Rechemscience Co., Ltd.) and cultured for 2 h at 37°C, 5% CO₂, as described previously (38). Transfections were then performed at 37°C with 5% CO₂ for 24 h.

RT-qPCR was used to determine the differences in miR-139-5p and HOXB2 expression in tumor tissues and paracarcinoma tissues, and to investigate the mRNA expression levels of PI3K, AKT and caspase-3 in cells. Total RNA was extracted with TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and the concentration of RNA was determined using a NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). Subsequently, the RNA was reversed-transcribed into complementary DNA using a PrimeScript™ RT reagent kit (Takara Bio, Inc.) at 42°C for 10 min. To detect mRNA expression, RNA was reverse-transcribed into cDNA using a Mir-X™ miRNA First Strand Synthesis kit, and RT-qPCR was performed with a Mir-X™ miRNA RT-qPCR SYBR^® kit (both from Takara Bio, Inc.). SYBR-Green (10 µl), molecular grade water (6 ml), 1 µl each of the forward and reverse primers and 2 µl cDNA were mixed in each reaction on the PCR plate. Amplification was performed using the following thermocycling conditions: 10 min at 95°C, 50 cycles at 95°C for 15 sec, and 60°C for 60 sec. Each reaction was performed three times. Primer sequences used in the present study are shown in Table I. The comparative cycle quantification (Cq) method was used to analyze the RT-qPCR data, and 2^−ΔΔCq values were chosen to reflect the mRNA difference (39). The U6 gene was selected as an internal control for miR-139-5p, and GAPDH was used as an internal control for HOXB2, PI3K, AKT and caspase-3. All experiments were performed in triplicate. All primers were displayed in Table I.

The binding sites of miR-139-5p and HOXB2 were determined using the TargetScan online tool (40). To observe the binding of miR-139-5p to HOXB2 mRNA, the 3′-untranslated region (3′-UTR) segment of HOXB2 mRNA was amplified by PCR as aforementioned and inserted into the pGL3/luciferase vector (Promega Corporation). The wild-type 3′-UTR of HOXB2 mRNA was obtained by PCR amplification as aforementioned and cloned and inserted into pGL3/luciferase, as described for the wild-type 3′-UTR plasmid. Co-transfections of the HOXB2 3′-UTR or mutated HOXB2 3′-UTR plasmid with miR-139-5p mimics into the cells were performed using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Luciferase activity was measured 48 h after transfection using the Dual-Luciferase Reporter assay system (Promega Corporation) and normalized to Renilla luciferase. Experiments were performed in triplicate.

Cell proliferation was measured using a Cell Counting Kit-8 (CCK-8; ApexBio Technology LLC) assay according to the manufacturer's protocol. Transfected cells were seeded into 96-well plates at a density of 5,000 cells/well, and the cells were cultured for 6 h. Aliquots of 10 µl CCK-8 were added into each well, and the cell mixture was incubated at 37°C for 4 h. Optical density (OD) was detected at a wavelength of 450 nm.

Apoptosis of transfected A549 cells (early and late apoptosis) was measured by flow cytometry using Annexin V/propidium iodide (PI) double staining. Cells were seeded into 600 mm-diameter culture dishes at a density of 4×10⁴ cells/well. Cells were harvested, and washed twice with ice-cold PBS. Subsequently, the cells were resuspended in 300 ml binding buffer containing Annexin V (3 µl) and PI (3 µl) for 15 min at room temperature in the dark. Finally, cell apoptosis was quantified using an FACScan flow cytometer (Becton-Dickinson and Company) and analyzed using CellQuest software version 5.1 (BD Biosciences). During the course of these experiments, at least 10,000 cells for each sample were analyzed.

Total protein of the tissues and transfected cells was extracted using a protein extraction kit (Beyotime Institute of Biotechnology), and protein concentration was determined using a bicinchoninic acid kit (Thermo Fisher Scientific, Inc.). Equal amounts of protein (20 µg) were separated via SDS-PAGE on 10% gel, and subsequently transferred to a polyvinylidene difluoride membrane. After blocking for 2 h in 5% skimmed milk at room temperature, the membranes were incubated with the following primary antibodies overnight at 4°C: Anti-HOXB2 (cat. no. ab220390; Abcam), anti-phosphorylated (p)-PI3K (cat. no. ab32089; Abcam) anti-p-AKT (cat. no. 4058; Cell Signaling Technology, Inc.), anti-caspase-3 (cat. no. 9662; Cell Signaling Technology, Inc.), anti-cleaved-caspase-3 (cat. no. 9664; Cell Signaling Technology, Inc.) and anti-GAPDH (cat. no. 5174; Cell Signaling Technology, Inc.). The primary antibodies were used at 1:1,000 dilution. After washing three times with TBS with 0.3% Tween-20, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (1:5,000; cat. no. 7074; Cell Signaling Technology, Inc.) at 37°C for 30 min. Protein expression was visualized using ECL-Plus reagent (Santa Cruz Biotechnology, Inc.) and analyzed with the ChemiDoc™ XRS imaging system (Bio-Rad Laboratories, Inc.). GAPDH was used as the loading control.

All data are expressed as the mean ± standard error of the mean. Student's t-test was performed to test differences between two groups, whereas one-way analysis of variance, followed by Tukey's post hoc analysis, was used to examine differences among multiple groups. P<0.05 was considered to indicate a statistically significant difference.

The mRNA and protein expression levels of miR-139-5p and HOXB2 in tissues were determined using RT-qPCR and western blotting. As shown in Fig. 1A and B, miR-139-5p was significantly downregulated, whereas HOXB2 mRNA was significantly upregulated, in NSCLC tissues compared with paracarcinoma tissues (P<0.01). Furthermore, the expression of HOXB2 protein was significantly upregulated in tumor tissues, as shown in Fig. 1C and D.

As shown in Fig. 2, miR-139-5p was upregulated in the mimics group compared with the Control and NC groups (P<0.01) after transfection with miR-139-5p mimics.

Based on putative target sequences at nt positions 312–319 of the HOXB2 3′-UTR (Fig. 3A), HOXB2 was selected as a potential target of miR-139-5p. A luciferase reporter assay was used to confirm HOXB2 as a direct target of miR-139-5p. As shown in Fig. 3B, miR-139-5p led to a significant decrease in the luciferase activity of the wild-type HOXB2 3′-UTR, but not that of the mutant 3′-UTR, in A549 cells. Compared with the Control group, the expression of HOXB2 was significantly decreased in the cisplatin group (P<0.01). The protein expression of HOXB2 in DDP-treated A549 cells transfected with miR-139-5p mimics was significantly downregulated compared with the DDP + miR-NC group (P<0.01) (Fig. 3C and D).

Transfection with miR-139-5p mimics in DDP-treated A549 cells inhibits NSCLC cell proliferation and promotes apoptosis. As shown in Fig. 4A, cell proliferation was significantly suppressed by DDP treatment compared with the Control group. Furthermore, cell proliferation in the DDP + miR-139-5p group was significantly decreased compared with the DDP + miR-NC mimics group (P<0.01).

The results of the flow cytometry analysis are shown in Fig. 4B and C. Significantly increased rates of cell apoptosis were observed in the DDP group compared with the Control group. The apoptosis rate of A549 cells treated with miR-139-5p mimics combined with DDP was significantly elevated compared with the DDP + miR-NC mimics group (P<0.01).

The relative expression levels of PI3K, AKT and caspase-3 in cells were determined by RT-qPCR. As shown in Fig. 5A-C, the mRNA expression levels of AKT and PI3K were not altered, whereas caspase-3 mRNA expression was significantly upregulated in DDP-treated A549 cells following miR-139-5p overexpression.

As shown in Fig. 6A-D, the expression levels of p-AKT and p-PI3K were significantly decreased in the DDP group compared with the Control group (P<0.01). In addition, cells treated with DDP + miR-139-5p mimics revealed a significant decrease compared with the DDP + miR-NC mimics group (P<0.01). Furthermore, increased expression of cleaved-caspase-3 protein was also found. Therefore, miR-139-5p may be able to inhibit the expression of p-AKT and p-PI3K, while increasing cleaved-caspase-3 expression to enhance DDP sensitivity.