Paired human LAC samples and adjacent normal tissues (≥3 cm away from the tumor) were obtained from 72 patients (male=40; female=32; average age, 43.79±6.33; range 35–61 years) who received surgical resection between January 2013 and May 2014 at the Department of Respiratory, The First Hospital Affiliated to the Xinxiang Medical College (Weihui, China). All surgical specimens were snap-frozen in liquid nitrogen and stored at −80°C following resection until RNA extraction was performed. All patients did not receive chemotherapy or radiotherapy prior to surgery. The diagnoses of these tissue samples were confirmed by pathologists in The First Hospital Affiliated to the Xinxiang Medical College. Written informed consent was obtained from all patients prior to enrolment in the present study the study protocol was approved by the Ethics Committee of The First Hospital Affiliated to the Xinxiang Medical College.

Total RNA from fresh tissues was isolated using an RNA Extraction Kit (Qiagen, Inc., Valencia, CA, USA), according to the manufacturer's protocol. Complementary DNA was obtained using specific miRNA primers for miR-383-5p (reverse transcription primer: 5′-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACAGCCAC-3′; protocol: 30 min at 16°C, 30 min at 42°C, and 5 min at 85°C) Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) using the miScript Reverse Transcription Kit (Qiagen, Inc.). The expression level of miR-383-5p was quantified using miRNA specific TaqMan miRNA Assay kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). qPCR was performed using the SYBR PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) and ABI 7500 Fast (Applied Biosystems; Thermo Fisher Scientific, Inc.). PCR was performed as follows: 25 cycles of 10 min at 98°C, 10 sec at 98°C, 10 sec at 55°C and 20 sec at 72°C, with a final extension at 72°C for 5 min. The PCR primers used were as follows: miR-383-5p forward, 5′-GGGAGATCAGAAGGTGATTGTGGCT-3′ and reverse, 5′-CAGTGCGTGTCGTGGAGT-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′. The relative quantification of miR-383-5p was determined using the 2^−ΔΔCq method (13), with U6 small nuclear (sn)RNA used as the endogenous control to normalize the data.

A549 and H1299 human lung adenocarcinoma cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA) and grown in RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Inc.) and 100 U/ml penicillin/streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.) in a 37°C humidified incubator containing 5% CO₂.

The miR-383-5p mimic and control miRNA mimic were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). The cells were treated with 50 nM GMR-miR™ mixed with the miRNA mimic (30 nM) using Lipofectamine™ 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol, when the density of cells was <70%. All the assays were performed 48 h after transfection.

A549 and H1299 cells were plated onto 24-well plates at a density of ~1×10⁴ cells/well. Cells were serum-starved for 12 h at 37°C followed by addition of serum and [³H]thymidine (2 Ci/mM) for 4 h. Subsequently, the cells were fixed in 0.3 ml 10% trichloroacetic acid and lysed in 100 µl 0.2 M NaOH/0.2% SDS for 10 min at 25°C. The radioactivity was detected using a liquid scintillation counting system (Beckman Coulter, Inc., Brea, CA, USA).

A total of 1×10⁶ A549 or H1299 cells were harvested, washed with ice-cold PBS and fixed in 70% ice-cold ethanol at 4°C overnight. The fixed cells were washed with PBS and resuspended in 1 ml PBS supplemented with 100 µg/ml bovine pancreatic RNase A (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and 40 µg/ml propidium iodide (PI; Sigma-Aldrich; Merck KGaA) for 30 min at 4°C, cell cycle analysis was performed with a Becton Dickinson FACSCalibur cytometer (BD Biosciences, Inc., Franklin Lakes, NJ, USA). Cell cycle analysis was performed using ModFit software (version 3.2.1, Verity Software House, Topsham, ME, USA).

Cell apoptosis were detected using the Annexin V-Fluorescein Isothiocyanate (FITC) Apoptosis kit (Merck KGaA), according to the manufacturer's protocol. Briefly, cells were washed with ice-cold PBS and incubated in 500 µl ice-cold 1X binding buffer containing 2.3 ml Annexin V-FITC for 10 min at 4°C, followed by 10 min of incubation at room temperature in the dark. Subsequently, all cells were resuspended in 500 ml ice-cold 1X binding buffer supplemented with 5 ml PI at room temperature for 15 min. Annexin V-FITC and PI signals were detected using a flow cytometer (FACSCalibur™;BD Biosciences, San Jose, CA, USA).

Total protein lysates were extracted using radioimmunoprecipitation assay lysis buffer (Sigma-Aldrich; Merck KGaA) at 4°C for 30 min and detected using a bicinchoninic acid kit (Pierce; Thermo Fisher Scientific, Inc.). Protein (30 µg) was separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Subsequently the membranes were blocked by 5% BSA (Sigma-Aldrich, Merck KGaA) at room temperature for 1 h, washed with TBST 3 times and then probed with primary antibodies against the following: CIP2A (cat no. NB110-59722; dilution, 1:1,000; duration, 4°C overnight, Novus Biologicals, LLC, Littleton, CO, USA) and GAPDH (cat. no. 5174; dilution, 1:2,000; duration, 4°C overnight; Cell Signaling Technology, Inc., Danvers, MA, USA), which was used as a control. The membranes were washed 3 times with TBST and incubated with horseradish peroxidase-linked secondary goat anti-rabbit antibody (cat. no. 1662408; dilution, 1:3,000; duration, 37°C for 1 h; Bio-Rad Laboratories, Inc., Hercules, CA, USA). The bands were visualized using an enhanced chemiluminescence detection reagent by the ChemiDoc XRS system (Bio-Rad Laboratories, Inc.).

For the immunohistochemistry assay, 10% formalin-fixed paraffin-embedded tissue sections (5 µm thick) were deparaffinized and rehydrated in graded alcohol (50, 65, 75, 85, 95 and 100%) at room temperature for 1 h. An endogenous antigen-retrieval procedure was performed using 10 mM citrate buffer, pH 6.0, at 95°C for 10 min. Subsequently, slides were washed with PBS and incubated with primary antibodies at 4°C in a humidified chamber overnight. The primary anti-CIP2A monoclonal antibody (cat. no. NB110-59722; dilution, 1:400; Novus Biologicals, LLC) were incubated at 4°C overnight. This was followed by incubation with biotinylated goat anti-rabbit serum IgG (cat. no. 21537; dilution, 1:500; Novus Biologicals, LLC, Littleton, CO, USA). Subsequently, the antigen-antibody reaction was visualized using diaminobenzidine serving as the chromogen under an Olympus CX41 microscope and counted in 5 high-power fields (magnification, ×200).

The 3′UTR region of CIP2A was amplified from human genomic DNA and inserted into the pmirGLO vector (Promega Corporation, Madison, WI, USA) with HindIII and EcoRI restriction sites at the 3′ end of the luciferase gene in order to construct the luciferase reporter plasmids. For sequence point mutation, site-directed mutagenesis of potential target sites in the CIP2A 3′UTR were performed using a QuikChange Site-Directed Mutagenesis kit (Promega). The CIP2A recombinant plasmid (lacking 3′UTR) was amplified by PCR with the following primers: Forward, 5′-CTGCCATCATGCCGATGTTCAT-3′ and reverse, 5′-CGGCTCTTAGGCGAAGGTG-3′ and the PrimeSTAR GXL DNA Polymerase (Takara Biotechnology Co., Ltd., Dalian, China). PCR thermocycling conditions were as follows: 30 cycles of 30 sec at 98°C, 90 sec at 56°C and 45 sec at 72°C with a final extension at 72°C for 5 min. A LightCycler^® instrument (Roche Diagnostics GmbH, Mannheim, Germany) was used for the PCR. The resulting PCR amplicons of CIP2A were cloned into the T vector (Promega). The correct clones were confirmed by sequencing.

The following online miRNA target prediction algorithms were used to evaluate the potential target genes of miR-383-5p: TargetScan 6.2 database (http://www.targetscan.org/vert_71/). The target prediction runs were performed with a context percentile of 95% and a conserved method (14). The list of potential target gene and binding site was available by searching its database.

For the luciferase reporter assay, A549 and H1299 cells were seeded into a 24-well plate at density of 10⁵ and co-transfected with 50 nM miR-338-5p mimic or control mimic and 200 ng reporter recombinant plasmid using Lipofectamine™ 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. At 48 h after transfection, luciferase activity was determined using a dual-luciferase system kit (Promega). Firefly luciferase activity was normalized against Renilla luciferase gene activity.

Data analyses were performed using SPSS software (version 15.0; SPSS, Inc., Chicago, IL, USA). Results are presented as the mean ± standard deviation. Differences between two groups were tested by Student's t-test, and differences among three or more groups were measured by one-way analysis of variance. Count data were analyzed using Fisher's exact tests. Univariate survival analysis was performed using the Kaplan-Meier estimator method and the log-rank test. P<0.05 was considered to indicate a statistically significant difference.

The expression levels of miR-383-5p in 72 LAC tissues and adjacent non-tumorous tissues were determined using RT-qPCR. The associations of miR-383-5p expression level with various clinicopathological parameters of patients with LAC are summarized in Table I. These results indicated that miR-383-5p was significantly downregulated in 58.3% (42/72) of the LAC tissues examined in comparison with the matched adjacent non-cancerous tissues, 31.9% (23/72), from the same patients. The results demonstrated that there were significant associations between miR-383-5p downregulation and unfavorable variables, including tumor size (P=0.0309) and differentiation (P=0.0299). Furthermore, patients with low miR-383-5p expression levels had a significantly decreased overall survival and disease-free survival rate compared with those with high miR-383-5p expression levels (Fig. 1A and B).

In order to investigate the role of miR-383-5p in human LAC proliferation and apoptosis, miR-383-5p was overexpressed in human A549 and H1299 LAC cells by transfection with an miR-383-5p mimic. The expression level of miR-383-5p in transfected cells revealed a significant increase compared with transfected control cells, which indicated that mir-383-5p was successfully transfected into LAC cells (Fig. 2A). [³H]thymidine incorporation assays and miR-383-5p overexpression significantly inhibited A549 and H1299 cell proliferation (Fig. 2B). To further elucidate the anti-proliferative mechanism underlying miR-383-5p in LAC cells, the cell cycle and apoptosis were analyzed. As presented in Fig. 2C and D, miR-383-5p overexpression in LAC cells significantly increased the proportion of cells in the G₀/G₁ cell cycle phase and decreased the proportion of cells in S phase compared with the control group. Furthermore, promotion of cell apoptosis was observed in LAC cells following transfection with miR-383-5p.

Using open access software TargetScan 6.2 database (http://www.targetscan.org/vert_71/), CIP2A was selected as a preferred candidate target gene of miR-383-5p. Immunohistochemistry analysis demonstrated that the expression levels of CIP2A in LAC with high miR-383-5p expression levels were significantly decreased compared with those with low miR-383-5p expression level (Fig. 3A). Western blotting revealed that miR-383-5p mimic significantly decreased the expression level of CIP2A protein in LAC cells (Fig. 3B). A target prediction program (TargetScan) was used to identify putative miRNA-binding sites in the 3′UTR of CIP2A. The potential wild-type and mutant CIP2A 3′UTR fragment were cloned into a luciferase reporter gene system (Fig. 3C). LAC cells were co-transfected with a vector containing wild-type/mutant 3′UTR of CIP2A and miR-383-5p mimic (or control mimic). Overexpression of miR-383-5p in the two LAC cell lines induced a significantly decreased luciferase activity for wild-type, whereas no alteration in luciferase activity was detected with the mutant CIP2A 3′UTR luciferase reporter plasmid (Fig. 3D).

To further investigate miR-383-5p repression of LAC cell proliferation mediated by CIP2A, A549 and H1299 cells were co-transfected with miR-383-5p mimic (or control mimic) with CIP2A constructs lacking the respective 3′UTR or empty vector. Western blotting was performed to evaluate the expression levels of CIP2A protein. As presented in Fig. 4A, the co-transfection rescued the decreased expression level of CIP2A protein in LAC cells that was induced by miR-383-5p. Additionally, restoration of CIP2A expression level reversed the inhibitory effects of exogenous miR-383-5p on proliferation, resulting in a significant increase in DNA synthesis (Fig. 4B). Similarly, re-expression of CIP2A exhibited an apparent rescued S cell cycle phase and decreased the apoptosis rate in LAC cells (Fig. 4C and D).