The lung cancer HCC827, A549, SK-LU-1 and A427 cell lines and non-cancerous MRC-5 cell line were purchased from the American Type Culture Collection (Manassas, VA, USA). All cell lines were maintained in Dulbecco's modified Eagle's medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.), antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin) and 2 mM glutamine. The cells were cultured in a CO₂ incubator (Thermo Fisher Scientific, Inc.) at 37°C with 98% humidity and 5% CO_2.

Total RNA was extracted from the lung cancer cells using RNeasy kits, mini RNA isolation kit (cat. no. 74104). (Qiagen GmbH, Hilden Germany). To reverse transcribe the cDNA, the Omniscript Reverse Transcriptase (RT) kit (cat. no. 205110; Qiagen GmbH) was employed using 1 µg extracted RNA. The cDNA was then used as template for qPCR, using the Taq PCR Master Mix kit (Qiagen GmbH), according to the manufacturer's protocol. The cycling parameters were 95°C for 20 sec, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. The relative quantification method (2^−ΔΔCq) was used to evaluate quantitative variation between the replicates examined as described previously (14). The amplification of actin was used as an endogenous control to normalize all data. Primer sequences of miR-204 are 5′-GCC AGA TCT GGA AGA AGA TGG TGG TTA GT-3′ (forward) and 5′-GGC GAA TTC ACA GTT GCC TAC AGT ATT CA-3′ (reverse), PCNA1 5′-GGC CGA AGA TAA CGC GGA TAC-3′ (forward) and 5′-GGC ATA TAC GTG CAA ATT CAC CA-3′ (reverse) and for actin 5′-AGA GCT ACG AGC TGC CTG AC-3′ (forward) and 5′-AGC ACT GTG TTG GCG TAC AG-3′ (reverse).

As the lung cancer A549 cells reached 80% confluence, they were transfected with 10 pmol negative control (NC) mimics (5′-UUC CCU UUG UCA UCC UAU GCC U-3′), miR-204 mimics (5′-UUC CCU UUG UCA UCC UAU GCC CU-3) and miR-204 inhibitor (5′-AAG AAA CCU GUC GCG AUA GCC AAC-3′), from Shanghai GenePharma (Shanghai, China; 10 pmol), small interfering (si)-negative control (si-NC) (5′-CGA ACU CAC UGG UCU GAC C-3′). (si) RNA-PCNA1 (5′-GGC ATT GCT AGA AAT TGA GAA-3) and pcDNA-PCNA1 (2 µg; Taijin Saier Biotechnology, Inc., Xiaozhan, China) using Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Further experiments were performed 24 h post transfection.

The cell viabilities of the A549 lung cancer cells were assessed using WST-1 colorimetric assays. Briefly, the lung cancer cells were seeded in 96-well plates at the density of 2×10⁵ cells/well. The cells were then incubated with 10 µl of WST-1 reagent (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 37°C for 4 h. The absorbance at 450 nm was then measured by a microplate reader at different time intervals (0, 12, 24, 48 and 96 h) to determine the viability of lung cancer cells. The colony formation assay was performed as described previously (9).

The nuclear morphology of the A549 lung cancer cells was assessed by fluorescence microscopy following treatment of the cells to cell-permeable Hoechst 33342 dye. A total of 10 fields with 100 cells/field were selected randomly for measurement of the cells with condensed nuclei. Then, Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining kit (BD Biosciences, San Jose, CA, USA) was used for the determination of the percentage of the apoptotic lung cancer cells, as described previously (10) A flow cytometer, (BD Biosciences) and BD FACSuite software version 1.0 (BD Biosciences) for were used for analysis.

The prediction of the miR-204 targets was performed with the online software TargetScan Version 7.2 (http://www.targetscan.org) using default parameters.

The cell migratory and invasive capabilities of the lung cancer cells were determined by Boyden Chamber assays as described previously (15).

A total of 36 BALB/c nude mice (4-week-old, male) weighing 18.25±1.5 g were obtained from the animal house of the Shengli Oilfield Central Hospital, (Dongying, China) maintained following the National Institutes of Health standards for the care and use of laboratory animals (16). The animals had ad libitum access to a pellet diet and water. Animals were maintained in well-ventilated rooms with a controlled environment, with a light: Dark (12-h) cycle and temperature of 28±2°C. The study was approved and supervised by the Ethics Committee of Shengli Oilfield Central Hospital (approval no. SOC-A77-204/17). The mice were randomly divided into two groups (n=18 in each group). A549 cells (~1.0×10⁷ cells/mouse), stably transfected with miR-204 or miR-NC, were subcutaneously injected into the back of the mice. Tumor volumes were monitored every 10 days after the tumors became visible. At the end of the study (65 days), the mice were sacrificed, and the weight and volume of the tumors were measured. The tumor volume was measured using the formula V = (W × W × L)/2, where W represents the width of the tumor and L represents the length of the tumor. The longest diameter observed for any tumor was 2 cm. Tumor tissues were then subjected to protein isolation for western blot analysis.

Immunohistochemical analysis was performed to examine the proliferation marker protein Ki-67 (Ki-67) protein expression in the xenograft tumors. Sections were deparaffinized by successive immersions in 100% xylene, 100% ethanol, 96% ethanol and 70% ethanol for 10, 10, 5 and 5 min, respectively. Endogenous peroxidase activity was inactivated with peroxidase blocking reagent (S2001; Dako; Agilent Technologies GmbH, Waldbronn, Germany) for 10 min. Antigen retrieval was achieved by exposure to 10 mM citrate buffer (pH 6.0) and autoclaving at 121°C for 15 min. Following blockade with 50 µl of 1% bovine serum albumin (Sigma-Aldrich, Merck KgaA) in TBS Tween 20 (TBST buffer; 50 mM Tris-HCl, 300 mM NaCl, 0.1% Tween-20) for 5 min at room temperature, the sections were incubated overnight with 40 µl of Ki-67 antibody (cat. no. 9449; 1:200; Cell Signaling Technologies, Inc., Danvers, MA, USA) pre-diluted 1:100 in TBST at 4°C in a humidified chamber. The sections were then washed with TBST and incubated with one drop of secondary antibody conjugated with horseradish peroxidase (HRP; cat. no. K4061; Dako; Agilent Technologies GmbH) for 60 min at room temperature. Following washing, the sections were incubated with one drop of chromogenic 3,3′-diaminobenzidine substrate (K3468; Dako; Agilent Technologies GmbH) for 15 min at room temperature. Slides were examined under a Leica, DM300 light microscope (Leica Microsystems, Wetzlar, Germany).

The lung cancer A549 cells were lysed using ice-cold hypotonic buffer (Invitrogen; Thermo Fisher Scientific, Inc.). Following estimation of the protein concentrations in each of the cell extracts by BCA assay, 40 µg of proteins from each sample were loaded and separated by SDS-PAGE (10%). This was followed by transference to nitrocellulose membranes. The membranes were blocked in blocking buffer (10 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20) containing 5% non-fat milk for 1 h at room temperature and then incubated with the primary antibody (PCNA-1; cat. no., sc-56; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; 1:1,000) for 24 h at 4°C. The membranes were then incubated with HRP-conjugated anti-rabbit secondary antibody (cat. no. sc-2372; Santa Cruz Biotechnology, Inc. 1:1,000) for at 24°C for 1 h. The visualization of the proteins was performed using an enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc.).

Statistical analysis was performed using a one way analysis of variance followed by Tukeys's post hoc test using SPSS software package v9.05 (SPSS, Inc., Chicago, IL, USA). Data are presented as the mean ± standard deviation, and P<0.05 was considered to indicate a statistically significant difference.

The expression of miR-204 was assessed in 4 different lung cancer cell lines and 1normal cell line by RT-qPCR analysis. The results revealed that the expression of miR-204 was significantly downregulated in all the lung cancer cell lines (P<0.05). The expression of miR-204 in lung cancer cells lines was decreased by 4–8-fold compared within the normal MRC-5 cells (Fig. 1). Among the lung cancer cell lines, the highest expression was observed in the SK-LU-1 cell line, followed by A427 and HCC827 cell lines. The expression of miR-204 was highly downregulated in A549 lung cancer cells; it was decreased by almost 8-fold compared with the normal MRC-5 cells. As this was the lowest expression of miR-204 observed, the A549 cell line was used for subsequent experiments.

To elucidate the role of miR-204 in lung cancer, it was overexpressed in A549 lung cancer cells by the transfection of miR-NC and miR-204 mimics. The overexpression of miR-204 was confirmed by RT-qPCR, which demonstrated a ~6-fold increase in the expression of miR-204 in miR-204 mimic-transfected cells (Fig. 2A). It was then identified that the overexpression of miR-204 inhibited the proliferation of lung A549 cancer cells (Fig. 2B). These results were also complemented by the results of the colony formation assays, wherein it was observed that miR-204 overexpression suppressed the colony-forming potential of the A549 lung cancer cells (Fig. 2C). Additionally, miR-204 over-expression caused an inhibition of the migratory and invasive capabilities of the A549 lung cancer cells (Fig. 2D and E). It was also observed that the overexpression of miR-204 in A549 cells triggered apoptosis, as indicated by Hoechst 33342 dye and Annexin V-FITC/PI staining (Fig. 3A and B).

To elucidate the target of the miR-204 in lung cancer cells, miR-204 was subjected to target scanning. A total of five targets for miR-204 were identified, which included tyrosine-protein kinase 2 (JAK2), runt-related transcription factor 2, B-cell lymphoma 2, ubiquitin carboxyl-terminal hydrolase 47 and PCNA-1. The majority of the five targets exhibited similar rankings, but most of these target proteins have been studied in detail, with the exception of PCNA-1. Therefore, PCNA-1 was selected for additional analysis as a prospective target of miR-204 (Fig. 4A). It was demonstrated that the expression of PCNA-1 was significantly upregulated in all the lung cancer cell lines, and that the overexpression of miR-204 caused significant downregulation of PCNA-1 in A549 (Fig. 4B and C). To confirm PCNA-1 as a target of miR-204, the expression of PCNA-1 was suppressed in A549 lung cancer cells by transfection with small interfering (si)-NC or si-PCNA-1, which resulted in a 5-fold decrease in the expression of PCNA-1 in A549 cells (Fig. 5A). The results revealed that the suppression of PCNA-1 caused an inhibition of A549 cell proliferation, migration and invasion (Fig. 5B–E). Additional suppression of miR-204 in A549 cells transfected with si-PCNA-1 did not rescue the effects of PCNA-1 suppression on the proliferation, migration and invasion of the cells (Fig. 6A–C). However, the overexpression of PCNA-1 in lung cancer A549 cells transfected with miR-204 mimics promoted the proliferative, migratory and invasive capabilities of these cells (Fig. 6D and E).

Next, the effect of miR-204 overexpression was also examined on lung tumor growth in vivo. The miR-NC or miR-204 mimic-transfected A549 cells were subcutaneously injected into male BALB/c nude mice. The results revealed that miR-204 overexpression significantly suppressed the tumor weight and volume in vivo (Fig. 7A–C). In addition, miR-204 overexpression in lung tumors caused a considerable inhibition of PCNA-1 expression (Fig. 7D). Ki-67 immunostaining revealed that the miR-204 overexpression group exhibited decreased numbers of proliferative cells compared with the NC group, which indicated the antiproliferative effects of miR-204 overexpression (Fig. 7E).