The present study was approved by the Medical Research Ethics Committee of The First Affiliated Hospital of Zhengzhou University for use of patient tissues as well as for the use of animals in research. All patients who participated in the present study provided written informed consent.

In the present study, 30 tissue samples from patients with advanced lung adenocarcinoma were obtained from CT-guided percutaneous puncture or bronchoscopic biopsy. Tissue samples were preserved in liquid nitrogen for the next isolation of RNA or protein. All patients accepted a chemotherapy regimen of pemetrexed combined with DDP (both from Qilu Pharmaceutical Co., Ltd., Jinan, China). The gender, age, level of differentiation, tumor-node-metastasis (TNM) stage and the reaction to two cycles of chemotherapy were recorded and evaluated according to WHO standards.

TRIzol (Invitrogen, Carlsbad, CA, USA) was used to isolate the total RNA of tissues and cells. Quantitative real-time PCR (qRT-PCR) was used to assess the relative expression level of miR-203 and DKK1 mRNA with the 7500 Fast PCR instrument (Applied Biosystems, Bedford, MA, USA). U6 (Invitrogen) and β-actin (Beijing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China) were used as internal controls for miR-203 and DKK1 mRNA, respectively. Primer sequences (Shanghai Biological Technology Co., Ltd., Shanghai, China) used for qRT-PCR are listed as follows: miR-203 RT-primer, 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCACTTTA-3′; forward, 5′-TCCGAGATCACCAGGATTTG-3′ and reverse, 5′-GTGCAGGGTCCGAGGT-3′; U6 RT primer, 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACGATT-3′; forward, 5′-TCCGATCGTGAAGCGTTC-3′ and reverse, 5′-GTGCAGGGTCCGAGGT-3′; DKK1 forward, 5′-TGTGCTAGACACTTCTGGTCCAA-3′ and reverse, 5′-TGATCTTTCTGTATCCGGCAAG-3′; β-actin forward, 5′-ATGATGATATCGCCGCGCTC-3′ and reverse, 5′-TCGATGGGGTACTTCAGGGT-3′. The relative expression levels in the tissues and cells were both assessed using ABI Power SYBR^®-Green PCR Master Mix (Applied Biosystems) and were calculated by evaluating the expression in normal human bronchial epithelium (NHBE) cells under internal control calibration represented by 2^−ΔΔCt.

The protein to be detected was obtained from lysed cells through the action of RIPA lysis buffer (Solarbio, Beijing, China). A BCA kit (Solarbio) was used to detect the protein concentration. SDS-PAGE gel (10%) was made for electrophoreses after protein specimens were added, and then the gel was transferred to a polyvinylidine fluoride (PVDF) membrane (Invitrogen) with a sandwich structure. Skimmed milk powder was used as a blocking agent to block the nonspecific binding site on the PVDF membrane for 2 h at room temperature. Subsequently, anti-human DKK1 rabbit polyclonal antibody at a 1:1,000 dilution (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was incubated overnight at 4̊C. Then, the PVDF membrane was washed 4 times with phosphate-buffered saline with 0.1% Tween-20 (PBST) (10 min/time). Then, goat anti-rabbit secondary antibody at a dilution of 1:10,000 (Santa Cruz Biotechnology, Inc.) was incubated for 2 h at room temperature. After antibody incubation and washing, ECL substrate was used to detect immunoreactive protein by a chemical luminescence imaging system (FluorChem E; ProteinSimple, San Jose, CA, USA). Quantified data were analyzed using IPP image analysis software. β-actin (Beijing Dingguo Changsheng Biotechnology Co., Ltd.) was used as an endogenous protein.

The human lung adenocarcinoma cell lines A549 and H460, and the NHBE cell line were purchased from the Cell Bank of Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences. All the cells were cultured in an incubator (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at constant temperature of 37̊C in 5% CO₂ in a water-saturated atmosphere. The cell culture medium was Dulbeccos modified Eagles medium (DMEM) high glucose medium with 10% fetal bovine serum (FBS) (both from Gibco Life Technologies, Carlsbad, CA, USA), and double antibody concentration of penicillin (100 U/ml) and streptomycin (100 µg/ml).

Dual luciferase reporter gene assay was used to verify whether miR-203 targets DKK1. First, point mutations at the seed region of the DKK1 3′ untranslated region (3′UTR) were established. These had complementary base pairing between miR-203 and the DKK1 mRNA 3′UTR sequence. Then, overlap PCR was used to collect mutant copies of the DKK1 3′UTR (Fig. 2A). Wild-type DKK1 3′UTR was amplified from human genomic DNA. Secondly, recombinant reporter gene vectors pmirGLO-wt-3′UTR DKK1 and pmirGLO-mut-3′UTR DKK1 were constructed (the combined vectors were purchased from Shanghai Biological Technology Co., Ltd.), and then co-transfected with scramble or miR-203 mimics (Shanghai GenePharma Co., Ltd., Shanghai, China) into A549 and H460 cells, respectively, using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer's recommendations. The confluence of the cells was 70–80%. The luciferase activity of the cells was determined using a luciferase assay kit (Promega, Madison, WI, USA) 24 h after transfection.

Pre-miR-203 and negative control lentivirus vectors were constructed and packaged by Shanghai GenePharma Co., Ltd. and transfected into A549 and H460 cells into 6-well plates, respectively. After transfection, the cells demonstrated puromycin resistance, and different concentrations of puromycin (Sigma-Aldrich, St. Louis, MO, USA) were used to select transfected and stably expressed cells 48 h after transfection. We finally used puromycin at the concentration of 2 µg/ml for 14 days to select the stably transfected cells. qRT-PCR was used to verify the expression of miR-203 in the three groups (miR-203, NC and blank).

A Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) assay was performed to examine cell growth after transfection in the three groups in accordance with the manufacturer's recommendations. After 24 h of treatment with different concentrations of DDP ranging from 4 to 20 µM, absorbance of the cells was measured using a fluorescence microplate reader, representing the proliferative activity of the cells. The inhibition rate was calculated using the following formula: Inhibition rate = (control group - experimental group)/control group × 100%. IC₅₀ is the half inhibitory rate, and stands for the concentration of DDP when the inhibition rate is 50%.

The apoptosis of A549 and H460 cells was measured by flow cytometry (BD Biosciences, San Diego, CA, USA) using an Annexin V-FITC/propidium iodide (PI) staining assay. Each group of cells (miR-203, NC and blank) was divided into two subgroups, one treated with 4 µM DDP and the other not treated with DDP. Apoptotic cells were calculated by gating PI- and Annexin V-positive cells using flow cytometry with fluorescence-activated cell-sorting (FACS). The activity of caspase-3/−7 in each subgroup was determined using a caspase-3/−7 kit (Promega).

Mice were bred in a temperature-controlled sterile environment with a 12-h light and dark cycle. Twenty BALB/c nude mice (female, 4–6 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Center (Beijing, China). Two groups of cells (5×10⁶ in 0.2 ml of DMEM) after lentivirus transfection (miR-203, NC) were subcutaneously injected under the left forelegs of the mice which were randomly distributed (n=10/group). In each group, half of the mice randomly received DDP (2 mg/kg in 0.2 ml of normal saline for 5 days) by intraperitoneal injection one week after implantation, and the others received 0.2 ml of normal saline alone. Thus, 4 groups were generated and the growth conditions of the xenografts were measured and recorded using an in vivo small animal imaging instrument every week for a total of 4 weeks, represented by the luciferase signals. Then, the mice were sacrificed by cervical dislocation. Each tumor was extracted and weighed.

siRNA-DKK1 was purchased from Shanghai GenePharma Co., Ltd. DKK1 lacking the 3′UTR (pcDNA3.1-DKK1 vector) was constructed by Shanghai Biological Technology Co., Ltd. siRNA-DKK1, miR-203 mimics, scramble and pcDNA3.1-DKK1 were transfected into A549/H460 cells alone or the combination using Lipofectamine™ 2000 in accordance with the manufacturers recommendations. The samples were divided into corresponding groups and CCK-8 assays (IC₅₀) were performed to assess the effects of miR-203 and DKK1 on DDP sensitivity in the A549/H460 cell lines.

Data were analyzed using SPSS 21 software. Data showing a normal distribution are expressed as mean ± standard deviation (SD). One-way ANOVA was used for comparison across three or more groups; a two-tailed unpaired Student's t-test was used to compare measurement data from pairs of independent samples. Pearson correlation analysis was used to evaluate the correlation of two groups. P<0.05 was considered to indicate a statistically significant result.

qRT-PCR was used to examine the expression levels of DKK1 mRNA and miR-203 in lung cancer tissues from 30 patients with advanced-stage lung adenocarcinoma; the clinical characteristics of the patients are shown in Table I. The results showed that in DDP-sensitive NSCLC samples, miR-203 was expressed at a higher level when compared with the level noted in the DDP-insensitive samples (P<0.05; Fig. 1B), while DKK1 mRNA was expressed at a lower level in the DDP-sensitive NSCLC samples (P<0.05; Fig. 1A). In addition, statistical analysis revealed that expression of DKK1 mRNA was negatively correlated with miR-203 (P<0.05; Fig. 1C; R²=0.532). The different levels of expression of miR-203 and DKK1 in the DDP-sensitive and -insensitive samples suggest that miR-203 is associated with the mechanism of cisplatin resistance.

In the present study, DKK1 was predicted to be a target gene of miR-203 by searching target gene prediction databases (microRNA.org, miRDB and TargetScan). A dual luciferase reporter gene assay was used to determine whether miR-203 targets DKK1. Western blot analysis showed that there was lower DKK1 expression in the miR-203 group than that in the blank and scramble groups of the A549 and H460 cells (P<0.05; Fig. 2B), which suggests that upregulation of miR-203 may lower the expression level of DKK1 protein. Under a dual reporter gene assay, the relative luciferase activity showed a nearly 1-fold decrease in wild-type DKK1 with upregulated miR-203. However, the relative luciferase activity showed no significant difference in the group with mutant DKK1 with upregulated miR-203 (P>0.05; Fig. 2C). The results were observed both in the A549 and H460 cells, which indicate that miR-203 may downregulate the expression level of DKK1 by binding to the 3′UTR of DKK1 mRNA.

qRT-PCR was used to examine the relative expression of miR-203 in the different groups after A549 and H460 cells were transfected with the lentiviral vectors. Cells in the group transfected with miR-203 revealed a ~7- to 8-fold higher expression of miR-203 when compared with the level in the blank and NC groups (Fig. 3A and B). Following treatment with different concentrations of DDP, ranging from 4 to 20 µM, the cells with miR-203 overexpression demonstrated a significant inhibition of cell growth not observed in the blank and NC groups (P<0.05). Inhibition of cell growth became more extensive upon increases in the concentration of DDP (Fig. 3C and D). Furthermore, the IC₅₀ value for the miR-203 group was significantly lower than that in the other two groups (P<0.05; Fig. 3E and F). IC₅₀ reflects the sensitivity of DDP in lung cancer cells. These results indicate that upregulation of miR-203 increased the sensitivity of lung adenocarcinoma A549 and H460 cells to DDP.

Cell apoptosis was detected using flow cytometry, and caspase-3/−7 activity was measured using a caspase-3/−7 kit. The results indicated that overexpression of miR-203 significantly increased cell apoptosis of the A549 and H460 cells with and without DDP treatment (P<0.05; Fig. 4A and B). Similar results were observed when the activity of caspase-3/−7 was examined (P<0.05; Fig. 4C and D). The increase in caspase-3/−7 activity was more significant following treatment with DDP. Upregulation of miR-203 was found to increase the activity of caspase-3/−7 and promote apoptosis in the A549 and H460 cells.

A nude mouse tumor xenograft model was established to study the effects of miR-203 on tumor growth. The luciferase signal detected by in vivo small animal imaging was in the present study considered representative of tumor growth. The tumor growth curve (Fig. 5A) clearly showed that overexpression of miR-203 inhibited the tumor growth, and this function was also observed under the treatment with DDP. Over time, the tumor inhibition effect of miR-203 and miR-203 combined with DDP was more significant. Moreover, tumors with upregulated miR-203 were more sensitive to DDP and showed a more pronounced decline in tumor weight (P<0.05; Fig. 5B). In this way, upregulation of miR-203 inhibits tumor growth and increases the sensitivity to DDP of the nude mouse tumor xenograft.

A great deal is known concerning the effects of miR-203 on the inhibition of cell growth and DDP sensitization. More experiments were designed to determine how miR-203 exerts its function. Both overexpression of miR-203 and knockdown of DKK1 resulted in the sensitization to DDP with a lower IC₅₀. In response to DKK1 knockdown, overexpression of miR-203 had no added effects on the sensitivity of the cells (P<0.05; Fig. 6A and C). Overexpression of miR-203 increased the sensitivity of the A549 and H460 cells to DDP compared to the control group, while DKK1 lacking 3′UTR decreased the sensitivity (P<0.05). In addition, miR-203 was not found to sensitize cells to DKK1 lacking the 3′UTR (P>0.05; Fig. 6B and D). In summary, miR-203 was found to exert the function of sensitization to DDP exclusively by targeting the 3′UTR of DKK1.