Human NSCLC tissues (n=28) and adjacent non-tumorous lung tissues (n=7) were obtained from NSCLC patients admitted to the Tumor Hospital of Hunan Province (Changsha, China) between March 2010 and September 2011. These 28 NSCLC patients included 20 males and 8 females, with a mean age of 62 years; 12 were at T1 stage while 16 were at T2-T4 stage (21). The current study was approved by the Ethics Committee of Hunan Province (Hunan, China). Written informed consent was obtained from all participants. Histomorphology was confirmed using hematoxylin and eosin staining by the Department of Pathology, Tumor Hospital of Hunan Province. Tissues were then immediately snap-frozen in liquid nitrogen following surgical removal and stored at −80°C.

NSCLC cell lines (SK-MES-1, A549, SPCA-1, H460, H1229 and HCC827) and the non-tumorous human bronchial epithelium cell line BEAS-2B, were all obtained from the Cell Bank, China Academy of Sciences (Shanghai, China). All cell lines were cultured in RPMI-1640 medium (Life Technologies; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Life Technologies; Thermo Fisher Scientific, Inc.) at 37°C in 5% CO₂.

Total RNA was extracted from the tissues or cells using TRIzol (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) according to the manufacturer's instructions. qPCR was used to examine the relative miR-17-5p expression using a mirVana™ qRT-PCR microRNA detection kit (Life Technologies; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions and U6 was used as an internal reference. The specific primers for miR-17-5p and U6 were purchased from Genecopoeia, Inc., (Guangzhou, China). Primer sequences were not available. mRNA expression was detected using the standard SYBR-Green RT-PCR kit (Takara Bio, Inc., Otsu, Japan) according to the manufacturer's instructions and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal reference. The specific primers for TGFβR2 were as follows: Forward, 5′-AAGATGACCGCTCTGACATCA-3′ and reverse, 5′-CTTATAGACCTCAGCAAAGCGAC-3′. The specific primers for GAPDH were as follows: Forward, 5′-CAGCCACCCGAGATTGAGCA-3′ and reverse, 5′-TAGTAGCGACGGGCGGTGTG-3′. The reaction conditions were 95°C for 3 min and 45 cycles of denaturation at 95°C for 15 sec followed by an annealing/elongation step at 58°C for 30 sec. Fold-change was calculated using the relative quantification (2^−ΔΔCq) method (22).

The predicted miR-17-5p binding sites on the 3′-untranslated region (3′UTR) of TGFβR2 were cloned into the pGL3 vector (Promega Corporation, Madison, WI, USA) named pGL3-TGFβR2-3′UTR. The mutant miR-17-5p binding sites on the 3′UTR of TGFβR2 were constructed using a QuikChange Site-Directed Mutagenesis kit (Stratagene; Agilent Technologies, Inc., Santa Clara, CA, USA), in accordance with the manufacture's protocol. TGFβR2 was also inserted into the pGL3 vector and named pGL3-TGFβR2-mut-3′UTR.

H460 cells (5×10⁵ cells per well) were seeded in a 6-well plate with RPMI-1640 medium and incubated at 37°C overnight to 60–70% confluence. For miR-17-5p overexpression or knockdown, miR-17-5p mimic or inhibitor (Genecopoeia, Inc.) was diluted in serum-free Opti-minimal essential medium (MEM) and transfected with Lipofectamine^® 2000 (both Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's instructions. For TGFβR2 silencing, the NC small interfering (siRNA) (forward: 5′-UUCUCCGAACGUGUCACGUTT-3′ and reverse: 5′-ACGUGACACGUUCGGAGAATT-3′) and TGFβR2-specific siRNA (forward: 5′-GACCUCAAGAGCUCCAAUATT-3′ and reverse: 5′-UAUUGGAGCUCUUGAGGUCTT-3′) were purchased from Amspring (Changsha, China), diluted in serum-free Opti-MEM and transfected with Lipofectamine^® 2000. Levels of miR-17-5p and TGFβR2 expression were examined 48 h after transfection. For the luciferase reporter assay, H460 cells (1×10⁵ cells per well) in 24-well plates were co-transfected with 500 ng pGL3-TGFβR2-3′UTR or pGL3-TGFβR2-mut-3′UTR, and 50 nM miR-17-5p mimic or miR-negative control (miR-NC). Additionally, pRL-SV40 (Promega Corporation) was co-transfected as the control. Reporter assays were performed at 48 h after transfection using a Dual-Luciferase^® assay system (Promega Corporation).

An MTT assay was used to examine cell proliferation. H460 cells (5×10⁴ per well) were plated into a 96-well plate and cultured at 37°C with 5% CO₂ for 12, 24, 48 or 72 h. Subsequently, 20 µl MTT (5 mg/ml, Life Technologies; Thermo Fisher Scientific, Inc.) was added. Following incubation at 37°C for 4 h, 150 µl dimethyl sulfoxide was added. The control was treated with dimethyl sulfoxide without MTT. Following incubation at room temperature for 10 min, formazan production was detected by determining the optical density at 570 nm using a Multiskan FC enzyme immunoassay analyzer (Thermo Fisher Scientific, Inc.).

Cell apoptosis was examined using the Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (556547; BD Pharmingen, San Diego, CA, USA) according to the manufacturer's instructions. H460 cells were re-suspended in 1X binding buffer solution (BD Pharmingen) with Annexin V-FITC and propidium iodide (PI) and incubated for 15 min at room temperature in the dark. Apoptotic cells were analyzed using a BD Accuri C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) with BD Accuri C6 software 1.0 (BD Biosciences).

Cells were lysed in cold radioimmunoprecipitation assay buffer (Sigma-Aldrich, Merck KGaA). The protein concentration was determined using the bicinchoninic acid protein assay kit (Pierce Biotechnology, Inc., Thermo Fisher Scientific, Inc.). Protein was separated with 10% SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane (Life Technologies; Thermo Fisher Scientific, Inc.). The membrane was then blocked in 5% powdered milk dissolved in PBS (Life Technologies; Thermo Fisher Scientific, Inc.) containing 0.1% Tween-20 (Sigma-Aldrich, Inc., Merck KGaA) at room temperature for 3 h. Subsequently, the PVDF membrane was incubated with mouse anti-human monoclonal TGFβR2 (1:200; ab78419; Abcam, Cambridge, MA, USA) or mouse anti-human GAPDH (1:200; ab8245; Abcam) primary antibodies for 3 h at room temperature, and then washed with Dulbecco's phosphate-buffered saline for 10 min. The PVDF membrane was then incubated with rabbit anti-mouse secondary antibody (1:5,000; ab175743; Abcam) at room temperature for 1 h. An enhanced chemiluminescence western blotting kit (Pierce Biotechnology, Inc.; Thermo Fisher Scientific, Inc.) was used to detect the protein bands according to the manufacturer's protocols. Protein bands were quantified by densitometric analysis using Image Lab analysis software 2.1 (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and expressed as the density ratio vs. GAPDH.

Results were expressed as the group means ± standard error of the mean and analyzed using Student's t-test for two-group comparisons and one-way analysis of variance for multiple-group comparisons. SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA) was used for the analysis. P<0.05 was considered to indicate a statistically significant difference.

To determine the role of miR-17-5p in NSCLC, the expression of miR-17-5p was examined in 28 NSCLC tissues and 7 non-tumorous lung tissues using qPCR. The results showed that miR-17-5p was significantly downregulated in NSCLC tissues compared with non-tumorous lung tissues (P<0.01; Fig. 1A). Additionally, miR-17-5p levels were significantly lower in T2-T4 stage NSCLC tissues compared with T1 stage NSCLC tissues (P<0.05; Fig. 1B), suggesting that low miR-17-5p levels were associated with an advanced pathological stage of NSCLC. A significant downregulation of miR-17-5p in all NSCLC cell lines was also observed compared with non-tumorous bronchial epithelium BEAS-2B cells (P<0.01; Fig. 1C). H460 cells exhibited the greatest downregulation of miR-17-5p (Fig. 1C), thus this cell line was used for all subsequent experiments.

H460 cells were transfected with miR-NC, miR-17-5p mimic or miR-17-5p inhibitor. At 48 h after transfection, qPCR was conducted to examine miR-17-5p expression. Transfection with miR-17-5p mimic was found to significantly increase miR-17-5p expression (P<0.01), while transfection with miR-17-5p inhibitor significantly decreased miR-17-5p expression (P<0.01) compared with the control group (Fig. 2A). The effects of miR-17-5p overexpression or downregulation on H460 cell proliferation were further studied by performing an MTT assay. The data showed that the absorbance in miR-17-5p-overexpressing H460 cells was significantly lower than in the control group (P<0.01; Fig. 2B). On the contrary, knockdown of miR-17-5p led to a significant upregulation of H460 cell proliferation compared with the control group (P<0.01; Fig. 2B). These results indicated that miR-17-5p has a role in suppressing role in regulating H460 cell proliferation.

Furthermore, the apoptotic levels were examined further as apoptosis may be a direct cause of proliferation inhibition. H460 cells were stained with Annexin V-conjugated FITC (indicating cell apoptosis) and PI (indicating cell necrosis) and flow cytometry was performed. Annexin V-FITC positive/PI negative cells were at an early apoptotic stage, while Annexin V-FITC positive/PI positive cells were at late apoptotic/necrotic stage. As indicated in Fig. 2C, the rate of apoptosis in the control group was 5.9%, but increased to 9.4% in miR-17-5p-overexpressing H460 cells, indicating that miR-17-5p induces H460 cell apoptosis. On the contrary, the apoptosis rate in H460 cells transfected with miR-17-5p inhibitor was 4.8%, indicating that knockdown of miR-17-5p inhibits H460 cell apoptosis (Fig. 2C). In addition, the apoptosis rate in the miR-NC-transfected H460 cells was 6%, and did not differ significantly from that in the control group (Fig. 2C).

The putative targets of miR-17-5p in NSCLC were studied further by conducting a bioinformatical analysis. It was predicted that TGFβR2 is a direct target gene of miR-17-5p (Fig. 3A) and that this target relationship is evolutionally conserved (Fig. 3B). To confirm this, the wild-type or mutant type of the miR-17-5p binding sequence in the 3′UTR of TGFβR2 was subcloned downstream of the firefly luciferase reporter gene in a pGL3 vector, named as pGL3-TGFβR2-3′UTR and pGL3-TGFβR2-mut-3′UTR (Fig. 3C and D), respectively. These vectors were were then co-transfected with miR-17-5p mimic or miR-NC into H460 cells. A luciferase reporter assay was further conducted 48 h following transfection. Relative luciferase activity in H460 cells co-transfected with pGL3-TGFβR2-3′UTR and miR-17-5p mimic was significantly reduced, compared with the control group, which was co-transfected with pGL3-TGFβR2-3′UTR and miR-NC (P<0.01; Fig. 3E). However, relative luciferase activity in H460 cells co-transfected with pGL3-TGFβR2-mut-3′UTR and miR-17-5p mimic did not differ from that in the control group (Fig. 3E). There results indicate that miR-17-5p can directly bind to the 3′UTR of TGFβR2 mRNA.

The effect of miR-17-5p on TGFβR2 expression at the transcriptional and translational levels was investigated further using RT-qPCR and western blot analysis. The results of the present study indicated that levels of TGFβR2 were significantly reduced in miR-17-5p-overexpressing H460 cells (P<0.01) and significantly increased following knockdown of miR-17-5p (P<0.01), compared with the control group (Fig. 3F and G). However, the data indicated that levels of TGFβR2 mRNA were unaffected by miR-17-5p expression (Fig. 3H). These data indicate that miR-17-5p negatively mediates the expression of TGFβR2 at the post-transcriptional level by directly binding to the 3′UTR of TGFβR2 mRNA.

Levels of TGFβR2 mRNA were assessed in human NSCLC tissues and cell lines. The results of the present study indicated that TGFβR2 was significantly upregulated in NSCLC tissues compared with non-tumorous lung tissues (P<0.01; Fig. 4A). Additionally, levels of TGFβR2 mRNA were significantly higher in the T2-4 stage NSCLC tissues, compared with those in non-tumorous lung or T1 stage NSCLC tissues (P<0.01), suggesting that low miR-17-5p levels are associated with an advanced pathological stage of NSCLC (Fig. 4B). Accordingly, the expression profile of TGFβR2 was in contrast with that of miR-17-5p in NSCLC tissue. It was further found that the TGFβR2 mRNA levels were also significantly increased in certain NSCLC cell lines including SK-MES-1, H1229, H460 and HCC827, compared with normal bronchial epithelium BEAS-2B cells (P<0.01; Fig. 4C).

To further investigate whether TGFβR2 is involved in miR-17-5p-mediated NSCLC cell proliferation and apoptosis, TGFβR2-specific siRNA was used to knockdown TGFβR2 expression in H460 cells. Following transfection with TGFβR2 siRNA, levels of TGFβR2 mRNA and protein were significantly downregulated (P<0.01) compared with the control siRNA-transfected H460 cells (Fig. 5A-C). An MTT assay was conducted to assess cell proliferation and the data indicated that TGFβR2 knockdown led to a significant decrease in cell proliferation compared with control siRNA-transfected H460 cells (P<0.01; Fig. 5D). Subsequently, the cell apoptotic rate was determined by flow cytometry. Rates of apoptosis in TGFβR2 siRNA-transfected cells were significantly higher than in control siRNA-transfected cells (P<0.01), indicating that inhibition of TGFβR2 enhanced apoptosis in H460 cells (Fig. 3E). Altogether, these data indicated that TGFβR2 knockdown showed similar effects to miR-17-5p overexpression on H460 cell proliferation and apoptosis, and suggests that TGFβR2 is involved in miR-17-5p-mediated H460 cell proliferation and apoptosis.