The NSCLC cell lines A549, H1299, H23 and SK-MES-1, and the normal bronchial epithelium BEAS-2B were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). The cells were cultured in Dulbecco's modified Eagle's medium (Gibco™ DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.), 100 U/ml of penicillin and 100 µg/ml of streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂. For cell transfection, 20 nM control miRNA (5′-CGGUACGAUCGCGGCGGGAUAUC-3′) or 20 nM miR-221-3p (5′-AGCUACAUUGUCUGCUGGGUUUC-3′) was transfected into 1×10⁴ NSCLC cells using Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The cells were harvested for the following experiments after transfection for 48 h.

The 50-paired NSCLC tissues and matched adjacent normal lung tissues were collected from patients (age range, 35–75 years; female to male ratio, 1:0.78) who were diagnosed at Longnan Hospital (Daqing, Heilongjiang, China) between April 2011 and August 2013. None of these patients received radiotherapy or chemotherapy prior to surgical resection. All samples were confirmed by histopathological examination and stored in liquid nitrogen before use. Written informed consent was obtained from all the participants. The sample collection was approved by the Institutional Review Board of Longnan Hospital (Daqing, Heilongjiang, China). The experiments using these samples were performed according to the Declaration of Helsinki.

Total miRNA was extracted from the tissues using the miRcute miRNA isolation kit (Tiangen, Beijing, China) according to the manufacturer's instructions. Reverse transcription (RT) PCR was performed using the One Step Prime Script miRNA cDNA Synthesis kit (Takara, Dalian, China). The quantitative PCR (qPCR) was conducted with TaqMan™ Multiplex Master Mix (Invitrogen; Thermo Fisher Scientific, Inc.) using the Applied Biosystems 7500 Real-Time RT-PCR system (Thermo Fisher Scientific, Inc.). The expression of U6 RNA was detected as the normalization control. The primers for miR-221-3p and U6 were designed as follows: miR-221-3p forward, 5′-ACACTCCAGCTGGGAGCTACA and reverse, 5′-CTCAACTGGTGTCGTGGA; U6 forward, 5′-ATTGGAACGATACAGAG and reverse, 5′-GGAACGCTTCACGAATTT. The thermocycling conditions were as follows: Initial denaturation at 95°C for 5 min followed by 40 cycles at 95°C for 10 sec and 60°C for 1 min. The relative expression of miR-221-3p was calculated with the 2^−∆∆Cq method (20). The experiments were performed in triplicate.

Both A549 and H1299 cells were seeded into a 96-well plate at a density of 2×10³ cells per well. The cell proliferation was quantified by adding 10 µl of Cell Counting Kit-8 reagent (CCK-8, Dojindo, Kumamoto, Japan) and incubated at 37°C for 2 h. The absorbance of each well at 450 nm was measured with a microplate reader.

Cells transfected with the corresponding miRNAs were cultured in 60-mm dishes. When the cell confluence reached approximately 70%, the cells were harvested and fixed with pre-cold 70% ethanol overnight at 4°C. The cells were washed twice with PBS and incubated with 1 mg/ml RNase A at 37°C for 30 min. Afterwards, cells were stained with 50 µg/ml propidium iodide (PI) for 40 min at 37°C. Cells were then passed through a 300-mesh nylon net and the cell cycle distribution was determined by flow cytometry (Becton Dickinson FACS Calibur; BD Biosciences, Franklin Lakes, NJ, USA).

NSCLC cells were cultured in 6-well plates and transfected with the indicated miRNA. After transfection, the cells were incubated for 36 h, and the wound was generated using a 200 µl pipette tip. Cells were washed twice with PBS buffer. The wound region was detected after 24 h by light microscopy (magnification, ×40).

The cell invasion assay was performed using 24-well polycarbonate Transwell chambers with 8-µm pores (Corning, Inc.). In total, 1×10³ NSCLC cells/well transfected with the control miRNA or miR-221-3p were plated in serum-free DMEM in the upper chamber. Inserts were precoated with Matrigel. DMEM with 10% FBS was added to the lower chambers. After incubation for 36 h at 37°C, invaded cells were stained with 0.1% crystal violet for 10 min at room temperature and quantified. Cells were visualized and counted using a light microscope (magnification, ×40).

The 3′-UTR of p27 containing the targeting sites of miR-221-3p was amplified and cloned into the p-MIR-reporter plasmid (Ambion; Thermo Fisher Scientific, Inc.). NSCLC cells cotransfected with miR-221-3p and the luciferase reporter vector of p27 were plated (1×10³ cells/well) in 96-well plates. The Renilla luciferase vector was also transfected with Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) as control of the transfection efficiency. After transfection for 48 h, the luciferase activity was measured with the Dual-Luciferase Reporter Assay System (Promega Corporation) according to the manufacturer's protocol. The p-MIR-firefly (Ambion; Thermo Fisher Scientific, Inc.) luciferase activity was normalized to p-MIR-Renilla (Ambion; Thermo Fisher Scientific, Inc.) activity.

The databases of TargetScan (http://www.Targetscan.org) and miRBase (http://www.mirbase.org) were used to predict the potential targets of miR-221-3p by inputting the name of miRNA in the query.

After transfection for 48 h, cells were harvested and lysed with the NP-40 buffer [150 mM NaCl, 1% NP-40, 50 mM Tris-HCl (pH 8.0), 1 mM EDTA] containing 0.15 U/ml aprotinin, 20 mM leupeptin and 1 mM phenylmethylsulfonyl fluoride. Proteins were loaded onto the 15% SDS-PAGE and transferred onto nitrocellulose filter membranes (Pall Life Sciences, Port Washington, NY, USA). The membrane were initially blocked with 5% non-fat milk for 1 h at room temperature (RT) and then incubated with the primary antibody overnight at 4°C. The membranes were then incubated with the secondary antibody for 1 h at RT. The western blot bands were visualized with the Amersham™ ECL Plus Western Blotting Detection System (GE Healthcare, UK). The antibodies used in this study included anti-p27 (cat. no. sc-1641, Santa Cruz Biotechnology, Inc., Dallas, TX, USA; dilution ratio: 1:2,000), anti-GAPDH (cat. no. 3H12, MBL, Japan; dilution ratio: 1:3,000) and anti-Flag (cat. no. ab1257; Abcam, Cambridge, MA, USA; dilution ratio: 1:2,000) which were purchased from the mentioned companies. The intensities of the protein bands were analyzed using the Image J software (version D1.47; National Institutes of Health).

The percentage of cell apoptosis was assessed using PI/Annexin V-based flow cytometry with the Annexin V-FITC Apoptosis Detection kit (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. Briefly, cells were harvested and washed with pre-cold PBS. Cells were re-centrifuged and resuspended to a final density of ~1×10⁶ cells/ml with the Annexin-binding buffer. 5 µl of FITC/Annexin V and 1 µl of 100 µg/ml PI working solution was added to each 100 µl of cell suspension. After incubation for 15 min at RT, 400 µl of 1X Annexin-binding buffer was added into the cells and mixed gently. The cell apoptosis was analyzed by flow cytometry as soon as possible.

Data are presented as mean ± standard deviation (SD). Statistical analysis was examined with SPSS 19.0 software version (IBM Corp., Armonk, NY, USA). Student's t-test was used to analyze the difference between two groups. One-way analysis of variance followed by Dunnett's test was adopted when comparing more than two groups. P<0.05 was considered to be statistically significant.

To investigate the involvement of miR-221-3p in NSCLC, the expression of miR-221-3p in 50-paired NSCLC tissues and matched corresponding normal lung tissues was detected with RT-qPCR. The data showed that the expression of miR-221-3p was significantly increased in NSCLC tissues compared with that in the adjacent normal tissues (Fig. 1A). Additionally, the abundance of miR-221-3p in NSCLC cell lines including A549, H1299, H23 and SK-MES-1 and normal bronchial epithelium BEAS-2B cells were also investigated. As presented in Fig. 1B, a significantly higher level of miR-221-3p was obtained in the NSCLC cell lines than that noted in the normal cells. These results indicated the overexpression of miR-221-3p in NSCLC.

As the expression of miR-221-3p was overexpressed in NSCLC, we investigated the influence of miR-221-3p on the growth of NSCLC cells. Thus, miR-221-3p was downregulated by transfecting miR-221-3p antagomir into A549 and H1299 cells. The downregulation efficiency of miR-221-3p was validated by RT-qPCR analysis (Fig. 2A). The effect of miR-221-3p on the proliferation of NSCLC cells was detected with the CCK-8 assay. As presented in Fig. 2B and C, suppression of miR-221-3p significantly inhibited the proliferation of both A5459 and H1299 cell lines in comparison with the cells transfecting with the negative control miRNA. To further confirm these results, an in vitro wound-healing assay was performed with A549 and H1299 cells expressing downregulated miR-221-3p. Decreased expression of miR-221-3p markedly reduced the migration of both A549 and H1299 cell lines (Fig. 2D). Furthermore, the effect of miR-221-3p downregulation by antagomir transfection on the apoptosis of NSCLC cells was also investigated. As shown in Fig. 2E, depletion of miR-221-3p significantly increased the relative cell apoptosis percentage of both A549 and H1299 cell lines. The effect of miR-221-3p on the invasion of NSCLC cells was also detected by transfecting negative control miRNA or miR-221-3p antagomir into A549 and H1299 cells. The result showed that downregulation of miR-221-3p significantly inhibited the invasion of NSCLC cells (Fig. 2F). Collectively, these data demonstrated that depletion of miR-221-3p suppressed the growth of NSCLC cells.

The prediction of RNA-associated interactions with computational prediction algorithms plays important roles in characterizing the function of miRNAs (21). To further understand the molecular mechanisms by which miR-221-3p regulates the growth of NSCLC cells, the potential targets of miR-221-3p were predicted with the bioinformatics resource TargetScan (http://www.Targetscan.org) and miRBase (http://www.mirbase.org) (Tables SI and SII). The data showed that p27, the inhibitor of cell cycle progression, which plays important roles in the initiation and progression of cancers (22,23), ranked highly among all the possible targets. The putative binding sites of miR-221-3p at the 3′-UTR of p27 are presented as Fig. 3A. To detect the binding between miR-221-3p with the 3′-UTR of p27, luciferase assay was performed by transfecting luciferase reporter plasmids containing wild-type (WT) or mutant 3′-UTR of p27 in the presence of miR-221-3p mimics in A549 and H1299 cells. The results showed that the luciferase activity of WT but not the mutant 3′-UTR of p27 was significantly decreased with the co-transfection of miR-221-3p mimics (Fig. 3B and C). This result suggested the binding between miR-221-3p and the 3′-UTR of p27. To detect the consequence of the binding, the mRNA and protein levels of p27 in the NSCLC cell lines with overexpression of miR-221-3p were examined, respectively. As presented in Fig. 3D, high expression of miR-221-3p significantly decreased the mRNA level of p27. Consistently, the protein level of p27 was also inhibited upon the overexpression of miR-221-3p in the A549 and H1299 cell lines (Fig. 3E). These results indicated that miR-221-3p was able to bind the 3′-UTR of p27 and suppress the expression of p27 in NSCLC cells.

An increased level of p27 has been reported to control the cell cycle progression at G1 phase (24). As miR-221-3p was found to negatively regulate the expression of p27, to ascertain the influence of miR-221-3p on the cell cycle progression of NSCLC cells, both A549 and H1299 cell lines were transfected with miR-221-3p mimics or control miRNA, and the cell cycle progression was assessed by flow cytometry. As presented in Fig. 4A and B, compared with the control cells, overexpression of miR-221-3p significantly promoted the cell cycle progression from G1 to S phase in the A549 and H1299 cell lines. This result was consistent with the decreased expression of p27 with the ectopic expression of miR-221-3p in NSCLC cells. To further confirm this observation, endogenous miR-221-3p was downregulated by inducing miR-221-3p antagomir into the NSCLC cells. RT-qPCR and western blot analysis were performed to detect the expression level of p27. The data showed that depletion of miR-221-3p increased the abundance of p27 in the A549 and H1299 cell lines (Fig. 4C and D). The cell cycle progression of NSCLC cells expressing negative control miRNA or miR-221-3p antagomir was examined by flow cytometry. As shown in Fig. 4E and F, downregulation of miR-221-3p significantly induced cell cycle arrest at the G1 phase. These results collectively indicated that miR-221-3p is a novel regulator of the cell cycle progression of NSCLC cells. To detect whether the modulation of miR-221-3p on the proliferation of NSCLC cells was by regulating p27, both A549 and H1299 cell lines were transfected with Flag empty vector or Flag-p27 (Fig. 4G). The CCK-8 assay indicated that overexpression of miR-221-3p promoted the proliferation of the NSCLC cell lines, and transfection of p27 significantly decreased the growth of NSCLC cells expressing miR-221-3p (Fig. 4H and I). These results suggest that p27 mediates the role of miR-221-3p in NSCLC.