NSCLC tumor samples and paired adjacent normal lung tissue samples were obtained from twelve NSCLC patients undergoing primary surgical resection at the Second Affiliated Hospital of Medical School, Xi'an Jiaotong University. The resected tissues were immediately frozen in liquid nitrogen at −80°C. The patients that participated in this study provided signed informed consent. This study was approved by Institutional Human Experiment and Ethics Committee of the Second Affiliated Hospital of Medical School, Xi'an Jiaotong University and was conducted in accordance with the Helsinki Declaration.

Human NSCLC cell lines including A549, H1299, H1975 and NCI-H1264, and the normal human lung cell line BEAS-2B were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Rockville, MD, USA) containing 10% fetal bovine serum plus with 1% penicillin/streptomycin (Sigma, St. Louis, MO, USA). Cells were routinely grown in a humidified atmosphere containing 5% CO₂ at 37°C.

The miR-330-5p mimics and negative control (NC) were purchased from GenePharma (Shanghai, China) and transfected into cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The cDNA of NOB1 without 3′-UTR was inserted into pcDNA3 plasmid that was transfected into cells using Lipofectamine 2000 (Invitrogen).

Total RNA from tissues or cells was extracted using TRIzol reagent (Invitrogen) as per the manufacturer's instructions. First-strand cDNA was synthesized using the MMLV Reverse Transcriptase (for mRNA detection) or miRcute miRNA cDNA kit (Tiangen, Beijing, China). RT-qPCR was performed with the SYBR Green PCR kit (Applied Biosystems, Foster City, CA, USA) and specific primers in the ABI7500 Real-time PCR system (Applied Biosystems). GAPDH and U6 served as internal controls. Data were calculated using the 2^−∆∆Ct method. Fold changes in gene expression were obtained by normalization to the control group.

Cells were plated into 96-well plates at a density of 1×10⁴ cells/well and cultured overnight. After transfection with miR-330-5p mimics for 72 h, cells were treated with 20 µl of MTT (5 mg/ml; Sigma) for 4 h. The formazan crystals were solubilized in 200 µl of dimethyl sulfoxide (Sigma). The absorbance at 490 nm was measured with a microplate reader (Bio-Rad, Hercules, CA, USA).

The BrdU assay was performed using a BrdU cell proliferation assay kit (Cell Signaling Technology, Danvers, MA, USA) according to the manufacturer's instructions. Briefly, cells were plated into 96-well plates at a density of 1×10⁴ cells/well and transfected with miR-330-5p mimics for 48 h. Then, cells were treated with 10 µl of BrdU solution for 2 h followed by incubation with 150 µl of denaturing solution. Afterwards, peroxidase conjugated anti-BrdU (1:1,000) was added and incubated for 90 min at room temperature. At the end of the incubation period, the solution was discarded and cells were washed with 200 µl of washing buffer. Finally, 100 µl of substrate solution was added and incubated for 15 min. The absorbance at 450 nm was measured with a microplate reader (Bio-Rad).

Cells transfected with miR-330-5p mimics for 48 h were diluted in 0.33% low-melting agarose and seeded into 6-well plates (1,000 cells/well) on the bottom of 0.5% agar layer. After growing for 2 weeks, the colonies were fixed with methanol for 30 min and then stained with 0.1% crystal violet (Sigma). Then, the visible colonies were counted under a microscope (Olympus, Tokyo, Japan).

After synchronization by serum-starvation for 24 h, cells were transfected with miR-330-5p mimics for 48 h. Then, cells were harvested, digested with trypsin and washed with ice-cold phosphate buffered saline (PBS). After fixation with 70% ethanol at 4°C overnight, cells were treated with 50 µg/ml RNase A for 30 min at room temperature. Then, cells were treated with 50 µg/ml of propidium iodide (PI) for 30 min in the dark. Cell cycle distribution was analyzed by FACS (BD Biosciences, San Jose, CA, USA).

Apoptosis was detected using an Annexin V-FITC/PI kit (Beyotime Biotechnology, Haimen, China) according to the manufacturer's instructions. Briefly, cells were transfected with miR-330-5p mimics for 48 h and then harvested. Cells were digested by trypsin and washed with PBS. Cells were re-suspended in 500 µl of binding buffer containing 1.25 µl of Annexin V-FITC and 10 µl of PI for 20 min in the dark. Cells were then assessed by a BD flow cytometer (BD Biosciences).

Capase-3 activity was detected using a caspase-3 activity assay (Beyotime Biotechnology). Briefly, after cells were transfected with miR-330-5p mimics for 48 h cells, cells were harvested, lysed and incubated with Ac-DEVD-pNA for 2 h at 37°C. The absorbance at 405 nm was determined using a microplate reader (Bio-Rad).

The 3′-UTR of NOB1 containing the wild-type or mutant binding sites of miR-330-5p were inserted into the pmirGLO vector (Promega, Madison, WI, USA) following by transfection into A549 and H1299 cells along with miR-330-5p mimics using Lipofectamine 2000 (Invitrogen). After 48 h, the relative luciferase activity was detected by a dual-luciferase assay kit (Promega).

Total protein was extracted using RIPA buffer and the protein concentration was measured using a BCA kit (Beyotime Biotechnology). An equal amount of protein (50 µg) was loaded onto a sodium dodecyl sulfate polyacrylamide gel for separation. The separated proteins were transferred to a polyvinylidene fluoride membrane (Millipore, Boston, MA, USA). The membrane was then blocked using 5% non-fat milk for 1 h at 37°C followed by incubation with primary antibodies (anti-NOB1 and anti-GAPDH; Abcam, Cambridge, UK) with appropriate concentrations at 4°C overnight. Then, the membrane was probed with horseradish peroxidase conjugated secondary antibodies (1:1,000, Beyotime Biotechnology) for 1 h at 37°C. The immunoreactive proteins were developed by using enhanced chemiluminescence system (Millipore). The intensity of the protein blots on the membrane was analyzed by Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

All results were depicted as means ± standard deviation and the statistical analyses were conducted by SPSS version 18.0 software (SPSS Inc., Chicago, IL, USA). Differences were analyzed by Student's t-test or one-way analysis of variance with a Bonferroni correction. Correlation analysis were performed by Spearman's correlation analysis. Differences were regarded as statistically significant at P<0.05.

To investigate the potential role of miR-330-5p on the progression of NSCLC, we first examined the expression of miR-330-5p expression in NSCLC tissues by RT-qPCR. The results showed that miR-330-5p was significantly decreased in NSCLC tissues compared with adjacent normal lung tissues (Fig. 1A). Moreover, we detected miR-330-5p expression in NSCLC cell lines including A549, H1299, H1975 and NCI-H1264. The results showed that miR-330-5p was markedly decreased in NSCLC cell lines compared with the normal lung cell line BEAS-2B (Fig. 1B). Taken together, these results suggest that miR-330-5p may be involved in the development and progression of NSCLC.

To investigate the exact biological function of miR-330-5p in NSCLC, we overexpressed miR-330-5p in A549 and H1299 cells by transfection with miR-330-5p mimics. We then detected the effect of miR-330-5p overexpression on cell growth of NSCLC cells by MTT, BrdU, colony formation and cell cycle assays. The MTT assay showed that overexpression of miR-330-5p significantly reduced the viability of A549 and H1299 cells (Fig. 2A). The BrdU assay showed that miR-330-5p overexpression markedly inhibited the proliferation of A549 and H1299 cells (Fig. 2B). Moreover, the colony-forming capacity of A549 and H1299 cells was also significantly decreased by miR-330-5p overexpression (Fig. 2C). In addition, overexpression of miR-330-5p significantly induced cell cycle arrest in G0/G1 phase (Fig. 2D). Overall, these results suggest that miR-330-5p suppresses NSCLC cell growth.

To further investigate the biological function of miR-330-5p in NSCLC, we assessed the effect of miR-330-5p overexpression on NSCLC cell apoptosis by Annexin V/PI apoptosis assay and caspase-3 activity assay. The Annexin V/PI apoptosis assay showed that overexpression of miR-330-5p significantly induced apoptosis in NSCLC cells (Fig. 3A). Furthermore, caspase-3 activity was markedly increased by miR-330-5p overexpression (Fig. 3B). These results suggest that miR-330-5p promotes apoptosis in NSCLC cells.

To investigate the underlying mechanism of miR-330-5p in regulating NSCLC cell growth, we performed bioinformatics analysis to predict the potential target genes of miR-330-5p. Interestingly, we found that NOB1 was a potential target gene of miR-330-5p (Fig. 4A). To investigate whether miR-330-5p directly binds to NOB1 3′-UTR, we performed dual luciferase reporter assays. The results showed that luciferase activity in the luciferase reporter vector containing the NOB1 3′-UTR was significantly reduced by overexpression of miR-330-5p (Fig. 4B and C). However, this inhibitory effect of miR-330-5p overexpression on luciferase activity was abolished by mutating binding sites in the NOB1 3′-UTR (Fig. 4B and C). Furthermore, we detected a direct regulatory effect of miR-330-5p on NOB1 expression by RT-qPCR and western blot analysis. The results showed that the mRNA (Fig. 5A and B) and protein (Fig. 5C and D) expression levels of NOB1 were significantly decreased with miR-330-5p overexpression in A549 and H1299 cells. Taken together, these results indicate that NOB1 is a target gene of miR-330-5p in NSCLC cells.

Previous studies have reported that NOB1 regulates NSCLC cell growth associated with regulating cyclin D1 and CDK4 (22). Considering the effect of miR-330-5p on NOB1 expression, we speculated that miR-330-5p may regulate the expression of cyclin D1 and CDK4. To test this hypothesis, we measured the effect of miR-330-5p overexpression on mRNA expression of cyclin D1 and CDK4 by RT-qPCR. The results showed that the expression of cyclin D1 (Fig. 6A) and CDK4 (Fig. 6B) was significantly reduced by miR-330-5p overexpression, implying that miR-330-5p regulates the expression of cyclin D1 and CDK4.

To further investigate whether miR-330-5p inhibits NSCLC cell growth by targeting NOB1, the pcDNA3-NOB1 vector and miR-330-5p mimics were cotransfected into A549 and H1299 cells. The results showed that NOB1 expression was significantly increased in cells cotransfected with the pcDNA3-NOB1 vector and miR-330-5p mimics compared with cells transfected with miR-330-5p mimics only (Fig. 7A). Moreover, the inhibitory effect of miR-330-5p on NSCLC cell growth was significantly reversed by NOB1 overexpression. (Fig. 7B and C). NOB1 overexpression also markedly abrogated miR-330-5p-induced NSCLC cell apoptosis (Fig. 7D). Collectively, these results suggests miR-330-5p suppresses cell growth and induces apoptosis by inhibiting NOB1 in NSCLC cells.

To further confirm the relevance of miR-330-5p/NOB1 in the progression of NSCLC, we analyzed the correlation between miR-330-5p expression and NOB1 mRNA expression in NSCLC tissues by RT-qPCR. The results showed that NOB1 mRNA was highly expressed in NSCLC tissues (Fig. 8A). Correlation analysis showed that miR-330-5p expression was inversely correlated with NOB1 mRNA expression in NSCLC tissues (Fig. 8B). The data indicate that decreased miR-330-5p expression may contribute to the increased expression of NOB1 in NSCLC.