Human NSCLC cell lines NCI-H157 and A549 were obtained from American Type Culture Collection (Manassas, VA, USA). Human keratinocyte cell line HaCaT and human embryonic kidney cell line HEK293T were purchased from Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). These cells were maintained in DMEM (Invitrogen, Carlsbad, CA, USA) plus 10% fetal calf serum (FCS; Gibco, Rockville, MD, USA) and 1% penicillin/streptomycin (Sigma, St. Louis, MO, USA) and maintained in a humidified incubator containing 95% air/5% CO₂ at 37°C.

Total RNA was extracted using miRNeasy mini kit (Qiagen, Dusseldorf, Germany) according to the manufacturer's instructions. For mRNA expression analysis, cDNA was reverse transcribed by M-MLV reverse transcriptase (Takara, Dalian, Shanghai). For miRNA expression analysis, cDNA was synthesised with miScript reverse transcription kit (Qiagen). Primer sequences were as follows: NSBP1, forward 5′-GCAGTCAGGCAGTGACTGCCTTCG-3′ and reverse 5′-CCCTTTTCTGTGGCATCTTC-3′; GAPDH, forward 5′-CAGTCAGCCGCATCTTCTTTT-3′ and reverse 5′-GTGACCAGGCGCCCAATAC-3′; miR-326, forward 5′-ACTGTCCTTCCCTCTGGGC-3′ and reverse 5′-AATGGTTGTTCTCCACTCTCTCTC-3′; U6 small nuclear RNA, forward 5′-GCTTCGGCAGCACATATACTAAAAT-3′ and reverse, 5′-CGCTTCACGAATTTGCGTGTCAT-3′. GAPDH was used as an internal control for mRNA quantification. U6 small nuclear RNA was used as an internal control for miRNA quantification. RT-qPCR analysis was performed using SYBR-Green Master Mix (Bio-Rad, Hercules, CA, USA). The relative gene expression was analysed using the 2^−ΔΔCt method and normalised to the internal controls.

Cells were lysed in RIPA lysis buffer, and the protein concentration was measured using a BCA kit (Beyotime Biotechnology, Haimen, China). Equal amounts of proteins from different samples were separated on 12.5% sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. The separated protein bands on SDS polyacrylamide gel were electro-transferred onto a nitrocellulose membrane (Bio-Rad). After blocking with 5% skim milk powder, the membrane was blotted with NSBP1, cyclin B1, MMP9 and GAPDH antibodies (Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at 4°C overnight. Horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc.) diluted in 1:2,000 were added and incubated for 1 h at room temperature. Finally, the immunoreactive bands were developed with the enhanced chemiluminescence method. The grey value of protein bands was quantified with Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA), and relative protein expression data were normalised to GAPDH.

NSBP1-specific siRNA and control non-specific siRNA (NC siRNA) were purchased from Cruz Biotechnology, Inc., and then transfected into cells according to the manufacturer's instructions. In brief, cells were plated into six-well plates at 2×10⁵ cells/well overnight. About 1 μg of siRNA was diluted into 100 μl of transfection medium, and 6 μl of transfection reagent was diluted into 100 μl of transfection medium. Both solutions were then mixed together for 45 min at room temperature. The siRNA transfection reagent mixture was added to each well with 0.8 ml of transfection medium and incubated for 7 h. The medium was replaced with fresh normal growth medium and cultured for another 48 h. The interfering efficiency was detected by western blot analysis. miR-326 mimics and non-specific controls (miR-NC) were obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China). NSCLC cells were seeded in six-well plates overnight and then transfected with 50 nM miR-326 mimics or miR-326 inhibitor using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instruction. The transfection efficiency was subsequently detected by RT-qPCR analysis after transfection for 48 h.

Cell proliferation of NSCLC cells was measured by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-di-phenyl-2-H-tetrazolium bromide (MTT) method. In brief, NSCLC cells were seeded into 96-well cell plates at a density of 1×10⁵ cells/well and grown for 24 h. The cells were then transfected with miR-NC or miR-326 mimics for 48 h. The old medium was replaced by fresh medium containing 20 μl of MTT (5 mg/ml in PBS; Sigma) and cultured for 4 h. The medium was discarded, and 200 μl of dimethyl sulphoxide was added to dissolve crystal formazan. The optical density (OD) of each reaction solution at 490 nm was measured with an enzyme immunoassay analyser (Bio-Tek Instruments, Winooski, VT, USA).

Tumour cell invasion ability was detected by Transwell invasion assay. The Transwell filter was precoated with Matrigel (BD Bioscience, San Jose, CA, USA) on the upper surface of a polycarbonic membrane. NSCLC cells were transfected with miR-326 mimics or miR-NC for 48 h and then starved overnight. A total of 1×10⁵ cells were added in the top chamber with serum-free medium, and the medium containing 10% FCS was added to the lower chamber. After 48 h, non-migrated cells in the upper chamber were removed by a cotton swab, and the migrated cells in the lower chamber were fixed with 95% ethanol and stained with 4 g/l trypan blue solution. Cells were observed under a microscope (Olympus, Tokyo, Japan), and positive staining cells were counted in five random fields.

Cell cycle distribution was detected by flow cytometry. In brief, NSCLC cells were serum-starved for 24 h and transfected with NSBP1 siRNA or miR-326 mimics for 48 h. Thereafter, cells were harvested, washed with ice-cold PBS and fixed with 70% ethanol. Propidium iodide containing RNase (Molecular Probes, Eugene, OR, USA) was added to the cells and incubated for 30 min in the dark. FACScan flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA) was used to detect the cell cycle distribution.

The cDNA fragments from NSBP1 containing an miR-326 binding site were inserted into pmirGLO vector (Promega, Madison, WI, USA). HEK293T cells were seeded into six-well plates at 2×10⁵ cells/well overnight. About 10 ng of pmirGLO-NSBP1 recombinant vectors and 50 nM miR-326 mimics were introduced into HEK293T cells using Lipofectamine 2000 (Invitrogen). The transfected cells were lysed after 48 h of transfection. Firefly luciferase activity and Renilla luciferase activity were quantified by the dual-luciferase reporter method (Promega).

Twenty pairs of NSCLC tissues and matched adjacent non-tumour tissues were provided by Xi'an Central Hospital of Xi'an Jiaotong University. The tissues were collected from NSCLC patients who had undergone surgical resections and stored at −80°C for use. The use of clinical tissues was approved by the hospital's Institutional Human Experiment and Ethic Committee with informed consent from the patients.

Data are presented as the mean ± standard deviation (SD). Statistical analyses were carried out using SPSS software (version 11.5; SPSS Inc., Chicago, IL, USA), and statistical differences were analysed using Student's t-test or one-way ANOVA. Correlation analysis was performed using Spearman's rank correlation coefficients. P<0.05 was considered to indicate a statistically significant difference.

To explore the potential role of NSBP1 in human NSCLC, the expression patterns of NSBP1 in the NSCLC cell lines were examined by RT-qPCR and western blot analysis. The results showed that the mRNA expression of NSBP1 was significantly higher in A549 and NCI-H157 cells than that in control HaCaT cells (Fig. 1A). Moreover, the NSBP1 protein expression was also highly upregulated in A549 and NCI-H157 cells (Fig. 1B and C). These results suggested a critical role of NSBP1 in NSCLC cells.

To understand the functional significance of NSBP1 in regulating the biological processes of NSCLC cells, we performed loss-of-function experiments of NSBP1 by transfection with specific siRNA targeting NSBP1. The transfection efficiency was detected by western blot analysis, which showed that NSBP siRNA effectively downregulated the expression of NSBP1 in A549 (Fig. 2A) and NCI-H157 (Fig. 2B) cells. Subsequently, we measured the effect of NSBP1 suppression on NSCLC cell proliferation. MTT assay showed that knockdown of NSBP1 significantly inhibited A549 (Fig. 2C) and NCI-H157 (Fig. 2D) cell proliferation. Furthermore, cell cycle distribution showed that knockdown of NSBP1 apparently induced G0/G1 cell cycle arrest of A549 (Fig. 3A) and NCI-H157 (Fig. 3B) cells. To further investigate the biological effect of NSBP1 in NSCLC cells, we examined the effect of NSBP1 knockdown on NSCLC cell invasion. We found that suppression of NSBP1 significantly repressed the invasive ability of A549 (Fig. 3C) and NCI-H157 (Fig. 3D) cells. In summary, these results indicated that NSBP1 participated in the regulation of NSCLC cell proliferation and invasion.

An increasing number of studies have suggested that miRNAs have the potential to treat lung cancer via targeting specific genes (21). To date, the miRNAs specifically targeting NSBP1 have not been well characterised. In the present study, we searched for specific miRNAs that could target and regulate NSBP, and investigated whether NSBP1 expression can be regulated by miRNAs. Bioinformatics analysis demonstrated that miR-326 had a predicted binding site in the 3′-UTR of NSBP1 (Fig. 4A). We then performed a dual-luciferase reporter experiment to verify their direct relationship. The results showed that overexpression of miR-326 markedly repressed the relative luciferase activity of pmirGLO-NSBP1, but it did not affect the relative luciferase activity of pmirGLO-NSBP1 (mut)-transfected cells (Fig. 4B).

To investigate whether miR-326 can regulate NSBP1 expression in NSCLC cells, we transfected miR-326 mimics into NSCLC cells and detected NSBP1 expression using RT-qPCR and western blot analysis. RT-qPCR analysis showed that miR-326 overexpression significantly suppressed the mRNA expression of NSBP1 in A549 (Fig. 4C) and NCI-H157 (Fig. 4D) cells. Western blot analysis further displayed that the protein expression of NSBP1 was also markedly repressed by miR-326 mimics in A549 (Fig. 4E) and NCI-H157 (Fig. 4F) cells. These results indicated that miR-326 could target the 3′-UTR of NSBP1 and repress its expression in NSCLC cells.

Given the inhibitory effect of miR-326 on NSBP1 expression, miR-326 might have a biological effect on NSCLC cells. To test this hypothesis, we examined the biological effect of miR-326 on cell proliferation and invasion in NSCLC cells. Interestingly, MTT assay demonstrated that transfection of miR-326 significantly inhibited cell proliferation of A549 (Fig. 5A) and NCI-H157 (Fig. 5B) cells. Moreover, miR-326 overexpression induced G0/G1 cell cycle arrest in A549 (Fig. 5C) and NCI-H157 (Fig. 5D) cells. The results of Transwell assay suggested that miR-326 mimic-transfected cells showed significantly decreased cell invasive capacity (Fig. 5E and F).

To further understand the molecular basis of NSBP1 gene silencing induced by NSBP1 siRNA or miR-326 in regulating NSCLC cell proliferation and invasion, we detected the regulatory effect of NSBP1 gene silencing on cyclin B1 and MMP9, which were involved in regulating cancer cell proliferation and invasion. We found that suppression of NSBP1 by siRNA or miR-326 inhibited the protein expression levels of cyclin B1 and MMP9 in A549 (Fig. 6A and B).

To investigate whether miR-326 overexpression suppressed cyclin B1 and MMP9 directly through NSBP1, we performed a rescue experiment by overexpression of NSBP1. We found that transfection of NSBP expression vectors harbouring no 3′-UTR of NSBP1 significantly restored NSBP1 expression, which was decreased by the transfection of miR-326 mimics (Fig. 7A and B). NSBP1 overexpression restored the decreased protein expression levels of cyclin B1 and MMP9 induced by miR-326 overexpression (Fig. 7A, C and D), indicating that miR-326 inhibited cyclin B1 and MMP9 expression through NSBP1.

To gain insight into the functional significance of miR-326 and NSBP1 expression in NSCLC, we uncovered their expression levels in clinical NSCLC tissues and elucidated their potential relationship. We found that NSBP1 mRNA expression was significantly increased in NSCCL specimens (Fig. 7E), whereas miR-326 expression was markedly decreased (Fig. 7F) compared with that in adjacent non-tumour tissues. We correlated NSBP1 mRNA expression and miR-326 expression using correlation analysis, which demonstrated that miR-326 expression was remarkably inversely correlated with NSBP1 expression in NSCLC tissues (Fig. 7G) (r=−0.8756, P<0.001), implying that decreased miR-326 might contribute to the increased NSBP1 expression during NSCCL development and progression.