A human normal lung cell line (MRC-5) and four human NSCLC cell lines (A549, H460, H1299 and H520) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). These cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; HyClone Laboratories; GE Healthcare Life Sciences, Logan, UT, USA), 100 U/ml penicillin, 100 µg/ml streptomycin and 1% glutamine (Invitrogen; Thermo Fisher Scientific, Inc.). All cells were maintained at 37°C with 5% CO₂.

For miR-150 downregulation or overexpression, miR-150 inhibitor (LNA-anti-miR-150) (Exiqon, Vedbæk, Denmark) or miR-150 mimics (Invitrogen; Thermo Fisher Scientific, Inc.) were respectively added to the culture medium as in a previous study (18). The transfection medium was replaced at 4 h post-transfection with regular culture medium.

For SIRT2 overexpression or knockdown, A549 and H1299 cell lines were infected with adenovirus expressing SIRT2 (Ad-SIRT2) or retrovirus expressing sh-STRT2, respectively, as in our previous study (19).

Real-time RT-qPCR was used to detect the expression level of miR-150 by a MiniOpticon™ Two-Color Real-Time PCR Detection system (Bio-Rad Laboratories, Hercules, CA, USA). miRNA was extracted from cultured cells by using an Applied Biosystems mirVana miRNA Isolation kit (Thermo Fisher Scientific, Inc.). U6 was used as the internal control.

Total protein isolated from 10⁵ A549 and H1299 cells was lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) and detected by a western blot assay as previously described (18). Antibodies against the following target proteins were used: Phosphorylated-AKT^Ser473 (dilution 1:1,000; cat. no. sc-7985-R), AKT (dilution 1:1,000; cat. no. sc-8312), JMJD2A (dilution 1:500; cat. no. sc-81302), SIRT2 (dilution 1:1,000; cat. no. sc-135793; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse PCNA (dilution 1:1,000; cat. no. ab-18197; Abcam, Cambridge, MA, USA) and GAPDH (dilution 1:5,000; Kangchen Bio-tech, Shanghai, China); the secondary antibodies were sheep anti-mouse IgG and anti-rabbit IgG (dilution 1:6,000; R&D Systems China, Shanghai, China). GAPDH was used as the internal control. All protein bands were semi-quantitatively detected with ImageJ software (National Institutes of Health, Bethesda, MD, USA).

A549 and H1299 cell viability was monitored using a Cell Proliferation kit I (MTT) (cat. no. 11465007001; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) according to the manufacturer's instructions.

NSCLC cell lines A549 and H1299 were collected following transfection, and 1×10³ cells were mixed with high-glucose DMEM supplemented with 10% fetal bovine serum (FBS) and cultured in 6-well tissue culture plates. The cells were continuously cultured for 2 weeks. Following culture, all cell colonies were stained with 0.005% crystal violet dye liquor. The stained cells were observed by microscopy (Leica DM2500; Leica Microsystems, Wetzlar, Germany) and 10 random fields were selected for cell colony counting. The experiment was conducted three times.

Cell mobility assays were performed with a Transwell chamber (EMD Millipore, Billerica, MA, USA) according to the manufacturer's guidelines. For an invasion assay, a total of 5×10⁴ A549 and H1299 cells at 24 h post-transfection were plated onto an 8-µm pore size Transwell insert pre-coated with extracellular matrix (ECM) (1:6 mix with DMEM) (BD Biosciences, San Jose, CA, USA). A cell migration assay was performed using the Transwell insert without the pre-coating with ECM. After conventional cell culture for 48 h, all cells which had adhered to the upper surface of the Transwell insert were wiped away gently with a cotton swab. The remaining cells which had passed through the Transwell insert were stained with 0.005% crystal violet dye liquor for 15 min. The stained cells were observed by microscopy (Leica DM2500; Leica Microsystems) and 10 random fields were selected for cell counting. The experiment was conducted three times.

Xenograft mouse experiments were performed as previously described (18). Twenty-four male BALB/c nude mice (20 g weight) at 8 weeks of age (Shanghai SLAC Laboratory Animal Center of Chinese Academy of Sciences, Shanghai, China) were randomly divided into four groups (n=6/group), subcutaneously injected the parental, LNA-anti-miR150-transfected, Ad-SIRT2-transfected and both transfected A549 cells, respectively, into the right and left flanks of male nu/nu mice. Mice were maintained in pathogen-free conditions and cared for according to the Laboratory Animal Care guidelines. They were given radiation-sterilized food pellets and distilled water. Tumor size was measured regularly, and tumor volume was estimated with the formula: a × b² × 0.5, in which a and b represent the maximal and minimal diameters. Mice were sacrificed by anesthetization at the end of the observation period and cancer tissues were harvested and the tumor weights were measured. Then, the tumors were fixed in 10% neutral formaldehyde for 6 h, and consecutive paraffin-embedded sections were cut to examine the expression of PCNA, JMJD2A and p-AKT^Ser473 by immunohistochemistry. The animal welfare guidelines for the care and use of laboratory animals were followed and the experimental protocol was approved by the Animal Care Committee of the Second Affiliated Hospital of Soochow University.

The experimental data from three independent experiments were presented as the mean ± standard deviation (SD) and analyzed using one-way analysis of variance (ANOVA) test with post hoc contrasts by the Student-Newman-Keuls test. The software package PASW Statistics 18.0 (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. A P<0.05 was considered to indicate statistical significance.

Our previous studies revealed that miR-150 was obviously upregulated in NSCLC tissues compared with that noted in normal tissues. To confirm the expression of miR-150 in NSCLC cells, we performed RT-qPCR to detect the expression of miR-150 in four NSCLC cell lines. As shown in Fig. 1A, compared with the normal cell line MRC-5, miR-150 was significantly upregulated in the four NSCLC cells lines. In particular, the relative expression of miR-150 was highly increased in the A549 and H1299 cells (6.12±0.23 in A549, 4.71±0.13 in H1299; P<0.01 vs. normal cell line). Meanwhile, significant suppression of SIRT2 was observed in the NSCLC cell lines, particularly in A549 and H1299 cells (P<0.01 vs. normal cell line; Fig. 1B). Accordingly, the A549 and H1299 cell lines, with obvious upregulation of miR-150 and downregulation of SIRT2, were selected for the following assays.

Previous studies have demonstrated that miR-150 expression is markedly positively related to the JMJD2A level, while being significantly negatively correlated with SIRT2 and JMJD2A expression in A549 cells (18,19). To further investigate the effect of miR-150 on H1299 cells, LNA-anti-miR-150 was used to silence miR-150, and miR-150 mimics were used to recover the level of miR-150. The knockdown of miR-150 in H1299 cells notably increased the expression of SIRT2 and reduced the level of JMJD2A (P<0.05; Fig. 2A). In turn, upon transfection with miR-150 mimics, the normal expression levels of SIRT2 and JMJD2A were recovered. To further explore the relationship between miR-150 and SIRT2, we knocked down or overexpressed SIRT2 in H1299 cells. As shown in Fig. 2B, re-expression of SIRT2 suppressed the expression of JMJD2A and miR-150 (P<0.05).

To explore the effect of miR-150 downregulation and SIRT2 re-expression on the proliferation of NSCLC cells, A549 and H1299 cell proliferation were measured after transfection with LNA-anti-miR-150 or Ad-SIRT2. At 48 h after transfection, the proliferation rate of cells transfected with LNA-anti-miR-150 or Ad-SIRT2 was decreased, relative to that of the negative controls. Moreover, combined treatment with LNA-anti-miR-150 and Ad-SIRT2 showed the most significant inhibitory effect on cell proliferation in A549 and H1299 cells (P<0.05; Fig. 3A). Next, the neoplastic capacity of NSCLC cells was examined by plate colony formation assay. The results demonstrated that the cells transfected with LNA-anti-miR-150 or Ad-SIRT2 formed less colonies than the corresponding control groups (P<0.05; Fig. 3B). Similarly, the A549 and H1299 groups co-transfected with LNA-anti-miR-150 and Ad-SIRT2 exhibited the most obvious suppression of neoplastic capacity (P<0.001; Fig. 3B). In addition, western blot analysis was used to test the ability of miR-150 downregulation and SIRT2 re-expression to regulate the expression of PCNA (proliferating cell nuclear antigen), which serves an important role in the proliferation of cells. The data indicated that PCNA protein expression was reduced in the LNA-anti-miR-150 or Ad-SIRT2-transfected cells, but remained to be expressed to a high level in the respective negative control groups (P<0.05; Fig. 5). Furthermore, significant reduction of PCNA expression was observed in the NSCLC cells co-transfected with LNA-anti-miR-150 and Ad-SIRT2 (P<0.001; Fig. 5). These results demonstrated that the combination of downregulated miR-150 and upregulated SIRT2 could strongly inhibit the proliferation and neoplastic capacity of NSCLC cells.

To investigate the effect of miR-150 downregulation and SIRT2 re-expression on cellular motility, the NSCLC cell lines A549 and H1299 were subjected to Transwell cellular mobility assays after transfection with LNA-anti-miR-150 and/or Ad-SIRT2. The results indicated that the relative migrated and invaded cell numbers were obviously decreased in the cell groups transfected with LNA-anti-miR-150 or Ad-SIRT2, compared with the negative controls (P<0.05; Fig. 4). In particular, the group transfected with both LNA-anti-miR-150 and Ad-SIRT2 showed the lowest numbers of migrated and invaded cells (P<0.001; Fig. 4).

It has been well demonstrated that NSCLC cells exhibit high expression of miR-150 and low expression of SIRT2. To further investigate the effect of miR-150 suppression and SIRT2 re-expression on NSCLC cells, LNA-anti-miR-150 and Ad-SIRT2 were transfected into A549 and H1299 cells, and the relative expression levels of AKT and JMJD2A were measured by western blot analysis. The results demonstrated that the expression of JMJD2A was decreased in the cell groups transfected with LNA-anti-miR-150 or Ad-SIRT2 (P<0.05; Fig. 5). The inhibition of JMJD2A expression was more marked in cells co-transfected with LNA-anti-miR-150 and Ad-SIRT2 (P<0.001; Fig. 5). In addition, reduction of miR-150 or re-expression of SIRT2 decreased the expression of p-AKT^Ser473 in A549 and H1299 cells (P<0.05; Fig. 5). Co-treatment to reduce miR-150 expression and re-express SIRT2 further inhibited p-AKT^Ser473 expression in the NSCLC cells (P<0.001; Fig. 5). These results suggested that reduction of miR-150 and re-expression of SIRT2 leads to suppression of JMJD2A and effectively inactivates the AKT signaling pathway.

To further explore the effect of miR-150 on the NSCLC cells, a xenograft tumor growth assay was performed. The results showed that miR-150 knockdown or SIRT2 overexpression significantly suppressed NSCLC tumor growth in vivo (Fig. 6A and B; P<0.05). Moreover, co-transfection of A549 cells with miR-150 knockdown and SIRT2 re-expression vectors could further inhibit NSCLC tumor growth in vivo (Fig. 6A and B; P<0.001). Next, the paraffin-embedded tumor sections were examined immunohistochemically. The results showed that the expression of JMJD2A, PCNA and p-AKT^Ser473 were decreased in the groups injected with cells transfected with LNA-anti-miR-150 or Ad-SIRT2. Moreover, the expression of JMJD2A, PCNA and p-AKT^Ser473 were more significantly inhibited in the group injected with cells co-transfected with LNA-anti-miR-150 and Ad-SIRT2 (Fig. 6C). These results suggested that reduction of miR-150 and re-expression of SIRT2 could inhibit NSCLC growth in vivo. It also provided evidence that miR-150 may play a tumor-promoting role in lung cancer.