Normal human lung epithelial cell line BEAS-2B, and NSCLC cell lines (H1299 and H460) were purchased from the Institute of Biochemistry and Cell Biology, CAS (Shanghai, China). BEAS-2B cells were grown in Dulbecco's modified Eagle's medium (DMEM) and the NSCLC cell lines were grown in RPMI-1640 supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) and 100 U/ml penicillin-streptomycin. The cultured cells were maintained at 37°C in a humidified environment with 5% CO₂ in air.

miR-19a overexpression and knockdown lentiviral vectors were prepared by GenePharma Co., Ltd. (Shanghai, China). H1299 and H460 were cultured in a 12-well plate for a confluence of 60%. The previous culture medium was discarded, and the cells were incubated with fresh RPMI-1640 for 2 h. After infecting the cells with lentivirus, the cells with a stable expression or knockdown of miR-19a were screened with puromycin.

The cell proliferation level was detected using the CCK-8 method. The cells were cultivated in a 96-well plate at a density of 2×10³/well and incubated for 24, 48 and 72 h at 37°C, 5% CO₂. Subsequently, the cells were incubated with CCK-8 for 1 h and then detected at 450 nm with a microplate reader (Bio-Tek Instruments Inc., Winooski, VT, USA).

The cells were collected and washed with cold PBS, then stained with PI (BD Biosciences, San Diego, CA, USA) according to the production manuals. Cells were analyzed using flow cytometry (Coulter, Luton, UK).

The cells were incubated with the culture medium containing FBS at a density of 1×10⁵/ml, and 200 µl was added in the upper chamber. RPMI-1640 supplemented with 20% FBS was added in the lower chamber with 400 µl. After incubation for 24 h at 37°C, 5% CO₂, cells on the upper surface of the filters were removed and cells adhering to the undersurface of the filter membrane were stained with 0.5% crystal violet for 30 min. The crystal violet was washed with PBS three times. The cells in the lower chamber were counted under a microscope for four fields randomly, and the mean cell numbers were recorded and analyzed.

Total RNA was extracted from the cells at a density of 75% confluence using TRIzol total RNA isolation reagent (Gibco, Waltham, MA, USA) as per the manufacturers instructions. Total RNA (2 µg) was transcribed with stem-loop RT primers. The primer sequences used were: miR-19a (5-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGCGGAAC-3); U6 (5-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAAATATGGAAC-3).

The produced cDNA was quantified by SYBR Premix Ex Taq (Takara Bio, Inc., Otsu, Japan). The primer sequences used were: miR-19a, 5-TGCGGTTCACAGTGGCTAAG-3; U6 forward, 5-TGCGGGTGCTCGCTTCGGCAGC-3 and reverse, 5-CCAGTGCAGGGTCCGAGGT-3. The reactions were incubated at 95°C for 5 min (95°C for 30 sec, 56°C for 30 sec and 72°C for 30 sec) for 40 cycles and maintained at 10°C. The expression of mRNA was quantified by 2^−ΔΔCT.

The cells were harvested (1.5×10⁵/ml) and lysed by RIPA with PMSF. After incubation for 20 min, the cell lysate was centrifuged for 10 min at 2,580 × g and later separated on 10% SDS-PAGE (50 µg/lane) and transferred onto nitrocellulose membranes. After blocking with 5% BSA, the target proteins were probed with the primary antibodies at 4°C overnight. After three 5-min washes with TBST, the bound antibodies were detected using HRP-conjugated secondary antibodies and were visualized using the enhanced chemiluminescent reagent. The relative levels of each protein were determined by densitometric scanning (Amersham WB System, GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). The antibodies used for the study were: MXD1 antibody (rabbit polyclonal, IgG, dilution 1:1000, Proteintech, Rosemont, IL, USA; catalog no.: 17888-1-AP), GAPDH antibody (rabbit polyclonal, IgG, dilution 1:20000, Proteintech, Rosemont, IL, USA; catalog no.: 10494-1-AP). The secondary antibody was goat F(ab')2 anti-rabbit IgG (Fab')2 (HRP) preadsorbed (Abcam, Cambridge, MA, USA; catalog no.: ab6112).

Data were analyzed using SPSS 19.0 software (IBM, Armonk, NY, USA). Data were presented as means ± standard deviation. Comparisons were conducted using one-way ANOVA followed by the post-hoc (LSD) test. P<0.05 indicated a statistically significant difference.

RT-PCR was used to detect the expression of miR-19a in lung cancer and normal cells to determine the association between miR-19a and lung cancer. The results showed a significant difference between the normal and lung cancer cells (Fig. 1).

To determine the effects of miR-19a on proliferation in the different cell lines, we constructed the lentivirus vectors to establish the cells lines with overexpressed and knocked down miR-19a. The results of RT-PCR indicated that the cells lines infected by lentivirus were successfully established (Fig. 2). Subsequently, we detected cell proliferation using CCK-8 to determine the effects of miR-19a on proliferation. The cells were cultivated in a 96-well plate at a density of 2×10³/well and incubated for 24, 48 and 72 h. There were no significant differences in proliferation at 24 and 48 h, whereas a significant difference was detected by 72 h, as observed by the inhibition of proliferation of the miR-19a knockdown cell lines, compared with the related control cell line and the overexpressed one (Fig. 3).

In addition to the proliferative function of miR-19a, the migratory influence of miR-19a was examined using an invasion assay. As shown in Fig. 4, miR-19a overexpressed cells had a significant increasing number of cells in the lower chamber compared with the non-transfected control cells. The invasion data indicated that miR-19a likely played an important role in promoting lung cancer metastasis.

The aforementioned results identified the upregulation of miR-19a in lung cancer cells and a higher expression of miR-19a may function as a promoter in cell proliferation and migration. Thus, the effect of miR-19a on the cell cycle was assessed. Using flow cytometry and PI staining, G2/M arrest in cell lines with miR-19a knockdown was identified, compared with the non-transfected control. However, the cell lines with miR-19a overexpression did not show any significant difference between the non-transfected control (Fig. 5).

MXD1 was capable of binding with MAX competitively, which inhibited the formation of the c-Myc/MAX heterodimers, and the transcriptional activity of c-Myc was downregulated (16). Our results found that following the overexpression of miR-19a in cancer cells, MXD1 expression was significantly downregulated, which indicated that miR-19a may regulate MXD1 expression negatively, and thus promote the formation of c-myc/max heterodimers indirectly, thereby enhancing cell proliferation. (Fig. 6).