NSCLC cell lines A549, NCI-H1299 and human HEK-293T cell lines were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). The cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (S/N:16000-044; Gibco) and antibiotics (100 U/ml penicillin and streptomycin; Invitrogen) at 37°C in a humidified 5% CO₂ atmosphere. Cell transfection was performed using Lipofectamine™ 2000 (Invitrogen). Cells were harvested at 48 h post-transfection for protein analysis or luciferase activity assay. The related sequences are shown in Table I.

The full length 3′UTR of human MMP-14 was PCR-amplified from human genomic DNA and cloned into psiCHECK™-2 dual luciferase reporter plasmid (Promega, Madison, WI, USA) immediately downstream of the stop codon of the Renilla luciferase gene with Xhol/NotI to generate psiCHECK™-2-MMP-14-3′UTR-wt. The mutant human MMP-14 3′UTR reporter, designated as psiCHECK™-2-MMP-14-3′UTR-mut, was created within the predicted hsa-miR-133a seed sequence binding site (GGACCAAA to GCTGGTAA) by site-directed PCR mutation. The construction was confirmed by DNA sequencing.

For luciferase reporter assay, HEK-293T cells (4.0×10³ cells/well) were plated in a 96-well plate (Corning, USA) 24 h before transfection. Cells were co-transfected with 60 ng of either the psiCHECK™-2-MMP-14-3′UTR-wt, psiCHECK™-2-MMP-14-3′UTR-mut or psiCHECK™-2 vector and 20 nM (final concentration) of either miRNA mimics negative control or miR-133a mimics or miR-133a inhibitor, which is antisense oligonucleotides with 2′O-methyl modification (RiboBio, Guangzhou, China). Forty-eight hours after transfection, the cells were washed with PBS twice and lysed in 100 μl Passive Lysis Buffer (Promega) and the luciferase activities were measured from 20 μl lysate using the Dual-Luciferase Reporter Assay kit (Promega) following the manufacturer’s instructions on a luminometer (Lumat LB 9507; Berthold, Germany). The data were obtained by averaging the results from six independent repeats. Transfection efficiency was normalized to thymidine kinase-driven Renilla luciferase activity.

Total RNA was isolated from cultured cells using TRIzol Reagent (Invitrogen). cDNA was synthesized by reverse transcription using oligo (dT) as the primer and proceeded to real-time PCR with gene-specific primers in the presence of 2X SYBR-Green Master Mix (DBI Bioscience, Shanghai, China). The relative abundance of mRNA was calculated by normalization to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Specific stem-loop RT primers were used for reverse transcription reaction of miR-133a. Normalization of miR-133a was performed by using RNU6 primers. All reactions were carried out in triplicate and all experiments were performed three independent times.

For the proliferation assay, 6 h after transfection, 3,000 cells were seeded in triplicate in 6-well plates. Cell Counting Kit-8 (CCK-8) reagent (C0038; Beyotime, China) was added at 12, 24, 48, 72 h after seeding and incubated at 37°C for half to 4 h according to the color change. The data of optical density (OD) value at 450 nm were read by a microplate reader (Synergy 2; BioTek, USA). Migration and invasion assays were performed according to the manufacturer's instructions. Cells (3×10⁴) in 0.5 ml of serum-free medium were seeded onto the top of each chamber containing BD BioCoat cell culture inserts (354578) or Matrigel Invasion Chamber (354480; both from BD Biosciences), and 0.75 ml of complete growth medium containing 10% FBS was added to each well in the lower chamber. Following incubation for 48 h at 37°C, non-invasive cells were removed from the upper chamber, the cells attached to the lower chamber were fixed with methanol, stained with 0.1% crystal violet and then counted under a light microscope.

Fifty micrograms of total proteins were loaded onto 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (PVDF; Millipore, Billerico, MA, USA). After blocking with 3% bovine serum albumin, the blots were incubated with antibodies against MMP-14 (Bioworld, Atlanta, GA, USA), GAPDH (Chemicon International, Temecula, CA, USA). Following incubation with red flurescence conjugated secondary antibody, protein bands were scanned using Odyssey bands scanner (S/N ODY-2792 model: 9120) The intensity of the bands was analyzed using Bandscan Software.

Analysis of variance and Student’s t-test were used to compare means of two or more different treatment groups. P<0.05 was considered to indicate a statistically significant difference, unless otherwise stated. Data are expressed as mean ± SE.

To identify potential targets of miR-133a both for experimental validation and functional studies in lung cancer, we performed in silico analysis of a range of miRNA target prediction databases. The target prediction of miR-133a was performed by using the following databases: TargetScan (http://www.targetscan.org), MicroCosm (http://www.ebi.ac.uk/), miRanda (http://www.microrna.org/microrna/getGeneForm.do) and miRGen (www.diana.pcbi.upenn.edu/cgi-bin/miRGen/v3/Targets.cgi). All databases analyzed presented MMP-14 as a converging target of miR-133a, which plays an essential role in the degradation of basement membranes and the expression of MMP-14 is correlated with metastasis. To validate MMP-14 as a de novo target of miR-133a, we constructed a luciferase reporter vector containing the entire wild-type 3′UTR of MMP-14 (psiCHECK™-2-MMP-14-3′UTR-wt) or the mutant 3′UTR (psiCHECK™-2-MMP-14-3′UTR-mut) with a 5-nucleotide mutation previously mentioned (Fig. 1A). HEK-293T cells, which exhibit low levels of miR-133a expression, were used for transient transfections with psiCHECK™-2-MMP-14-3′UTR-wt or psiCHECK™2-MMP-14-3′UTR-mut. Co-transfection with miR-133a (a synthetic miR-133a mimic) resulted in a significant decrease (to 63%) in luciferase gene expression from the reporter vector containing the wild-type 3′UTR of MMP-14 when compared with a scrambled control (Fig. 1B), demonstrating direct targeting by miR-133a (P<0.01). Consistent with the data, no decrease in luciferase activity was observed when miR-133a was co-transfected with the mutant 3′UTR of MMP-14 reporter. Furthermore, miR-133a specific inhibitor, which is the antisense oligonucleotides of miR-133a can almost restore the inhibition effect (P<0.05) (Fig. 1B). These results indicate that MMP-14 is a direct target of miR-133a and is responsible for miR-133a targeting in the 3′UTR.

To characterize the functional impact of miR-133a on lung cancer cell behavior such as growth rate, we transfected A549 and NCI-H1299 cells with either miR-133a mimics or mimics control and transferred the cells into 96-well plates at 3.0×10³ cells/well post transfection. We harvested and detected the OD450 value of each well by CCK-8 kit at indicated times after seeding. These results showed that the growth rates of the two lung cancer cell lines were suppressed in miR-133a mimics transfected groups compared to control groups at 72 h (P<0.01 and P<0.05 at A549 and NCI-H1299 cell lines, respectively) (Fig. 2A). To confirm the transfection, we assessed the expressions of miR-133a at 72 h post transfection. As shown in Fig. 2B, miR-133a was upregulated to 63.6- and 52.3-fold at A549 and NCI-H1299 cells, respectively (both P<0.001) (Fig. 2B).

We next assessed the effect of miR-133a overexpression on MMP-14. Transfection of miR-133a into A549 and NCI-H1299 cells resulted in a significant decrease of MMP-14 mRNA levels compared with control-transfected cells. As measured by qRT-PCR, the MMP-14 mRNA levels were decreased to 41 and 38% in A549 and NCI-H1299, respectively (both P<0.01) (Fig. 3A). Subsequent western blot analysis of MMP-14 showed that miR-133a overexpression caused a reduction in MMP-14 protein levels, which can be reversed by miR-133a specific inhibitor (Fig. 3B). Measured by Bandscan Software according to grayscale of each band, MPP-14 protein is downregulated to 37 and 31% in A549 and NCI-H1299 cells, respectively. These results indicated that miR-133a can inhibit lung cancer cell proliferation, which may be mediated by downregulation of direct target MMP-14 at mRNA and protein levels.

MMP-14 is an MT1-MMP as a primary regulator of interstitial collagenolysis and there is evidence to suggest that MMP-14 is enhanced in metastatic PDAC lesions compared with the primary tumors and myogenic tumors (31,32). The downregulation of MMP-14 by miR-133a overexpression prompted us to examine whether miR-133a could modulate migration and invasion in lung cancer cell lines. We performed the transwell migration and Matrigel invasion assays in A549 and NCI-H1299 cells transfected with either miR-133a mimics or control mimics. As shown in Fig. 3A, miR-133a mimics suppressed cell migration and invasion in both A549 and NCI-H1299 cells (migration and invasion, P<0.05 in the NCI-H1299 cell line and P<0.01 in the A549 cell line) (Fig. 4A). The specific inhibitor of miR-133a almost restored the inhibition effect in both cell lines (Fig. 4A and B). Moreover, the MMP-14 protein levels were also restored (Fig. 3B). Collectively, these results clearly indicate that miR-133a suppresses cell migration and invasion in the A549 and NCI-H1299 cells lines by reducing MMP-14 expression.