A549 cells were propagated in DMEM (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 U/ml streptomycin. The cells were incubated at 37°C in a 5% CO₂ humidified atmosphere until 75% confluent.

All transfections were carried out using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The following 2′-O-methyl oligonucleotides were synthesized by Integrated DNA Technologies and purchased from Shanghai GeneChem: 2′-O-Me-sense-125a-3p: 5′-ACA GGU GAG GUU CUU GGG AGCC-3′, 2′-O-Me-antisense-125a-3p: 5′-GGC UCC CAA GAA CCU CAC CUGU-3′, and 2′-O-Me-scramble-125a-3p: 5′-GGU CGG UGC UCG AUG CAG GUAA-3′. Twenty-four hours prior to transfection, the cells were plated into 6-well plates for RNA extraction and into 10-cm dishes for protein extraction. A549 cells were transfected with 20 μM of 2′-O-methyl oligo-nucleotides, and RNA and protein were extracted 24 h after transfection. In addition, RhoA small interfering RNA (siRNA) or the negative control was used in co-transfection with or without antisense-125a-3p. The sequences of the RhoA siRNAs were: 5′-GAAGUCAAGCAUUUCUGUCTT-3′ (sense) and 5′-GACAGAAAUGCUUGACUUCTT-3′ (antisense) (17).

To block the function of RhoA, the Rho inhibitor CT04 (Cytoskeleton, Denver, CO, USA), was added to the culture medium at a final concentration of 2.0 μg/ml, as described previously (18). Migration was investigated in the presence of CT04 using a Transwell chamber as described in the following section.

A549 cells (5×10⁴ cells/chamber) were trypsinized, washed, resuspended in serum-free DMEM, and placed in the top portion of the chamber. The lower portion of the chamber contained 10% FBS as a chemoattractant. The chambers were incubated at 37°C, 5% CO₂ for 12 h, and then the cells remaining on the membrane were washed with PBS, fixed in 100% methanol, stained with haematoxylin, photographed and counted. Five random fields were selected randomly, then analyzed for each chamber. Each of 3 independent experiments was carried out in duplicate.

The plasmid used for these assays contained a full-length RhoA 3′UTR coupled to a luciferase reporter, and was a kind gift from Dr William Kong and Dr Jin Q. Cheng (H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA). The following primers were used to amplify the 3′UTR of the RhoA gene from human cDNA (NM_001664.2): forward primer 5′-GAC TAG TCA ATC TGG GTG CCT TGT CTTG-3′, reverse primer 5′-CCC AAG CTT GGG TGC CTT TAT TCT ATT AGT AGT TGG AAA-3′. The PCR fragment were digested and cloned into the pMIR-Report vector (Ambion) at the SpeI and HindIII sites. The mutant plasmid containing a full-length RhoA 3′UTR mutant coupled to a luciferase reporter was constructed by Sauer Biotechnology Inc. (China).

A549 cells were seeded onto 24-well plates the day prior to transfection. They were transfected with 0.4 μg of the luciferase expression construct, 20 μM 2′-O-methyl sense or scramble oligonucleotides, and 0.02 μg of the Renilla luciferase vector pRL-TK (Promega) for normalization. Mock transfected cells were transfected with the luciferase constructs alone. After 24 h, luciferase assays were performed using the Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity. All transfection experiments were conducted in triplicate.

Total RNA was prepared using TRIzol (Invitrogen). RhoA cDNA was generated by PrimerScript RT reagent kit (Takara, Dalian, China) and amplified using RhoA primers with SYBR Premix EX Taq II (Takara). The primer sequences for RhoA were: 5′-CGA CAG CCC TGA TAG TTT A-3′ (forward) and 5′-GTG CTC ATC ATT CCG AAG A-3′ (reverse), and the primer sequences for β-actin were F: 5′-AGCACAGAGCCTCGCCTTTG-3′ (forward), R: 5′-ACATGCCGGAGCCGTTGT-3′ (reverse). The relative quantity of RhoA, normalized to β-actin, calculated based on the following equation RQ = 2^−ΔΔCt.

Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed using the M-PER reagent (Pierce Biotechnology) containing 1 mM PMSF and phosphatase inhibitors for 1 h at 4°C. The supernatants were centrifuged at 12,000 × g for 30 min at 4°C, and collected for analysis. Aliquots of supernatant containing 80 μg of total protein were separated on 12 or 10% SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked using 5% fat-free milk, and incubated with mouse anti-RhoA (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), or mouse anti-GAPDH (1:1,000, Santa Cruz Biotechnology). Membranes were incubated at 4°C overnight and incubated with corresponding secondary antibodies (1:4,000, Chemicon, Temecula, CA, USA) at room temperature for 1 h. Immunoreactive bands were identified using Super ECL reagent (Pierce Biotechnology), according to the manufacturer’s protocol. Specific bands for RhoA, F-actin and GAPDH were identified using prestained protein molecular weight markers (SM0441, MBI Fermentas). The ECL Imaging System (UVP Inc.) was used to visualize the specific bands and the optical density of each band was measured using Image J software.

When cell confluence was less than 90% at 48 h after transfection, wounds were created in confluent cells using a 200-ml pipette tip. The cells were then rinsed with medium to remove any free-floating cells and debris. Medium was then added, and culture plates were incubated at 37°C. Wound healing within the scrape line was observed at different time points, and representative scrape lines for each cell line were photographed. Duplicate wells for each condition were examined for each experiment, and each experiment was repeated 3 times. The optical the distance of wound were measured using Image J software.

The Rho-GTP pull-down assay was performed using an RhoA/Rac1/Cdc42 Activation Assay Combo Kit (STA-405, Cell Biolabs Inc., San Diego, CA, USA). Medium was aspirated off cells cultured to approximately 80 to 90% confluence, and the cells were rinsed twice with ice-cold PBS before being treated with ice-cold lysis buffer (0.5–1 ml per 100-mm tissue culture plate). The cell culture plates were subsequently placed on ice for 10–20 min. The cells were centrifugated at 14,000 × g for 10 min at 4°C, and then 20 μl of 0.5 M EDTA was added to each aliquot sample. The positive and negative controls received 10 μl of 100X GTPγS and 10 μl of 100X GDP, respectively. All tubes were incubated with agitation for 30 min at 30°C. Reactions were stopped using 65 μl of 1.0 M MgCl₂. Rhotekin RBD or PAK PBD Agarose beads were added to the cell lysates and incubated for 1 h at 4°C. After centrifugation for 10 sec at 14,000 × g, the beads were washed with lysis buffer and Rho-GTP was eluted in Laemmli sample buffer. Antibody against RhoA was used to analyze eluted sample on western blots.

When cell confluence was less than 5×10⁵ cells at 48 h after transfection, cells were serum-starved for 4 h and fixed in 2% formaldehyde/PBS for 7 min at room temperature. After permeabilization with 0.1% Triton X-100 and blocking with 1.5% normal goat serum, the cells were stained with rhodamine-phalloidin for 30 min at 37°C to label F-actin, and mounted using Gelvatol mounting medium.

All data are presented as mean ± SD of 3 independent experiments. A p-value ≤0.05 was considered significant. All statistical analyses were performed using the SPSS13.0 software package.

To study how miR-125a-3p might regulate migration, we first proceeded to identify potential targets known to play a role in cell mobility. Among the candidates surveyed previously (14), we found that the 3′UTR of the RhoA gene contains highly conserved regions that may serve as binding sites for miR-125a-3p, as determined at microrna.org (Fig. 1).

Since results of our previous study showed that miR-125a-3p expressed moderately in A549 NSCLC cells, we chose A549 cells for this study. To confirm whether suppression of migration by miR-125a-3p is associated with changes in RhoA expression, we measured the level of both RhoA mRNA and protein using qRT-PCR and western blot analysis in the presence of upregulation and downregulation of miR-125a-3p. Upregulation of miR-125a-3p was achieved using transfected sense-miR-125a-3p; while RhoA mRNA remained unchanged (p=0.309, Fig. 2A), the RhoA protein concentration decreased (p<0.001, Fig. 2B). Downregulation of miR-125a-3p was achieved using transfected antisense-miR-125a-3; while RhoA mRNA remained unchanged (p=0.942, Fig. 2C), the RhoA protein concentration increased (p<0.001, Fig. 2D). These results indicate that miR-125a-3p may post-transcriptionally regulate RhoA expression.

To further demonstrate that RhoA is a potential target of miR-125a-3p, we generated luciferase reporters that contained the 3′UTR of the RhoA gene (Fig. 3A). Results from 3 independent experiments showed that reporter activity was reduced by the ectopic expression of miR-125a-3p (p<0.001, Fig. 3B). We also generated luciferase reporters that contained a mutated sequence within the predicted target sites of the 3′UTR of the RhoA gene (Fig. 3A) to further demonstrate the interaction between miR-125a-3p and the 3′UTR of RhoA. Data showed that reporter activity was not reduced by the ectopic expression of miR-125a-3p (Fig. 3B). Taken together, these results indicated that in A549 cells, the RhoA gene was a functional target of miR-125a-3p.

Previously we found that downregulation of miR-125a-3p induced increased migration of A549 cells in transwell experiments (14). Fig. 4A shows that the number of migrating cells decreased after transfection with sense miR-125a-3p. In contrast, the number of migrating cells increased after transfection with antisense miR-125a-3p. A similar trend was seen in the wound healing assays (Fig. 5A). To further validate whether miR-125a-3p inhibits migration in a Rho-dependent manner, we blocked RhoA activity using the Rho inhibitor CT04 at a final concentration of 2.0 μg/ml. The number of migrating cells was significantly decreased after treatment with CT04 compared with the untreated cells in a Transwell experiment without matrigel (p<0.001, Fig. 4B and C), and as seen in the wound healing assay (p<0.001, Fig. 5B and C). Moreover, after treatment with CT04, the migratory ability of A549 cells transfected with the antisense-miR-125a-3p or the sense miR-125a-3p was neither decreased nor increased (p<0.001, Figs. 4B, C, 5B and C). These results suggest that miR-125a-3p cannot regulate RhoA when the Rho pathway is blocked.

Downregulation of RhoA by the inhibitor CT04 may directly affect the function of RhoA, independent of the concentration of protein. To rule out this effect, siRNA was used for specific downregulation of the expression of RhoA to confirm that miR-125a-3p mediated NSCLC cell migration through RhoA. The knockdown efficacy was identified using western blot analysis (p<0.001, Fig. 6A). Knockdown of the expression of RhoA (p<0.001, Fig. 6B), abolished the effect of antisense-miR-125a-3p on cell migration (Fig. 6C and D).

We investigated whether RhoA expression affected the migration of A549 cells via organization of the actin cytoskeleton, and also affected the levels of RhoA-GTP. The levels of RhoA-GTP were quantified using a pull-down assay after transfecting A549 cells with sense-miR-125a-3p or antisense-miR-125a-3p. The results showed that the levels of RhoA-GTP were decreased (p<0.001, Fig. 7) along with decreased expression of RhoA when sense-miR-125a-3p was transfected. In addition immunofluorescence microscopy demonstrated that accumulative actin filaments were lower (Fig. 8). By contrast, the levels of RhoA-GTP increased (Fig. 7) with increase in RhoA expression when antisense-miR-125a-3p was transfected. Moreover, immunofluorescence microscopy results demonstrated that the accumulative actin filaments had grown (Fig. 8).