Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), TaqMan MicroRNA assay kit, BCA protein assay kit, ECL western blotting kit and Lipofectamine 2000 were purchased from Life Technologies (Carlsbad, CA, USA). Demethylation drug 5-aza-2′-deoxycytidine (5-Aza) and histone deacetylase inhibitor 4-phenylbutyric acid (PBA) were purchased from Sigma (St. Louis, MO, USA). A miRNeasy Mini kit was purchased from Qiagen (Valencia, CA, USA). The mouse anti-human antibodies and rabbit anti-mouse secondary antibody were purchased from Immunoway (Cambridge, UK). The Cell Invasion assay kit was purchased from Merck Millipore (Darmstadt, Germany).

Human SW620 and LOVO colon cancer cell lines, and a normal HCEpiC colon epithelium cell line were obtained from the Cell Bank of Central South University, Changsha, China. The cells were cultured in DMEM supplemented with 10% FBS in a humidified atmosphere containing 5% CO₂ at 37°C.

The tissue microarray (Auragene Bioscience Co., Changsha, China) covering 39 colon cancer tissues and their matched adjacent normal tissues was prepared for immunohistochemistry. Tissue microarray blocks were incubated with anti-PAK6 antibody or normal rabbit IgG as the negative control. Immunohistochemical staining was performed using the Moticam 3000 system with diaminobenzidine (Zhongshan Jinqiao Co., Beijing, China). Image-Pro Plus software was used to quantify the mean density of PAK6 staining, according to the manufacturer’s instructions.

miRNAs were isolated from cells using the miRNeasy Mini kit according to the manufacturer’s instructions. miRNA expression was then determined using the TaqMan MicroRNA assay kit on a 7500 Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA), in according to the manufacturer’s instructions. U6 was used as an endogenous reference. For each sample, independent experiments were repeated three times. The relative expression of miRNA was analyzed using the 2^−∆∆Ct method.

Human SW620 and LOVO colon cancer cells were treated with 5-Aza (15.55 nM) and PBA (1.5 nM) for 72 h.

Luciferase assay was performed to determine whether PAK6 was the direct target of miR-429. A wild-type or a mutant type of PAK6 3′-UTR was subcloned into the psiCHECK™2 luciferase miRNA expression reporter vector. The psiCHECK-PAK6–3′-UTR or psiCHECK2-mut PAK6–3′-UTR vector plus miR-429 or miR-429 inhibitors were cotransfected into colon cancer LOVO cells.

The cells were lysed in cold RIPA buffer. The BCA protein assay kit was used to determine the protein concentration. Protein was then separated with 10% SDS-PAGE, and transferred to a PVDF membrane. The PVDF membrane was blocked in 5% non-fat dried milk in PBS for 4 h. Subsequently, the PVDF membrane was incubated with specific primary antibodies for 3 h. After being washed with PBS three times, each time for 5 min, the PVDF membrane was incubated with the appropriate secondary antibody. After being washed with PBS three times, each time for 5 min, an ECL western blotting kit was used to detect the immune complexes on the PVDF membrane.

A wound-healing assay was performed to evaluate the cell migratory ability. Briefly, the cells were cultured to full confluence. Wounds of ~1 mm width were created with a plastic scriber, and the cells were washed and incubated in a serum-free medium. After wounding for 24 h, the cells were incubated in a medium including 10% FBS. Cultures at 0 and 48 h were fixed and observed under a microscope.

Cells treated with the indicated drugs for 72 h were starved in serum-free medium for 24 h, and then resuspended in serum-free medium. The cells were added to the upper chamber, while the lower chamber was filled with base medium containing 10% FBS. After incubation for 24 h, the cells attached to the bottom were stained with crystal violet for 20 min, washed and air dried. Invasive cells were observed under a microscope.

Data were presented as mean ± SD of three independent experiments. Statistical analysis was performed using SPSS 17.0 statistical software (SPSS, Inc., Chicago, IL, USA). The differences between groups were determined using the one-way ANOVA. P<0.05 was considered to indicate a statistically significant result.

We first examined the expression level of miR-429 in the SW620 and LOVO colon cancer cell lines, and normal HCEpiC colon epithelial cells. The expression level of miR-429 was significantly reduced in the SW620 and LOVO colon cancer cell lines, compared to the normal HCEpiC colon epithelial cells (Fig. 1).

After treatment with 5-Aza and PBA in SW620 and LOVO colon cancer cells, we determined the expression level of miR-429. As shown in Fig. 2A, miR-429 was significantly upregulated following treatment with 5-Aza and PBA. We also transfected the colon cancer cells with pre-miR-429 plasmid, and showed that the expression of miR-429 was increased after transfection (Fig. 2B). We then investigated the effect of miR-429 upregulation on colon cancer cell migration and invasion. The results showed that the upregulation of miR-429 markedly suppressed colon cancer cell migration (Fig. 3). We also found that the upregulation of miR-429 markedly suppressed colon cancer cell invasion (Fig. 4). These results suggested that miR-429 plays a suppressive role in colon cancer metastasis.

We focused on the targets of miR-429 in colon cancer cells, and the luciferase reporter assay data confirmed that PAK6 was a target gene of miR-429 in LOVO colon cancer cells (Fig. 5A). We also found that transfection with pre-miR-429 led to a decreased mRNA and protein expression of PAK6 (Fig. 5B and C), suggesting that miR-429 negatively mediated the expression of PAK6 in colon cancer cells.

We determined the expression of PAK6 in colon cancer tissues as well as their matched adjacent normal tissues. As shown in Fig. 6, the expression of PAK6 was positive in colon cancer tissues, but negative in their matched adjacent normal tissues, suggesting that PAK6 acts as an oncogene in colon cancer.

It has been established that cofilin plays a role in the regulation of cell motility (11). Accordingly, we investigated whether cofilin was involved in miR-429-mediated migration and invasion of SW620 and LOVO colon cancer cells. The antibody of p-cofilin was used to inhibit the activity of cofilin. As shown in Fig. 7, inhibition of cofilin activity markedly suppressed the migration of colon cancer cells, and enhanced the inhibitory effect of miR-429 upregulation on colon cancer cell migration. Similar results were observed in the cell invasion assay. As shown in Fig. 8, inhibition of cofilin activity also suppressed the invasion of colon cancer cells, even in colon cancer cells transfected with anti-miR-429. Taken together, the results suggested that cofilin acts a downstream effector in miR-429-mediated colon cancer cell migration and invasion.