Up-regulation of miR-224 promotes cancer cell proliferation and invasion and predicts relapse of colorectal cancer

A total of 108 stage I-II (UICC, 6th ed., 2002) colorectal patients received radical surgery at the First Department of General Surgery, the Affiliated Hospital of North Sichuan Medical College, from January 2004 to January 2009, were collected. All clinicopathological characteristics of patients with disease relapse (n=40) or without disease relapse (n=68) within 3 years after surgery were available for all patients. Disease relapse was defined as local recurrence or distant metastasis of colorectal cancer. All tissue specimens were derived from 108 patients who did not received neoadjuvant therapy before surgery. The patients who received postoperative adjuvant therapy were also excluded. To test whether miR-224 was differentially expressed between paired tumor and adjacent normal tissue in the same subject, we recruited a second cohort comprising 20 CRC patients. All tissue samples were immediately frozen in liquid nitrogen and stored at -80°C for subsequent analysis. The median follow-up time was 48.3 months (12.6–62.5 months) until June, 2012. Disease-free survival (DFS) was calculated from radical surgery to the first disease relapse. Informed written consent was obtained from each patient, and research protocols were approved by the Medical Ethics Committee of North Sichuan Medical College.

The human CRC cell line SW480 was purchase from American Type Culture Collection. The cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 u/ml penicillin and 100 mg/ml streptomycin, at 37°C in a humidified atmosphere of 5% CO₂.

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The PCR primers for miR-224 and U6 were purchased from Applied Biosystems (Applied Biosystems, Foster City, CA, USA). The PCR primers for SMAD4 were 5′-TGGCCCAGGATC AGTAGGT-3′ and 5′-CATCAACACCAATTCCAGCA-3′.The primers for β-actin: 5′-CCAAGGCCAACCGCGAGAAGATGAC-3′ and 5′-AGGGTACAT GGTGGTGCCGCCAGAC-3′. The first-strand cDNA was synthesized using the PrimeScript RT reagent Kit (TaKaRa, Dalian, China). Real-time PCR was performed using SYBR Premix Ex Taq (TaKaRa) and measured in a LightCycler 480 system (Roche, Basel, Switzerland). U6 or β-actin was used as internal control. Relative gene expression was calculated using 2^-ΔCT method, and fold change of gene was calculated using the equation 2^-ΔΔCT.

Ectopic expression of miR-244 in cells was achieved by transfection with Pre-miR-224 precursor (pre-miR-224) (Ambion, Foster City, CA, USA) using Lipofectamine 2000 (Invitrogen). 2 ×10⁵ cells were seeded into each well of a 6-well plate and transfected for 24 h or 48 h. Transfected cells were used in further assays or RNA/protein extraction.

2×10⁴ SW480 cells were plated onto 96-well plates for 24 h. The cells were then transfected with 50 nM pre-miR-224 or pre-miR-nc. At different time points (24 h, 48 h and 72 h), the culture medium was removed and replaced with culture medium containing 10μl of sterile MTT dye (5 mg/ml). After incubation at 37°C for 4 h, the MTT solution was removed, and 150μl dimethyl sulfoxide (DMSO) was added to each well followed by measuring the absorbance at 570 nm on an enzyme immunoassay analyzer (Bio-Rad).

For migration assays, 5×10⁴ cells transfected with either pre-miR-224 or pre-miR-nc were placed into Boyden chambers (Corning, Cambridge, MA, USA) with an 8.0mm pore membrane. For invasion assays, 5×10⁴ cells were placed into chambers coated with 150μg of Matrigel (BD Biosciences, Bedford, MD, USA). Medium containing 10% fetal bovine serum in the lower chamber served as the chemoattractant. After the cells were incubated for 48 h at 37°C in a humidified incubator with 5% CO₂, the cells remaining on the upper surface of the membranes were removed, whereas the cells adhering to the lower surface were fixed, stained with hematoxylin and counted under a microscope at a magnification of 400× to calculate their relative numbers.

Immunoblotting was performed to detect the expression of SMAD4 in CRC cell lines. Transfected cells were lysed in RIPA lysis buffer (ProMab Biotechnology). Protein was loaded onto a SDS-PAGE minigel and transferred onto PVDF membrane. After probed with 1:500 diluted mouse polyclonal SMAD4 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight, the blots were subsequently incubated with HRP-conjugated secondary antibody (1:5000). Signals were visualized using ECL Substrates (Millipore, MA, USA). GAPDH was used as an endogenous protein for normalization.

For luciferase reporter experiments, the wild-type and mutated 3′UTR of SMAD4 mRNA were subcloned into the Xho I and Not I site of the psicheck-2 vector (Promega) and the new vectors were named psicheck-2-SMAD4-WT and psicheck-2-SMAD4-MUT, respectively. The primers as shown in Table 1 were used to amplify specific fragments. For reporter assay, HEK 293T cells were plated onto 24-well plates at 2×10⁴ cells/well and transfected with 200 ng of psicheck-2-SMAD4-WT or psicheck-2-SMAD4-MUT and 40 nM pre-miR-224 or pre-miR-nc using Lipofectamine 2000 (Invitrogen). Firefly luciferase was used to normalize the Renilla luciferase. After transfection for 48h, cells were harvested and assayed with Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s protocols.

All data presented in this study have been repeated at least three times from three independent experiments. Continuous variables were expressed as the mean ± standard deviation. Measurement data were analyzed using Student's t-test, while categorical data were studied using chi-square test. Receiver operating characteristic (ROC) curve was used to determine the cut off value of miR-224 expression. The postoperative survival rate was analyzed with Kaplan–Meier method, and differences in survival rates were assessed with log-rank test. All statistical analyses were performed using SPSS 16.0 software (SPSS, Chicago, IL, USA). Two-sided P-values were calculated, and differences were considered significant at P-values of <0.05.

A total of 108 patients were included in this study with 40 patients in relapse group (relapse within 3 years after radical surgery) and 68 patients in non-relapse group (no relapse within 3 years after radical surgery). There were no differences between the two groups in terms of age, gender, tumor location, differentiation and TNM stage. The details were seen in Table 2.

In this study, we found that miR-224 expression in tumor tissues was significantly higher than that in normal tissues (P<0.01, Figure 1A). Using the samples from the second cohort, we found that the miR-224 expression levels were significantly up-regulated in the tissues of CRC patients with disease relapse (n=40) compared with those without disease relapse (n=68) (P<0.01, Figure 1B).The expression levels of the miR-224 were categorized as low or high in relation to the cutoff value (25.72) on the basis of ROC curve analysis. Therefore, 48 patients were included in the high expression group and 60 in the low-expression group. Among patients with miR-224 high expression, 27 patients relapsed (27/48, 56.3%), while only 13 patients relapsed (13/60, 21.7%) among patients with miR-224 low expression. Using chi-square test and Kaplan-Meier analysis, the results demonstrated that high miR-224 expression was significantly associated with disease relapse and a relative poorer disease-free survival rate (Figure 1B).

MiR-224 was upregulated in CRC, implicating its potential role in CRC cells biological properties. To further characterize the functional importance in CRC tumorigenesis, we examined the effect of miR-224 on the proliferation of CRC cells using MTT assay. Compared to pre-miR-nc, transfection with pre-miR-224 in SW480 cells led to an approximately 6-fold increase in miR-224 expression as detected by qRT-PCR (Figure 2A). We observed that overexpression of miR-224 significantly promoted the proliferation of SW480 cells, at 24, 48, 72 h after transfection (P<0.05, Figure 2B).

The potential roles of miR-224 in CRC cell migration and invasion were assessed using transwell migration and invasion assays. We observed that cell migration was significantly increased following transfection with pre-miR-224 compared with the negative control (P<0.01, Figure 3A,3B). We then examined the effect of miR-224 on cell invasion across an extracellular matrix and showed that in SW480 cells, the overexpression of miR-224 markedly enhanced the invasive potential compared with the control (P<0.05, Figure 3A,3B). These observations suggest that miR-224 plays an important role in promoting migration and invasive ability of CRC cells.

Analysis by using publicly available programs, TargetScan (http://​www.​targetscan.​org/​) and miRanda (http://​www.​microrna.​org/​microrna/​home.​do), indicates that SMAD4 is theoretically the target gene of miR-224 (Figure 4A). Therefore, in the current study, we further determined whether SMAD4 gene was an authentic target gene of miR-224 in CRC.

We performed a luciferase reporter assay to verify that miR-224 directly targets SMAD4.Sequences of the 3′UTR of the SMAD4 mRNA surrounding the two close miR-224 potential binding sites (site 1 and site 2) containing the wild-type (WT1).we cloned the regions of 3′UTR each containing one putative miR-224 binding site into the psicheck-2 vector and named as WT1 (site1, site2) and WT2 (site3) (Figure 4A). The reporter constructs harboring mutation of the miR-224 target sites were generated similarly (MUT1, MUT2 and MUT3) (Figure 4A). The luciferase reporter constructs were transfected into HEK 293T cells, along with pre-miR-224 or pre-miR-nc. Luciferase activites were then measured. The luciferase activity of WT1 (site1, site2) reporter transfected with pre-miR-224 was significantly decreased compared with control (P<0.01, Figure 4B), while the luciferase activity of the WT2 (site3) reporter was not interfered with after transfection with pre-miR-224 compared with control (P>0.05, Figure 4B). These data indicate that miR-224 may target SMAD4 gene through the seeding region of wild type 3′UTR (site1, site2). However, the luciferase reporter activity was not inhibited by miR-224 when the seeding sites were mutated.

To further confirm that SMAD4 was the downstream target of miR-224, we analyzed SMAD4 mRNA and protein levels in transfected SW480 cells by qRT-PCR and Western blot. Western blot analysis demonstrated that high expression of miR-224 dramatically suppressed the endogenous protein level of SMAD4 (P<0.01, Figure 5B), while mRNA remained unchanged (P>0.05, Figure 5A).Thus, SMAD4 is likely to be suppressed by miR-224 through translational inhibition.