miR‑20a is an independent prognostic factor in colorectal cancer and is involved in cell metastasis
- Authors:
- Published online on: April 15, 2014 https://doi.org/10.3892/mmr.2014.2144
- Pages: 283-291
Abstract
Introduction
Colorectal cancer (CRC) is the third most common malignancy and the fourth most frequent cause of cancer-related mortality worldwide (1). Despite numerous advances in treatment, patients with CRC still have an unfavorable prognosis, and 25–40% of patients develop recurrence following surgery (2). Therefore, it is crucial to enhance the understanding of the mechanisms involved in CRC metastasis and to identify potential prognostic biomarkers and therapeutic targets.
Epithelial-mesenchymal transition (EMT) is a key step towards cancer metastasis. Loss of E-cadherin expression is a hallmark of the EMT process and is likely to be required for enhanced tumor cell motility (3–5). Epithelial cells lose their epithelial characteristics and acquire mesenchymal characteristics by the downregulation of E-cadherin (6). EMT has a crucial role in tumor cell invasion and migration in numerous types of cancer, including CRC (7). Inducers of EMT include certain protein polypeptides, transcription factors, growth factors and microRNAs (miRNAs) (8).
miRNAs are small, noncoding RNA gene products of ~22 nucleotides that are found in a variety of organisms. miRNAs have key roles in regulating the translation and degradation of mRNAs through base pairing to partially complementary sites, predominantly in the 3′-untranslated regions (UTRs) of mRNAs (9–11). It is well known that miRNAs have important regulatory functions in basic biological processes, including development, cellular differentiation, proliferation and apoptosis, which affect major biological systems, including stemness, immunity and cancer (12,13). Data suggest that dysregulation of miRNAs is an important step in the pathogenesis, from initiation to metastasis, of numerous types of cancer, including CRC (14–16). Accumulating evidence has demonstrated that the induction of EMT and aberrant expression of miRNAs are associated with tumorigenesis, tumor progression, metastasis and relapse in certain cancers, including CRC (17,18).
Recently, miR-20a has been shown to be deregulated in several types of cancer (19–21). In particular, miR-20a has a key role in the regulation of tumor proliferation, metastasis and EMT (19–24). Chai et al (25) reported that the overexpression of miR-20a contributed to the resistance of colorectal adenocarcinoma to chemotherapeutics. However, the clinical and biological roles of miR-20a in CRC remain to be fully elucidated.
The aim of the present study was to assess the clinical significance of miR-20a in CRC and to investigate the effects of miR-20a on the migration, invasion and EMT of CRC cells, and to further discuss the mechanisms of action of miR-20a by identifying its potential target gene.
Materials and methods
Patients and tissue samples
Paired tissue specimens (tumor and adjacent normal mucosa) were obtained from 86 patients with primary CRC who underwent surgery without preoperative treatment at the First Department of General Surgery, the Affiliated Hospital of North Sichuan Medical College (Nanchong, China), from 2005 to 2008, following receipt of their informed consent. All tissue samples were immediately frozen in liquid nitrogen and stored at −80°C for subsequent analysis. Specimens were stained with hematoxylin and eosin and examined histopathologically. Sections that consisted of >80% carcinoma cells were used to prepare the total RNA. Clinicopathological information, including age, gender, tumor size, histological type, depth of invasion, location, lymph node metastasis, lymphatic invasion and distant metastasis, was available for all patients. The study was approved by the Medical Ethics Committee of North Sichuan Medical College.
Cell culture
The human SW480 CRC cell line was obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured 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.
Quantitative polymerase chain reaction (qPCR)
Total RNA was extracted using TRIzol® reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) for both miR-20a and SMAD family member 4 (SMAD4) mRNA analyses. For detection of miR-20a expression, qPCR was performed using the QuantiMir RT kit (System Biosciences, Mountain View, CA, USA) according to the manufacturer’s instructions with miR-20a-specific primers (Applied Biosystems, Foster City, CA, USA). The amplification profile was denaturation at 95°C for 10 min followed by 45 cycles of denaturation at 95°C for 15 sec, annealing at 60°C for 30 sec and extension at 72°C for 1 min. For the detection of SMAD4 mRNA expression, qPCR was performed using SYBR® Green PCR Master Mix (Applied Biosystems). The primers for SMAD4 were as follows: 5′-TGGCCCAGGATCAGTAGGT-3′ and 5′-CATCAACACCAATTCCAGCA-3′. The primers for β-actin were: 5′-CCAAGGCCAACCGCGAGAAGATGAC-3′ and 5′-AGGGTACATGGTGGTGCCGCCAGAC-3′. The amplification profile was denaturation at 95°C for 30 min, followed by 40 cycles of denaturation at 95°C for 10 sec, annealing at 60°C for 10 sec and extension at 72°C for 1 min. U6 and β-actin were used as internal controls. Relative gene expression was calculated using the equation 2−ΔCT. The fold-change of gene expression was calculated using the equation 2−ΔΔCT. All reactions were run in triplicate.
Transfection of miRNA
Ectopic expression of miR-20a in cells was achieved by transfection with Pre-miR™ miR-20a precursor (pre-miR-20a; Applied Biosystems) using Lipofectamine 2000 (Invitrogen Life Technologies). A total of 2×105 cells were seeded into each well of a six-well plate and transfected for 24 or 48 h. Transfected cells were used in further assays or RNA/protein extraction.
Migration and invasion assays
To assess cell migration and invasion, 5×104 SW480 cells transfected with either pre-miR-20a or the negative control miRNA precursor (pre-miR-nc; Applied Biosystems) were seeded into Transwell chambers (8.0-μm pore size; Corning, Inc., New York, NY, USA) uncoated or coated with Matrigel™ (BD Biosciences, Bedford, MA, USA). Medium containing 10% fetal bovine serum in the lower chamber served as the chemoattractant. Following incubation for 48 h at 37°C in a humidified incubator with 5% CO2, cells that did not migrate through the pores were mechanically removed using a cotton swab. The migrated cells attached to the bottom of the membrane insert were fixed with methanol at room temperature for 5 min and stained with hematoxylin. The number of migrated or invaded cells on the lower surface of the membrane was then counted under a microscope at a magnification of ×400.
Western blot analysis
To isolate the proteins, cells collected from the six-well plates were washed twice with phosphate-buffered saline and lysed in radioimmunoprecipitation assay lysis buffer (ProMab Biotechnologies, Inc., Richmond, CA, USA). Lysates were kept on ice for 30 min and then centrifuged at 13,000 × g for 30 min. The supernatant was collected and then 20 μg of each of the proteins was separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. Following blocking in 5% skimmed milk, the membranes were incubated with the respective antibodies: Mouse anti-E-cadherin (1:1,000; BD Biosciences, San Jose, CA, USA), mouse anti-vimentin (1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), mouse anti-SMAD4 (1:500; Santa Cruz Biotechnology, Inc.) and mouse anti-GAPDH (1:1,000; Santa Cruz Biotechnology, Inc.) overnight at 4°C. Following incubation with the appropriate secondary antibody, the bands were visualized using ECL-Plus™ reagents (GE Healthcare Bio-Science Corp., Piscataway, NJ, USA). The density of the SMAD4 and GAPDH bands was measured using Image J software (National Institutes of Health, Bethesda, MD, USA), and values were normalized to the densitometric values of GAPDH in each sample. Fold-changes in the amount of protein were then calculated for the experimental sets compared with the control.
Luciferase assay
For the luciferase reporter experiments, the wild-type and mutated 3′UTRs of SMAD4 mRNA were subcloned into the XhoI and NotI sites of the psicheck-2 vector (Promega Corp., Madison, WI, USA) and the new wild-type and mutant vectors were referred to as psicheck-2-SMAD4-WT and psicheck-2-SMAD4-MUT, respectively. The following primers were used to amplify specific fragments: WT sense, 5′-CACAACTCGAGCACTGTTCTGCAAAGGTGGC-3′ and WT antisense, 5′-AAGGAAAAAAGCGGCCGCGACCTT CTGAGCAAGGCAGT-3′; MUT sense, 5′-CTTTTCGTG AAATGAGTCCAATCTCAGTGATGAGG-3′ and MUT antisense, 5′-CTCATTTCACGAAAAGAAAAAAAAAAT CTTAAAAATCA-3′. For the reporter assay, HEK 293T cells (ATCC, Manassas, VA, USA) were seeded onto 24-well plates at 2×104 cells/well and transfected with 200 ng psicheck-2-SMAD4-WT or -SMAD4-MUT and 40 nM pre-miR-20a or -nc using Lipofectamine 2000 (Invitrogen Life Technologies). Firefly luciferase was used to normalize the Renilla luciferase. Following transfection for 48 h, cells were harvested and assayed with the Dual-Luciferase Reporter Assay System (Promega Corp.) according to the manufacturer’s instructions.
Bioinformatics analysis
The miRNA targets predicted by computer-aided algorithms were obtained from TargetScan (http://www.targetscan.org) and miRanda (http://www.microrna.org).
Statistical analysis
Values are expressed as the mean ± standard deviation from at least three separate experiments performed in triplicate. The gene expression levels in CRC tissue samples were compared with those in normal adjacent mucosa using the Wilcoxon signed-rank test. Measurement data were analyzed using the two-tailed Student’s t-test, while categorical data were studied using the χ2 test. The correlation between miR-20a levels and SMAD4 expression was analyzed using Pearson’s correlation. The postoperative survival rate was analyzed using the Kaplan-Meier method, and differences in survival rates were assessed using the log-rank test. A Cox proportional hazards model was used for multivariate analysis. The findings were considered significant at a P-value of <0.05. All statistical analyses were performed using the SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA).
Results
Expression of miR-20a in CRC tissue samples and normal adjacent mucosa
The expression levels of miR-20a in tumor and normal tissue samples from 86 patients are shown in Fig. 1. Most tumor tissue samples from patients with CRC (75/86; 87.21%) showed elevated levels of miR-20a, unlike the corresponding normal tissue samples (P<0.001).
Correlation between miR-20a expression and clinicopathological characteristics in CRC
The expression levels of miR-20a were categorized as low or high relative to the median value (4.65). High expression rates of miR-20a in CRC tissue samples with respect to several standard clinicopathological characteristics are listed in Table I. High expression of miR-20a was significantly correlated with lymph node and distant metastases (P<0.05, Table I). However, no significant correlation was observed between miR-20a expression and other clinicopathological features, including age, gender, tumor size, histological type, depth of invasion, tumor location and lymph node invasion (P>0.05, Table I).
 | Table IAssociation of miR-20a expression with clinicopathological factors of patients with colorectal cancer. |
Correlation between miR-20a expression and prognosis of patients with CRC
Overall survival curves were plotted according to miR-20a mRNA expression levels using the Kaplan-Meier method. As shown in Fig. 2, the overall survival rate was significantly lower in patients with high miR-20a expression than that in patients with low expression levels (P=0.021). Univariate analysis using the Cox proportional hazards model identified seven prognostic factors: Histological type, tumor size, depth of invasion, lymph node metastasis, lymph node invasion, distant metastasis and miR-20a expression. The other clinicopathological features (age, gender and location) were not statistically significant prognostic factors (Table II). A multivariate analysis of the prognostic factors using the Cox proportional hazards model confirmed that high miR-20a expression was a significant independent predictor of low survival rates of patients with CRC (P=0.045) in addition to the presence of lymph node metastasis (P=0.025) and distant metastasis (P<0.001) (Table III).
miR-20a regulates CRC cell invasion and migration in vitro
To investigate the role of miR-20a in CRC metastasis, the role of miR-20a in the migration and invasion of SW480 cells was examined (Fig. 3). Levels of miR-20a expression were observed to be significantly increased following transfection with pre-miR-20a compared with those following transfection with pre-miR-nc (Fig. 3A). Furthermore, cell migration was significantly increased following transfection with pre-miR-20a as compared with that following transfection with the negative control (P<0.01, Fig. 3B). The effect of miR-20a on SW480 cell invasion across an extracellular matrix was then assessed, revealing that the overexpression of miR-20a markedly enhanced cell invasion as compared with the control (P<0.01, Fig. 3C). These observations suggested that miR-20a may have an important role in promoting migration and the invasive potential of CRC cells.
miR-20a induces EMT-like changes
pre-miR-20a-transfected SW480 cells were observed to undergo EMT-like transformation, as evidenced by the loss of cell-cell adhesion and alterations in morphology from a round, compact shape to an elongated, fibroblast-like morphology with scattered distribution, which may facilitate cell migration (Fig. 4A). In addition, western blot analysis indicated that overexpression of miR-20a downregulated the epithelial marker E-cadherin, while it upregulated the mesenchymal marker vimentin (Fig. 4B). Therefore, overexpression of miR-20a may induce EMT in SW480 cells.
SMAD4 is a target of miR-20a
Since miR-20a has a pivotal function in the metastasis of CRC cells, its putative target genes with metastatic functions were investigated using the online search tools TargetScan (http://www.targetscan.org) and miRanda (http://www.microrna.org). Among these genes, SMAD4, one of the mediators of transforming growth factor β (TGF-β) signaling, was predicted to be a theoretical target of miR-20a based on putative target sequences at position 1,357–1,363 of the SMAD4 3′-UTR (Fig. 5A). Therefore, it was further investigated whether SMAD4 was the authentic target gene of miR-20a in CRC.
To directly address whether miR-20a bound to the 3′-UTR of the target mRNA, a luciferase reporter vector containing the SMAD4 3′-UTR with the putative miR-20a binding sites was generated. Correspondingly, a mutant reporter vector containing the SMAD4 3′-UTR with a mutation at the putative miR-20a binding site was produced. As shown in Fig. 5B, a marked reduction in luciferase activity was observed in cells transfected with pre-miR-20a as compared with pre-miR-nc-transfected cells (P<0.01). By contrast, no change in luciferase activity was observed in cells transfected with the mutant 3′-UTR constructs.
It was then determined whether the overexpression of miR-20a can lead to downregulation of SMAD4 protein expression. Western blot analysis showed that SMAD4 protein levels were markedly reduced in SW480 cells overexpressing miR-20a as compared with those in the control (P<0.01, Fig. 5C). To further explore the correlation between miR-20a and SMAD4 expression in CRC tissue samples, miR-20a and SMAD4 mRNA were examined in 10 sets of CRC tissue samples. Using Pearson’s correlation analysis, a significant inverse correlation between miR-20a and SMAD4 mRNA was observed (P=0.033, Fig. 5D). Taken together, the results suggest that miR-20a may attenuate the expression of SMAD4 by directly targeting the 3′UTR of SMAD4.
Discussion
Accumulating evidence has indicated that dysregulation of miR-20a is associated with the development of cancer. However, the function of miR-20a in the development and progression of cancer is controversial. High levels of miR-20a have been found to correlate with malignant phenotypes and unfavorable prognosis in hepatocellular and gallbladder carcinoma (19,20). Recently, it was shown that miR-20a encoded by the miR-17–92 cluster enhances the proliferation and metastatic potential of ovarian cancer and osteosarcoma cells (21,22). By contrast, miR-20a overexpression inhibited proliferation and metastasis in breast and pancreatic cancers (23,24). These controversial results may reflect the diverse roles of miR-20a in different types of cancer.
The present study demonstrated that miR-20a was frequently upregulated in CRC tissue samples as compared with normal adjacent mucosa, which was consistent with previous studies (26,27). Furthermore, the patient group with high miR-20a expression levels showed a greater incidence of lymph node and distant metastases compared with the low miR-20a expression group, strongly suggesting that miR-20a may be involved in the metastasis of CRC. In addition, patients whose tumors had higher miR-20a expression levels had a significantly lower overall survival rate, indicating that high miR-20a levels are a marker of unfavorable prognosis for patients with CRC. Multivariate analysis indicated that high miR-20a expression was an independent prognostic factor for survival. However, Yu et al (26) reported that miR-20a was not an independent prognostic biomarker among the miR-17–92 cluster in colon cancer. This inconsistency may be due to differences in sample origin, tumor clinicopathological characteristics or different detection methods of the studies. Therefore, multi-institutional, prospective randomized trials are required before a consensus can be reached.
Given that miR-20a was upregulated in CRC tissue samples, it was speculated that miR-20a may have an oncogenic effect in CRC. As expected, ectopic expression of miR-20a promoted the migration and invasion of SW480 cells. In addition, the present study revealed that cells with high expression of miR-20a had a spindle-like morphology and that the upregulation of miR-20a decreased the expression of E-cadherin, while it increased the expression of vimentin, suggesting that miR-20a is involved in the regulation of EMT. The above data suggest that miR-20a is involved in modulating cell metastasis in CRC.
To address the molecular mechanisms involved in the miR-20a-mediated changes in biological properties, SMAD4 was selected for further study as it was predicted to be a target of miR-20a by bioinformatics analysis. SMAD4 is a member of the evolutionarily conserved family of SMAD proteins, which are transmitters of signals from the TGF-β superfamily of cytokines (28). It has been suggested that SMAD4 can function as a tumor suppressor gene in gastrointestinal carcinoma (29,30). The results in the present study indicated that SMAD4 is a direct target gene of miR-20a in CRC, since overexpression of miR-20a downregulated SMAD4 protein expression. Furthermore, miR-20a expression was inversely correlated with SMAD4 expression in CRC tumors, and overexpression of miR-20a significantly reduced the activity of a luciferase reporter containing the 3′UTR sequence of SMAD4. Combining these experimental results with the bioinformatics analysis led to the conclusion that SMAD4 is a target gene of miR-20a in CRC.
Loss of SMAD4 is considered to be a genetic step associated with the advanced stages of the disease, and frequently occurs in metastatic CRC (31). SMAD4 inactivation is rarely detected in adenomas; however, it strongly increases in frequency at late, metastatic stages (32,33). Deckers et al (34) demonstrated that the knockdown of SMAD4 in breast cancer cells strongly attenuated the transformation of epithelial cuboidal cells into parallel aligned fibroblastic-like cells, as well as the downregulation of E-cadherin and the upregulation of N-cadherin, suggesting that SMAD4 acts as an EMT suppressor (34). Further studies have found a dependency of TGF-β-induced EMT on SMAD4 expression (35,36). A previous study reported that the invasion suppressor E-cadherin is a target gene in the SMAD4 signaling network and re-expression of SMAD4 in SW480 human colon carcinoma induced E-cadherin expression (37). Pohl et al (38) reported that loss of SMAD4 promotes migration and invasion and mediates EMT in the SW480 CRC cell line. In the present study, restoration of miR-20a induced EMT and promoted CRC cell migration and invasion, which may be attributed to miR-20a-mediated downregulation of SMAD4 expression.
In conclusion, the present study has provided novel insights into the role of miR-20a in CRC. The results showed that miR-20a is an independent prognostic factor for patients with CRC and provided a potential mechanism for SMAD4 dysregulation and its contribution to CRC cell migration, invasion and EMT. miR-20a may function as a potential oncogene in CRC and has a potential application in cancer therapy.
Acknowledgements
The present study was supported by the Scientific Research Fund of Sichuan Provincial Education Department of China (CBY12-A-ZD16).
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