Background
Colorectal cancer (CRC) is one of the most common malignancies in the world, with over one million new patients diagnosed every year [
1]. CRC progression is accompanied by the accumulation of mutations in tumor-suppressor genes and oncogenes, including
adenomatous polyposis coli (APC) [
2]. Inactivation of
APC is a major initiating event in colorectal tumorigenesis [
3]. Specifically, mutations in
APC are a leading cause of CRC [
4]. Mutations in
APC have been found in all patients diagnosed with familial adenomatous polyposis, as well as in almost 90% of patients diagnosed with CRC [
5]. Most of the mutations in
APC generate premature stop codons leading to truncated proteins that lack β-catenin binding sites. APC-free β-catenin stimulates the Wnt signaling pathway, leading to the active transcription of target genes such as c-Myc and cyclin D1, thereby promoting tumorigenesis [
6,
7].
APC and Axin serve as essential scaffolds for glycogen synthase kinase 3 beta (GSK-3β) and β-catenin, and impaired association of APC, Axin, with β-catenin leads to constitutive activation of the Wnt signaling pathway [
8,
9]. In the intestine, the canonical Wnt pathway maintains the proliferative cell layer in the crypts [
10]. Upon activation of the Wnt pathway, β-catenin is released from the cytoplasmic complex formed by APC, Axin, and GSK-3β. Consequently, β-catenin is then able to bind the T-cell factor/lymphoid enhancer factor binding factor (TCF/LEF) transcription factors, resulting in increased transcription of downstream targets such as c-Myc or cyclin D1. In contrast, in differentiated intestinal epithelial cells, APC acts as a negative regulator of the Wnt signaling pathway by binding to β-catenin in order to induce its degradation [
11].
MicroRNAs (miRNAs), are a class of naturally occurring small, noncoding RNAs comprising of 19 to 25 nucleotides, that are an important class of cellular regulators that modulate gene expression, and thereby influence cell fate and function [
12‐
16]. miRNAs function by binding to target mRNAs via sequence complementarity, and repress translation or induce degradation of their target mRNAs [
17,
18]. So far, a number of miRNAs have been ascribed oncogenic or tumor-suppressive functions, and they are involved in almost every type of cancer, including breast, lung, gastric carcinoma, and CRC [
19‐
22]. Some miRNAs have been studied for their roles in colorectal carcinogenesis [
23‐
26]. For example, miR-494 has previously been reported to be upregulated in CRC, and it promotes cell migration and invasion in CRC by directly targeting phosphatase and tensin homolog (PTEN) [
27]. The roles and potential mechanisms of miRNAs, mediated by APC, in CRC are still largely unknown.
Here, we report that miR-494 activates the Wnt/β-catenin signaling pathway by suppressing the expression of APC and consequently plays an important role in the development and progression of CRC.
Methods
Tissue specimens and immunohistochemistry (IHC) staining
The Ethics Committee of Institute of Zoology approved this study, and all patients gave their informed consent prior to surgery. Colon carcinoma tissues from human patients were obtained from Beijing 301 Military General Hospital (Beijing, China). The patients clinical characteristics are shown in Table
1. Detailed information including demography, clinical characteristics, histopathology, APC mutation status, and survival status were collected for all patients. Patients were followed after surgical treatment with a median follow-up of 61 months (range, 8–122 months). All patients were staged based on the Tumor-Node-Metastasis (TNM) classification. For mRNA extraction, samples were frozen in liquid nitrogen immediately after surgical removal and maintained at −80 °C until use. Additional samples were fixed in 10% neutral-buffered formalin overnight, processed, paraffin embedded, and sectioned. A patient tissue with one synonymous mutation in the codon encoding amino acid 1828 in APC, that did not cause a change in amino acid sequence, was selected for IHC analysis. IHC analysis was performed using 3 μm sections which were incubated with an anti-APC antibody (Abcam catalog no. 154906) overnight at 41 °C in a humidified chamber, followed by incubation with an HRP-conjugated secondary antibody for 2 h. Staining was completed after 5 to 10 min incubation with 3,3′-diaminobenzidine (DAB) as substrate, which resulted in a brown-colored precipitate at the antigen site.
Table 1
Clinical correlation between miR-494 expression and other clinicopathological characteristics in CRC
Age (years) | 46 | 20 | 0.476 |
≤ 60 | 24 | 11 | |
> 60 | 22 | 9 | |
Sex | | | 0.887 |
Men | 28 | 12 | |
Women | 18 | 8 | |
TNM stage | | | *0.004 |
I + II | 19 | 6 | |
III + IV | 27 | 14 | |
Differentiation | | | *0.034 |
Well | 35 | 14 | |
Poorly | 11 | 6 | |
APC mutation status | | | 0.567 |
Wild-type | 5 | 2 | |
Mutant | 41 | 18 | |
Survival status | | | 0.476 |
Dead | 22 | 9 | |
Alive | 24 | 11 | |
Cell lines and cell culture
Human colon cancer cell lines were obtained from American Type Culture Collection (Manassas, VA). HCT-116 and HT-29 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cells were maintained in a humidified incubator equilibrated with 5% CO2 at 37 °C.
Microarray analysis
Total RNA was extracted from 17 tumor tissues and paired normal colorectal tissues using TRIzol reagent (Invitrogen). miRNA microarray profiling was performed as previously described [
28]. Data analysis was performed using GeneSpring GX software (Agilent). An miRNA was designated as overexpressed if the expression in tumor tissues was greater than 2.0-fold that in normal colorectal epithelial tissues.
Real-time PCR detection of mature miRNAs
Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. All-in-One™ miRNA First-Strand cDNA Synthesis Kit (Genecopoeia) was used to transcribe 10 μL of purified RNA. Real-time PCR, using miR-specific primers and universal adaptor PCR primers (Genecopoeia), was performed with a Stratagene Mx3000P QPCR System (Genetimes Technology, Shanghai, China). The reactions were incubated in a 96 well plate at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s, and 60 °C for 1 min. All reactions were run in triplicate.
RNA extraction and real-time PCR
Total RNA was isolated as described above. For reverse transcription into cDNA, 1 μg of RNA from each sample was incubated with random primers and then subjected to real-time PCR. Real-time quantification was performed using a Stratagene Mx3000P quantitative PCR system (Genetimes Technology, Shanghai, China). The reaction solutions were incubated in a 96-well plate at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s, and 60 °C for 1 min. All reactions were run in triplicate. The primer pairs used were as follows: APC, 5′-CTGCGGACCGAGGTTGGCTC-3′ (forward) and 5′-CTTCCTGCCAGACGCTCGCC-3′ (reverse); and GAPDH, 5′-CTCTGCTCCTCCTGTTCGAC-3′ (forward) and 5′-CGACCAAATCCGTTGACTCC-3′ (reverse).
Oligonucleotide transfections
All commercial miRNAs and the APC targeted small interfering RNAs (siRNAs) were synthesized and purified by RiboBio Co. (Guangzhou, China). The APC siRNA sequences used were APC-siRNA1, 5′-GGATCAGCCTATTGATTAT-3′ and APC-siRNA2, 5′-GTACGCCAGTCAACTTTCA-3′. Transfections of the aforementioned miRNAs and siRNAs were performed using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions.
Western blot analysis
Western blotting was performed as previously described (31). The commercial antibodies used were anti-APC (Abcam catalog no. 154906), anti-β-catenin (Santa Cruz catalog no. 7963), anti-cyclin D1 (Cell Signaling Technology catalog no. 2978S), and anti-fibrillarin (Abcam catalog no. ab5812).
HCT-116 cells were transfected with the indicated long-lasting synthetic miRNAs. The commercial miRNAs used were a negative miR-control (Ribobio catalog no. miR04101–1-10), a miR-494 mimic (Ribobio catalog no. miR40002816–1-10), a negative anti-miR-control (Ribobio catalog no. miR03101–1-10), or an anti-miR-494 (Ribobio catalog no. miR30002816–1-10). At 48 h post-transfection, cells were plated in triplicate at 500 cells per 60 mm dish. After 7 days, the colonies were stained with 0.1% crystal violet solution and counted using a light microscope.
Flow cytometry
Cells were harvested by trypsinization, washed with ice-cold PBS, and fixed in 70% ice-cold ethanol in PBS. Before staining, cells were sedimented in a chilled centrifuge and resuspended in cold PBS. Bovine pancreatic RNase (Sigma–Aldrich) was added to a final concentration of 2 μg/mL, and the cells were incubated at 37 °C for 30 min, followed by incubation with 20 μg/mL propidium iodide (Sigma–Aldrich) for 20 min at room temperature. The cell cycle profiles of 2 × 104 cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences).
Statistical analysis
All experiments were repeated at least three times. Data shown are presented as mean ± SD of three or more independent experiments. A Student’s t-test was used for statistical analysis of data. Differences in clinicopathological characteristics between the two groups were examined by Fisher’s and chi-square tests. All P-values were determined using two-sided tests and statistical significance was based on a P-value of 0.05. Correlation analysis was performed using scatter plots and Pearson’s correlation analysis. The correlation coefficients and the corresponding P-values were calculated by the cor.test function of R.
Discussion
The key finding in this study is that miR-494 expression is markedly upregulated in CRC tissues compared to normal colorectal tissues. Furthermore, ectopic expression of miR-494 enhances the proliferation of CRC cells, whereas inhibition of miR-494 expression has the opposite effect. Moreover, we demonstrate that miR-494 upregulation in CRC cells activates the Wnt/β-catenin pathway by directly targeting APC. These findings suggest that this dysregulation of miR-494 levels may play an important role in promoting proliferation and tumorigenesis in CRC.
Aberrant Wnt/β-catenin signaling has been linked to the pathogenesis of various diseases, including cancer, and is thought to promote tumor progression [
23‐
28]. Multiple key negative regulators, such as APC, GSK-3β, and Axin, are suppressed in cancer, contributing to the promotion of tumor progression through regulation of the Wnt/β-catenin pathway. For instance, inactivation of GSK-3β via kinases promotes mammary tumorigenesis by activation of the canonical Wnt pathway [
31]. In mouse models, loss of APC function has been shown to lead to colorectal tumorigenesis through hyperactivation of Wnt/β-catenin signaling [
32]. Herein, we demonstrate that miR-494 suppresses APC expression by directly targeting the 3′-UTR of the APC mRNA. Consistent with the tumor-suppressive effects of APC, miR-494 was upregulated in CRC, and its overexpression dramatically promoted CRC cell proliferation.
The Wnt/β-catenin signaling pathway is controlled at multiple levels. β-catenin is the major cellular effector of Wnt signaling and is normally retained by a degradation complex containing Axin, APC, GSK-3β, and casein kinase 1a. In the absence of Wnt, the complexed β-catenin is targeted for proteasome-mediated degradation following its phosphorylation and ubiquitination [
33‐
35]. APC is crucial for the capture of β-catenin by the degradation complex, and binds β-catenin directly via its central armadillo repeats [
36]. Upon Wnt activation, Axin is removed from the destruction complex allowing the release of β-catenin. The accumulated β-catenin enters the nucleus and binds to the TCF/LEF family of transcription factors to induce target gene expression [
37,
38]. A key event therefore in both Wnt signal transduction and cancer cell proliferation is the regulation of β-catenin stability and activity. In this study, we have uncovered a mechanism for controlling APC expression, and thereby the activity of the Wnt pathway, through the action of miR-494, and suggest that miR-494 contributes to the pathogenesis of CRC.
miR-494 is a product of the
Dlk1-Dio3 region, which is located on human chromosome 14q32, and constitutes one of the largest miRNA clusters in the human genome. The expression patterns of miR-494 are not consistent across different types of tumors, suggesting that there is tissue-dependent regulation of expression of this miRNA, among other potential mechanisms [
39‐
44]. In this study, we demonstrated that miR-494 is upregulated in CRC tissues compared to adjacent, non-cancerous tissues from the same patient. Furthermore, we demonstrated that ectopic miR-494 expression increases the growth rate of HCT-116 cells compared to a control miRNA, whereas suppression of miR-494 expression inhibited cell proliferation and colony formation. The data strongly suggest that upregulation of miR-494 correlates with clinical CRC progression and that miR-494 may function as an onco-miRNA.
Conclusions
Our study has shown that elevated expression of miR-494 promotes cell proliferation and tumorigenesis in CRC by suppressing the expression of APC, an inhibitor of β-catenin signaling. These findings uncover a novel molecular mechanism for the hyperactivation of the Wnt/β-catenin signaling pathway in CRC, suggesting that miR-494 may serve as a potential therapeutic target for the treatment of CRC.
Acknowledgements
The authors wish to thank; the patients involved in the study and their guardians; Xin Tong for providing CRC tissue; Meng Wang for providing the APC 3′-UTR vector construct; and Haixi Sun for bioinformatic analysis. We also thank all members of our group for discussions and assistance with this study.
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