MicroRNA-130b regulates the tumour suppressor RUNX3 in gastric cancer
Introduction
RUNX3 has been identified as a critical tumour suppressor in many human cancer types through the work of our laboratory and others.1, 2, 3 RUNX3 knockout mice developed gastric hyperplasia, an early precursor of gastric cancer.2 Gastric epithelial cells derived from RUNX3–/–p53–/– mice induced the formation of tumours when inoculated into nude mice, while those derived from RUNX3+/+p53–/– mice did not. Tumourigenicity of human gastric cancer cell lines in nude mice was inversely related to their level of RUNX3 expression, and a mutation (R122C) occurring within the conserved Runt domain eliminates the tumour suppressive activity of RUNX3. RUNX3 is also a potent activator of many developmental and homeostatic signalling components linked to tumour suppression such as cyclin-dependent kinase inhibitor CDKN1A (p21WAF1/Cip1)4 and pro-apoptotic Bim.5 Recent results indicate that RUNX3 forms a ternary complex with β-catenin/TCF4 and attenuates the Wnt signalling pathway.1
Other studies have shown that RUNX3 expression is significantly reduced in many human tumour types including gastric cancers and gastric pre-neoplastic lesions2 as well as colorectal,6 breast,7 endometrial,8 oral squamous cell9 and oesophageal squamous cell carcinomas.10 Moreover, inactivation of RUNX3 has been correlated with advanced disease stage and poor prognosis.6, 11
The downregulation of RUNX3 has been attributed to a number of mechanisms, including hypermethylation of its promoter region,2 cytoplasmic mislocalisation,12 histone modification13 and hemizygous deletion.14 However, there remains a proportion of tumours that show reduced RUNX3 expression in the absence of these mechanisms, suggesting that other factors may be involved in the regulation of RUNX3.
MicroRNAs (miRNAs) are recently characterised, highly conserved, small RNA molecules of approximately 21–25 nucleotides encoded in the genomes of plants and animals.15 They suppress gene expression either by repressing translation or by direct sequence-specific cleavage through the action of the RNA-induced silencing complex (RISC) following binding to the 3′-untranslated region (3′UTR) of mRNA.16 Differential expression of miRNA between tumour tissue and normal tissue has been observed in various cancer types17 suggesting a possible link between miRNA expression and the development of cancer. miRNAs have been observed to regulate both oncogenes, such as ras,18 and tumour suppressor genes, such as PTEN.19
In exploratory analysis using bioinformatic algorithms, the RUNX3 3′UTR was found to contain numerous potential miRNA-binding sites, indicating that miRNA may play a role in regulating RUNX3 expression. The aims of this study were to determine whether RUNX3 is a target of miRNA regulation and to evaluate the role of this mechanism in gastric cancer.
Section snippets
Selection of candidate miRNAs
Candidate miRNAs for binding to RUNX3 were identified using four web-based bioinformatic algorithms. TargetScan, PicTar and miRanda predict miRNA-binding sites based on complementarity of nucleotide sequence to the 3′-untranslated region of RUNX3 mRNA. miRBase Target is based on miRanda algorithms.20
Cell lines
Eight gastric cancer cell lines were obtained from American-Type Culture Collection (Manassas, VA) (AGS, SNU1, SNU5 and SNU16) and Japanese Collection of Research Bioresources (Osaka, Japan) (AZ521,
Consensus from four bioinformatic algorithms identifies 10 candidate miRNAs that bind to RUNX3 3′UTR
The four algorithms (TargetScan, PicTar, miRanda, miRbase Target) used to predict miRNA binding to the 3′UTR of RUNX3 identified 32, 7, 21 and 17 candidate miRNAs, respectively (Supplementary Table 1). Greater specificity in miRNA prediction can be attained by using the consensus of multiple algorithms23 and hence miRNA predicted to bind to RUNX3 by at least two algorithms was selected for further study. Ten miRNA were selected using this criterion: miR-19a, 19b, 130a, 130b, 301a, 301b, 326,
Discussion
Accumulating evidence indicates that miRNA repression of tumour suppressor genes may be a common mechanism involved in tumourigenesis, including the regulation of RB1 by miR-106a24 and PTEN by miR-21.25 Aberrant expression of a growing number of miRNAs has also been linked to the development of gastric cancer. These include upregulation of miR-21 which targets RECK,26 an anti-metastasis factor, and upregulation of miR-27a which targets prohibitin,27 a negative regulator of cell proliferation.
In
Conflict of interest statement
None declared.
Acknowledgements
This study was supported by grants from the National Medical Research Council of Singapore (NMRC/TCR/001/2007), the Singapore Cancer Syndicate (SCS#BU51), and the Singapore National Research Foundation and the Ministry of Education under the Research Center of Excellence Programme.
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