MiR-34a as potential fecal biomarker Colitis-induced Colorectal cancer

Abstract

Colorectal cancer represents one of the most dreaded complications of chronic ulcerative colitis. Typical surveillance approaches (every other year colonoscopy with >30 random or targeted biopsies) have been suboptimal at protection against CRC. Our group has been developing biomarkers of field carcinogenesis for colorectal cancer risk stratification (reviewed in Gastroenterology, 2011). MicroRNAs (miRNAs) are being increasingly recognized as powerful cancer biomarkers given the critical role in regulation of gene expression. In particular miR34a has been shown to be a tumor promotor (oncomiR) involved in both initiation and progression phases of colon carcinogenesis. Our group has developed a novel modality of isolating and analyzing mucus layer fecal colonocytes(MLFC). MLFC are abraded from the normal colonic epithelium by passage of solid stool and therefore represents a potential means of field carcinogenesis. We have recently observed that miR 34a was overexpressed in the microscopically normal mucosa of both animal models (azoxymethane-treated rat) and patients harboring neoplasia elsewhere in the colon. In this study, we assess the potential translatability of fecal miR 34 to colitis-induced CRC. We used an in vivo and in vitro model using citrobacter rodentium as the model of inducing inflammation In the in vitro model, CRC cell line HT-29 was infected with C. rodentium and total RNA was processed for miRNA analysis. For the in vivo C57BL/J6 mice were infected with C. rodentium. Stool was collected with isolation of mucus layer fecal colonocytes (MLFC) and total RNA was extracted and processed for miRNA detection. Total RNA was processed for reverse transcription using miRNA reverse transcription kit (Applied Biosystems) and assayed for specific miRNAs using individual Taqman miRNA assays namely, miR-142-3p, miR-21, miR-34a, miR-146b, miR-148b-3p and miR-494 (Applied Biosystems) using manufacturer's instructions. The six miRNAs tested had been previously demonstrated to be upregulated in colonic field carcinogenesis. Out of the miRNAs tested, miR-34a was found to be significantly induced in HT-29 cells.(4.8 fold induction, p=0.02). We next both in the fecal colonocytes from C. rodentium infected mice and noted a dramatic induction (7.2 fold, p<0.05). To demonstrate the relevance to colon carcinogenesis, we used gold-standard model, the azoxymethane-treated rat (a carcinogen induced model). We noted that at premalignant time points there was a marked increase in mucus layer fecal colonocytes (4.2 to 6.2 fold, p<0.05) We present herein the first data that fecal miR 34a may be a marker for neoplastic risk of in both an inflammation model and a well-validated CRC models. Importantly, miR 34a is regulated by p53, one of the earliest events in colitis-induced CRC (found in flat dysplasia or histologically normal mucosa of patients harboring dysplasia). Thus, while confirmation for IBD dysplasia will be performed with standard DSS or transgenic (IL10 knockouts etc.) models, this work provides a powerful proof of concept that miR34a may serve as a biomarker for colitis-induced CRC. Furthermore, the development of mucus layer fecal colonocytes as a marker of field carcinogenesis may represent significant advance in the surveillance for IBD-related neoplasia.