The role of micro RNA in TLR2 signaling in colitis-associated Colon cancer

Abstract

Previous investigations from our laboratory have focused on defining how TLR2 signaling is involved in colitis and colitisassociated colon cancer. In a murine model of colitis-associated colon cancer, we observed an increased development of adenomas in TLR2-deficent (TLR2−/−) mice that was accompanied by an increased proliferation of intestinal epithelial cells after tissue injury. The chronic inflammation seen in TLR2−/− mice is thought to contribute to the development of colon cancer, but the molecular mechanisms are not completely understood. MicroRNAs (miRNA) are non-coding RNAs which bind to 3′ untranslated regions (UTR) of target mRNAs to repress protein translation or induce mRNA degradation. MiRNA have been shown to regulate multiple signaling pathways including development, differentiation, proliferation, and survival. MiRNA have been shown to play an important role in the differentiation and function of the intestinal epithelium and have been implicated in colorectal cancers and ulcerative colitis. To determine a role of miRNA in the development of intestinal adenomas we isolated intestinal epithelial cells (IEC) from WT and TLR2−/− mice and analyzed the expression of miRNA. TLR2−/− and wild-type (WT) littermate were either left untreated (=baseline) or injected with AOM and treated with DSS drinking water for one or three cycles (=AOM/DSS). Intestinal epithelial cells (IEC) were isolated using a previously published protocol. miRNA was extracted from isolated IEC of the colons of TLR2−/− or WT mice and miRNAs expression was analyzed using RT2 miRNA PCR Arrays. MiRNA expression data were analyzed using the ΔΔCt method. MiRNA expression data were confirmed by quantitative real-time PCR. A total of 88 different miRNAs were analyzed, 82 miRNA passed the quality control. 5 miRNA were down-regulated in IEC isolated from TLR2−/− mice compared to WT mice by more than 2.5-fold and 2 miRNA were up-regulated in TLR2−/− mice compared to WT mice by more than 2.5-fold at baseline. Additionally, 2 miRNA were non-detectable in IEC isolated from TLR2−/− mice but detectable in IEC from WT mice and 3 miRNA were detectable in TLR2−/− mice but not in IEC from WT mice at baseline. Surprisingly, only 3 miRNA were differentially expressed in TLR2−/− vs. WT IEC after one cycle of AOM/DSS treatment (early tumorigenesis) when changes >2.5-fold was used as a cut-off. Studies determining the target genes of differentially expressed miRNA are currently underway. Our data suggest that miRNA may play important roles in regulating intestinal homeostasis and responses to intestinal injury. Defining the role of miRNA and their target genes in TLR2 signaling, and their effect on the phenotype that we observed in TLR2−/− mice during intestinal tumorigenesis will allow us to contribute to our understanding of TLRs in the development of colon cancer.