694 Identification of novel epigenetic biomarkers in colorectal cancer, GLDC and PPP1R14A

Background: MicroRNAs (MiR) are small (16−29 nt), single-stranded, noncoding RNA molecules that regulate gene expression via cleavage of targeted mRNA or via translation repression. miRs are implicated in various physiological and pathological processes, including neoplasia. Production and function of miR requires a set of proteins, collectively referred as the miR machinery. Two ribonucleases, Drosha (in the nucleus) and Dicer (in the cytoplasm) process the primary transcripts (pri-miR) to generate mature miR. miR is incorporated into the RNA-induced silencing complex (RISC) that binds on target mRNA and mediates RNAi functions. Dicer, Argonaute-2, (Ago2) and TRBP are major constituents of RISC. Herein, we explored for the first time the expression and distribution of Drosha, Dicer, Ago2, TRBP in colon cancers and investigated their role in colon carcinogenesis. Materials and Methods: Three human colon cancer cell lines (Caco-2, DLD-1, HT-29) were examined. Western blotting was performed for Dicer, Ago2, TRBP and Drosha detection. Immunoprecipitation was used for identification of possible complexes between the RISC constituents. With immunofluorescence (confocal microscopy) distribution/colocalization of the assessed molecules was investigated. The 3 cells lines together with paraffin embedded tissue from 50 patients with colon cancer of various stages and grades as well as normal colonic tissues, were evaluated via qRT-PCR for Drosha, Dicer and Ago2 expression. Results: Ago2, Dicer, TRBP and Drosha were detected in all cell lines at both protein and mRNA levels. Ago2, Dicer and TRBP displayed both cytoplasmic and nuclear localization, whereas Drosha expression was primarily nuclear. Immunoprecipitation and immunofluorescence analyses uncovered the formation of Ago2/Dicer and Ago2/TRBP complexes. Ago2, Dicer and Drosha genes were expressed in all the normal colonic epithelia and in the majority of the carcinoma cases assessed. Ago2, Dicer and Drosha mRNA levels were significantly decreased in malignant compared to normal tissues (p < 0.05). Conclusions: (1) Drosha, Dicer, Ago2 and TRBP are expressed in colon cancer cells in a well-orchestrated fashion. (2) The significant downregulation of the tested genes in colon carcinomas compared to normal tissues implies that these genes may be implicated in colon carcinogenesis in humans. (3) Since the RISC complex proteins correlate with RNAi-based gene silencing it is possible that alterations of their expression levels might reflect the response of colon cancer to future RNAi-related therapies.