Roles of microRNAs in diseases of the human gastrointestinal tract

Abstract

Crohn’s disease (CD) and colorectal cancer (CRC) are major disorders of the intestine. Inflammation in CD often precedes fibrosis and stricture formation, and is linked to increased cancer risk. Hypoxia is a common feature of inflammation and CRC that can severely compromise the effectiveness of current therapy regimes including chemo-radiotherapy and maintenance of remission in CD patients. MicroRNAs (miRNAs) are key regulatory molecules involved in cellular proliferation, apoptosis and fibrosis, which are all modulated by hypoxia. This thesis aims to understand the role of miRNAs in these two intestinal diseases. Microarray profiling identified differentially expressed miRNA in the intestinal mucosa overlying strictured and non-strictured CD tissue samples and in six CRC cell lines cultured in hypoxic conditions compared to normoxia. Validation experiments using qRT-PCR confirmed the differential expression of miR-29a, -29b, -29c, -34a, -493* and -708 in CD mucosa and miR-21, -210, -30d, -320a, -320b and -320c in CRC cell lines. Functionally, over-expression of miR-29b in CD intestinal fibroblasts modulated the down-regulation of collagen I and III transcripts and collagen III protein in a TGF-β-dependent manner. Furthermore, miR-29b induced indirectly the expression of the anti-apoptotic protein Mcl-1 via the cytokines IL-6 and IL-8. A positive correlation between miR-210 and the hypoxia marker CAIX was found in CRC tissue in vivo. Furthermore, HCT116 cells cultured under hypoxia were more resistant to the chemotherapy drug 5-FU than cells grown under normoxia. Knockdown of miR-21 or miR-30d under hypoxia may sensitise CRC cells to 5-FU. CRC cell lines grown under hypoxic conditions present an altered cellular metabolic profile compared to their normoxic counterparts. This thesis has showed that critical miRNAs have a functional role in the progression of two important diseases of the intestine. The work presented highlights the potential of miRNAs as biomarkers and therapeutic targets to improve the clinical management of patients with digestive diseases.