MicroRNAs as Biomarkers for Inflammatory Bowel Disease

Download or read book MicroRNAs as Biomarkers for Inflammatory Bowel Disease written by Poorani Sivaraman and published by . This book was released on 2014 with total page 372 pages. Available in PDF, EPUB and Kindle. Book excerpt: MicroRNAs (miRNAs) are short non-coding RNAs of 19-22 nucleotides that regulate gene expression. They target the 3'-untranslated region (UTR), and sometimes the 5'-UTR, of messenger RNAs (mRNAs), and suppress protein translation and/or trigger mRNA degradation. Since their discovery, miRNAs have been implicated in various regulatory pathways and diseases. New miRNAs are constantly being discovered that are yet to be fully characterised or validated. Few miRNAs and their genes have been well studied and characterised. Inflammatory bowel disease (IBD) is a chronic inflammatory disorder, whose etiology appears to involve genetic, environmental, and immunological factors. The Krissansen laboratory recently obtained evidence for the overexpression of hsa-miR-595 and hsa-miR- 1246 in the sera of patients suffering from severe forms of IBD. It was hypothesized that both miRNAs might be involved in the maintenance of the epithelial barrier function of the intestine, and/or in immune regulation. There is a lack of information in the published literature regarding these miRNAs, their gene features, and their target mRNAs. Research was carried out to study the characteristics of the genes that encode hsa-miR-595 and hsa-miR-1246, to identify potential mRNA targets of these miRNAs, and determine whether the miRNAs are involved in disrupting the tight junctions formed by intestinal epithelial cells. Bioinformatics analyses were carried out to identify features of the genes encoding miR-595 and miR-1246 to provide evidence of the authenticity of these miRNAs. The NCBI GenBank database was searched for expressed sequence tags (ESTs) encoding the 5'- and 3'-flanking regions of the genes to provide evidence that the genes are transcribed. In silico screening of various miRNA target databases for mRNA targets of these miRNAs was carried out. 3'-UTR luciferase reporter assays were developed to determine whether selected targets were authentic. The ability of miR-595 and miR-1246 to inhibit the expression of selected mRNA targets expressed by the human epithelial colorectal adenocarcinoma (Caco- 2) cell line was examined. An attempt was made to get Caco-2 cells to form spheroids during 3D cell culture, in order to subsequently use the spheroids to demonstrate disruption of tight junctions by miR-595 and miR-1246. Bioinformatics analyses indicated that both hsa-miR-595 located at chromosome 7q36.3 and hsa-miR-1246 located at chromosome 2q31.1 potentially contain common gene features such as putative promoters, transcription start sites, CpG islands, and polyadenylation signal sites. The hsa-miR-595 gene is intragenic, being located within intron 1 of the protein tyrosine phosphate receptor type 2, N polypeptide (PTPRN2) gene. In contrast, hsa-miR-1246 is intergenic, being located between the metaxin 2 (MTX-2) and nuclear factor, erythroid 2-like 2 (NFE2L2) genes. Experimental work needs to be carried out to prove whether the predicted gene features identified are involved in the transcription and regulation of these miRNA genes. ESTs encoding each gene were identified in cDNA libraries prepared from a variety of different tissues, indicating that the miR-595 and miR-1246 genes are actively transcribed in different tissues. A literature search revealed that hsa-miR-1246 had been cross-mapped to chromosome 17, being located within the gene encoding the small nuclear RNA RNU2-1. The U2 locus consists of 6 to more than 30 tandem repeats indicating that this locus gives rise to multiple copies of hsa-miR-1246. It is not known whether the elevated levels of hsa-miR- 1246 in the sera of IBD patients arise from overexpression of hsa-miR-1246 on chromosome 2 or chromosome 17, or both. A search of various miRNA target databases indicated that either miR-595 or miR-1246, or both, targeted CDH2, PARD6A, NCAM1, FGFR2, AJAP1, and DYRK1A. MiR-595 had previously been reported to target the tight junction protein PARD6A, causing disruption of the permeability of epithelia, which might be expected to have important implications for IBD. MiR-1246 had previously been reported to target DYRK1A, causing increased expression of the transcription factor NFAT1c that induces the expression of inflammatory cytokines, and which might be expected to have important implications for IBD. An attempt was made to experimentally validate the above mRNA targets using a 3'-UTR luciferase reporter assay, but the assay was not successful due to technical issues and time constraints. Several attempts were made to form hollow spheroids from Caco-2 cells to mimic the gut wall and lumen, but the spheroids produced were not correctly formed in that they appeared to be deflated. In summary, the experimental approaches tackled were not successful in providing conclusive evidence about the authenticity of the mRNA targets, but will be useful for future validation. In conclusion, the results of the computational analyses and experimental approaches attempted provide a framework, and basis for future investigation of miR-595 and miR-1246, and their mRNA targets. In the future, these miRNAs could be investigated for their utility in diagnosing the severity of IBD. Inhibitors of the miRNAs could be investigated for their ability to prevent or attenuate disease symptoms.