Abstract
Introduction: In prior studies, we identified RNAs in non-dysplastic mucosa that distinguished patients with ulcerative colitis (UC) and concomitant neoplastic lesions from those without neoplasia. We sought to validate these findings in a larger independent cohort and identify miRNAs regulating these genes to discover biomarkers and elucidate neoplastic pathways in this high-risk population. Methods: Colonoscopic or surgical biopsies of noninflamed, non-dysplastic rectosigmoid mucosa were obtained from subjects with long-standing UC with (n=19) or without remote neoplasia (n=23). mRNAs were analyzed with the Illumina HumanHT-12 v4 BeadChip microarray, and miRNAs were analyzed by the miRCURY LNA microRNA array 7th gen (Exiqon). Following pre-processing, two-group comparison was performed using R/Bioconductor package ‘Limma'. Real-time PCR was performed for miR-4728-3p. HT-29 and HCT-116 were transfected with mature miR-4728-3p or scrambled controls and predicted target proteins were assessed by Westerns. Results: By unsupervised hierarchical clustering, UC subjects with neoplasia clustered by both miRNA and mRNA profiles compared to those without neoplasia. There were 35 significantly downregulated and 47 up-regulated miRNAs (adj. p 0.5, adj. p<0.01). Hierarchical clustering was used to assemble genes into modules, which were assessed for enrichment in Gene Ontology (GO) annotations. Genes with enriched functional GO annotations that were in the same module as predicted miRNA regulators were analyzed for their locations in KEGG regulatory pathways to assess their potential roles in regulatory sub-networks. miR-47283p was identified as a regulator of three genes (CAV1, THBS2, COL1A2) involved in in focal adhesion signaling, the most enriched pathway in UC subjects with neoplasia (p=2.2 x 10-4). miR-4728-3p was down-regulated (LogFC=-0.42, p=0.003) as confirmed by qPCR in preliminary studies, whereas all three target genes were significantly up-regulated in UCassociated neoplasia [CAV1 (LogFC=1.06, adj p=2.9 x 10-17), THSB2 (LogFC=1.33, adj. p= 1.7 x 10-18) and COL1A2 (LogFC=1.01 adj = 1.4 x 10-9)]. miR-4728-3p transfection into colon cancer cells down-regulated CAV1 as well as COL1A2 and study of its affects on THBS2 are in progress. We are also examining miR-4728-3p-induced changes on cell invasion, migration, and focal adhesion disassembly. Conclusion: Patients with long-standing ulcerative colitis who harbor remote neoplasia can be identified based on miRNA and mRNA profiles in non-dysplastic tissue. Using a newly developed algorithm to analyze miRNA and mRNA expression from the same tissues, we identified miR-4728-3p as a key regulator of focal adhesion and likely an important tumor-suppressor in UC-associated colon carcinogenesis.
Published Version
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