MicroRNA-10b Downregulates KIT Expression Through NCOR2 in Diabetic Interstitial Cells of Cajal

Abstract

Interstitial cells of Cajal (ICCs) are pacemakers in the gastrointestinal (GI) tract that regulate intestinal motility through spontaneous electrical slow waves. ICCs have been shown to express the receptor tyrosine kinase c-Kit, which is the receptor for stem cell factor. While recent studies have found that reduction of c-Kit expression in ICC induces abnormal contractile patterns in the GI tract in diabetic mouse models, further investigation are still needed in order to address underlying mechanisms. MicroRNAs (miRNAs) are a new class of transcriptional regulators that are involved in nearly all developmental and pathological processes in animals. The specific goals of this research project were: (1) to identify miRNA profiles in ICC from jejunum and colon smooth muscle through genome-wide miRNA sequencing (miRNA-seq) technology in both non-diabetic and diabetic mouse models, and (2) to characterize gene functions encoded in these miRNA circuits using microRNA target validation and pathway analysis programs. Genome-wide miRNA-seq identified 11,381,897 to 19,938,908 raw reads in total for non-diabetic and diabetic ICC isolated from jejunum and colon smooth muscle. Interestingly, miR-10b-5p expressed the most change in diabetic ICC through its significant downregulation. Target genes for miR-10b-5p were identified using the five databases found in miRbase which include: DIANAMICROT, MICRORNA.ORG, MIRDB, RNA22-MMU, and TARGETSCAN-VERT. A network was then graphically depicted in order to show gene relationships between diabetes mellitus, miR-10b and its gene targets, and c-Kit through the Ingenuity Pathway Analysis (IPA) program. From these two analyses, it was found that Bcl2l11 and Ncor2 were the most optimal target genes for miR-10b. Immunoblotting analysis showed that NCOR2 was clearly upregulated in diabetic smooth muscle, while BCL2L11 showed no expression in either diabetic or non-diabetic conditions. Finding novel molecular mechanisms relating to diabetic miRNAs could provide a better understanding of the disease, and may have significant therapeutic implications, especially in the prevention of ICC damage in patients with GI complications.