Abstract
We have reported that epithelial adenosine 2B receptor (A(2B)AR) mRNA and protein are up-regulated in colitis, which we demonstrated to be regulated by tumor necrosis factor alpha (TNF-alpha). Here, we examined the mechanism that governs A(2B)AR expression during colitis. A 1.4-kb sequence of the A(2B)AR promoter was cloned into the pFRL7 luciferase vector. Anti-microRNA (miRNA) was custom-synthesized based on specific miRNA binding sites. The binding of miRNA to the 3'-untranslated region (UTR) of A(2B)AR mRNA was examined by cloning this 3'-UTR downstream of the luciferase gene in pMIR-REPORT. In T84 cells, TNF-alpha induced a 35-fold increase in A(2B)AR mRNA but did not increase promoter activity in luciferase assays. By nuclear run-on assay, no increase in A(2B)AR mRNA following TNF-alpha treatment was observed. Four putative miRNA target sites (miR27a, miR27b, miR128a, miR128b) in the 3'-UTR of the A(2B)AR mRNA were identified in T84 cells and mouse colon. Pretreatment of cells with TNF-alpha reduced the levels of miR27b and miR128a by 60%. Over expression of pre-miR27b and pre-miR128a reduced A(2B)AR levels by >60%. Blockade of miR27b increased A(2B)AR mRNA levels by 6-fold in vitro. miR27b levels declined significantly in colitis-affected tissue in mice in the presence of increased A(2B)AR mRNA. Collectively, these data demonstrate that TNF-alpha-induced A(2B)AR expression in colonic epithelial cells is post-transcriptionally regulated by miR27b and miR128a and show that miR27b influences A(2B)AR expression in murine colitis.