Adenosine 2B receptor expression is post-transcriptionally regulated by microRNA.
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.
Kolachala
Vasantha L
VL
Division of Digestive Diseases, Emory University School of Medicine, Atlanta, Georgia 30322, USA. vkolach@emory.edu
Wang
Lixin
L
Obertone
Tracy S
TS
Prasad
Meena
M
Yan
Yutao
Y
Dalmasso
Guillaume
G
Gewirtz
Andrew T
AT
Merlin
Didier
D
Sitaraman
Shanthi V
SV
D020413:3' Untranslated Regions; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D003092:Colitis / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D035683:MicroRNAs / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D043704:Receptor, Adenosine A2B / Q000096:biosynthesis*; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D020413:3' Untranslated Regions; C545189:MIRN128 microRNA, human; C531388:MIRN27 microRNA, human; D035683:MicroRNAs; C550324:Mirn128 microRNA, mouse; C540674:Mirn27 microRNA, mouse; D043704:Receptor, Adenosine A2B; D014409:Tumor Necrosis Factor-alpha; D000242:Cyclic AMP
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