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Author Notes:

To whom correspondence should be addressed: Division of Digestive Diseases, Rm. 265, 615 Michael St., Whitehead Research Bldg., Emory University, Atlanta, GA 30322. Tel.: 404-712-2862; Fax: 404-727-5767; E-mail: vkolach@emory.edu.

Both authors contributed equally to this work.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) DQ 351340.

Subjects:

Keywords:

  • Diseases
  • G Proteins/Coupled Receptors (GPCR)
  • Gene/Regulation
  • Methods/PCR
  • Protein
  • Receptors/Regulation
  • Adenosine 2B Receptor
  • Colitis
  • miR27b
  • miR128a
  • TNF-α

Adenosine 2B Receptor Expression Is Post-transcriptionally Regulated by MicroRNA

Tools:

Journal Title:

Journal of Biological Chemistry

Volume:

Volume 285, Number 24

Publisher:

, Pages 18184-18190

Type of Work:

Article | Final Publisher PDF

Abstract:

We have reported that epithelial adenosine 2B receptor (A2BAR) mRNA and protein are up-regulated in colitis, which we demonstrated to be regulated by tumor necrosis factor α (TNF-α). Here, we examined the mechanism that governs A2BAR expression during colitis. A 1.4-kb sequence of the A2BAR 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 A2BAR mRNA was examined by cloning this 3′-UTR downstream of the luciferase gene in pMIR-REPORT. In T84 cells, TNF-α induced a 35-fold increase in A2BAR mRNA but did not increase promoter activity in luciferase assays. By nuclear run-on assay, no increase in A2BAR mRNA following TNF-α treatment was observed. Four putative miRNA target sites (miR27a, miR27b, miR128a, miR128b) in the 3′-UTR of the A2BAR mRNA were identified in T84 cells and mouse colon. Pretreatment of cells with TNF-α reduced the levels of miR27b and miR128a by 60%. Over expression of pre-miR27b and pre-miR128a reduced A2BAR levels by >60%. Blockade of miR27b increased A2BAR mRNA levels by 6-fold in vitro. miR27b levels declined significantly in colitis-affected tissue in mice in the presence of increased A2BAR mRNA. Collectively, these data demonstrate that TNF-α-induced A2BAR expression in colonic epithelial cells is post-transcriptionally regulated by miR27b and miR128a and show that miR27b influences A2BAR expression in murine colitis.

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© 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

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