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

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 ␣ (TNF-␣). 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-␣ 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-␣ 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-␣ reduced the levels of miR27b and miR128a by 60%. Over expression of premiR27b 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-␣-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.

Contributions of A2Aand A2Badenosine receptors in coronary flow responses in relation to the KATPchannel using A2Band A2A/2Bdouble-knockout mice

“…The occurrence of the upregulatory mechanism phenomenon in our study may be TNF-␣ induced and posttranscriptionally regulated (41) or due to DNA methylation, since this may regulate AR cell surface expression levels (9). Furthermore, HIF-1 has also been suggested to upregulate the expression of A 2B ARs under hypoxic conditions (76).…”

Regulation of IRS1/Akt insulin signaling by microRNA-128a during myogenesis

“…TNF-a expression is upregulated immediately following CTX injection in mouse muscles (Chen et al, 2007). Furthermore, TNF-a is also known to regulate miR-128a by targeting the adenosine 2B receptor (A2BAR) in epithelial cells (Kolachala et al, 2010), which in turn regulates insulin signaling and myoblast proliferation (Alter et al, 2008;Li, 2003;Yamasaki et al, 1996). We first examined the effects of TNF-a on miR-128a expression in myoblasts.…”

Regulation of miR106b cluster through the RB pathway

“…p21 and PTEN 3'UTRs were cloned in pMIR-vector as described, 26,37,46 and the primers are presented in Table S1. The 3'UTRs (25 nM) were transiently transfected in MCF7 cells using Lipofectamine 2000 (Invitrogen), and renilla luciferase was co-transfected as an internal control.…”