Kolachala Vasantha L, Wang Lixin, Obertone Tracy S, Prasad Meena, Yan Yutao, Dalmasso Guillaume, Gewirtz Andrew T, Merlin Didier, Sitaraman Shanthi V
Division of Digestive Diseases, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
J Biol Chem. 2010 Jun 11;285(24):18184-90. doi: 10.1074/jbc.M109.066555. Epub 2010 Apr 13.
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.
我们曾报道，上皮腺苷2B受体（A(2B)AR）的信使核糖核酸（mRNA）和蛋白质在结肠炎中上调，我们证明其受肿瘤坏死因子α（TNF-α）调控。在此，我们研究了结肠炎期间调控A(2B)AR表达的机制。将A(2B)AR启动子的1.4千碱基序列克隆到pFRL7荧光素酶载体中。抗微小核糖核酸（miRNA）是根据特定的miRNA结合位点定制合成的。通过将此3'-非翻译区（UTR）克隆到pMIR-REPORT中荧光素酶基因的下游，检测miRNA与A(2B)AR mRNA的3'-UTR的结合。在T84细胞中，TNF-α使A(2B)AR mRNA增加35倍，但在荧光素酶检测中未增加启动子活性。通过核转录分析，未观察到TNF-α处理后A(2B)AR mRNA增加。在T84细胞和小鼠结肠中，在A(2B)AR mRNA的3'-UTR中鉴定出四个假定的miRNA靶位点（miR27a、miR27b、miR128a、miR128b）。用TNF-α预处理细胞可使miR27b和miR128a的水平降低60%。前体miR27b和前体miR128a的过表达使A(2B)AR水平降低>60%。在体外，阻断miR27b可使A(2B)AR mRNA水平增加6倍。在A(2B)AR mRNA增加的情况下，小鼠结肠炎受累组织中的miR27b水平显著下降。总体而言，这些数据表明TNF-α诱导的结肠上皮细胞中A(2B)AR表达在转录后受miR27b和miR128a调控，并表明miR27b影响小鼠结肠炎中A(2B)AR的表达。
