Calretinin ELISA Kit

Calretinin (CALB2) is a diagnostic and prognostic marker in malignant pleural mesothelioma (MPM). We previously reported that calretinin expression is regulated at the mRNA level. The presence of a medium-sized (573 nucleotide) 3' untranslated region (3' UTR) predicted to contain binding sites for miR-30a/b/c/d/e and miR-9 as well as an adenine/uridine-rich element (ARE) in all three transcripts arising from the CALB2 gene, suggests that calretinin expression is regulated via posttranscriptional mechanisms. Our aim was to investigate the role of the CALB2-3' UTR in the posttranscriptional regulation of calretinin expression in MPM. CALB2-3' UTR was inserted downstream of the luciferase reporter gene using pmiRGLO vector and reporter expression was determined after transfection into MPM cells. Targeted mutagenesis was used to generate variants harboring mutated miR-30 family and ARE binding sites. Electrophoretic mobility shift assay was used to test for the presence of ARE binding proteins. CALB2-3' UTR significantly decreased luciferase activity in MPM cells. Analysis of mutation in the ARE site revealed a further destabilization of the reporter and human antigen R (HuR) binding to the ARE sequence was detected. The mutation of two miR-30 binding sites abolished CALB2-3' UTR destabilization effect; a transient delivery of miR-30e-5p mimics or anti-miR into MPM cells resulted in a significant decrease/increase of the luciferase reporter expression and calretinin protein, respectively. Moreover, overexpression of CALB2-3' UTR quenched the effect of miR-30e-5p mimics on calretinin protein levels, possibly by sequestering the mimics, thereby suggesting a competitive endogenous RNA network. Finally, by data mining we observed that expression of miR-30e-5p was negatively correlated with the calretinin expression in a cohort of MPM patient samples. Our data show the role of (1) adenine-uridine (AU)-binding proteins in calretinin stabilization and (2) miR-30e-5p in the posttranscriptional negative regulation of calretinin expression via interaction with its 3' UTR. Furthermore, our study demonstrates a possible physiological role of calretinin's alternatively spliced transcripts.

The short-chain fatty acid butyrate plays an essential role in colonic mucosa homeostasis through the capacity to block the cell cycle, regulate differentiation and to induce apoptosis. The beneficial effect of dietary fibers on preventing colon cancer is essentially mediated through butyrate, derived from luminal fermentation of fibers by intestinal bacteria. In epithelial cells of the colon, both in normal and colon cancer cells, the expression of several genes is positively or negatively regulated by butyrate likely through modulation of histone acetylation and thereby affecting the transcriptional activity of genes. Calretinin (CALB2) is a member of the EF-hand family of Ca2+-binding proteins and is expressed in a majority of poorly differentiated colon carcinoma and additionally in mesothelioma of the epithelioid and mixed type. Since CALB2 is one of the genes negatively regulated by butyrate in colon cancer cells and butyrate decreases calretinin protein expression levels in those cells, we investigated whether expression is regulated via putative butyrate-responsive elements (BRE) in the human CALB2 promoter. We identified two elements that act as butyrate-sensitive repressors in all colon cancer cell lines tested (CaCo-2, HT-29, Co-115/3). In contrast, in cells of mesothelial origin, MeT-5A and ZL34, the same two elements do not operate as butyrate-sensitive repressors and calretinin expression levels are insensitive to butyrate indicative of cell type-specific regulation of the CALB2 promoter. Calretinin expression in colon cancer cells is negatively regulated by butyrate via a bipartite BRE flanking the TATA box and this may he linked to butyrate's chemopreventive activity. J. Cell. Biochem. 109: 519-531, 2010. (C) 2009 Wiley-Liss, Inc.