Posttranscriptional Regulation Controls Calretinin Expression in Malignant Pleural Mesothelioma

Abstract

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.

Rabies Virus Infection is Associated with Variations in Calbindin D-28K and Calretinin mRNA Expression Levels in Mouse Brain Tissue.

“…Increased Calb2 expression as reported in our study could also be attributed to the same reasons as seen with Calb1, especially as both genes encode proteins with closely related functions [13,11]. Since Calretinin expression is normally regulated at post transcriptional level by the transcription factors NRF-1 and E2F2 respectively [20,21], the probability that cytoplasmic RABV replication interferes with normal regulation of Calb2 expression, is further strengthened.…”

Calretinin promotes invasiveness and EMT in malignant mesothelioma cells involving the activation of the FAK signaling pathway

“…A similar mechanism might be envisaged also for the PTK2 (FAK) promoter. The CALB2 gene is also subject to alternative splicing producing among others also the transcript CALB2b , likely acting as a long non-coding RNA [ 38 ]. Finally the interaction of CR with FAK at adhesion sites and/or in the nucleus as reported in this study might result in a protein complex (possibly together with additional proteins) involved in the regulation of FAK expression.…”

Heterogeneity of RNA editing in mesothelioma and how RNA editing enzyme ADAR2 affects mesothelioma cell growth, response to chemotherapy and tumor microenvironment

“…Therefore, the identification of clusters with different RNA editing in introns suggests the presence of different splicing in these clusters. Splice variants have been identified in mesothelioma with mutated SF3B1 splicing factor (5), but have not yet been systematically analyzed, although we have documented their occurrence in several mesothelioma relevant genes such as lncRNA GAS5, CALB2 and RBM8A (26, 67, 68) or in major mesothelioma tumor suppressors such as NF2 (69) and BAP1 (70). Splice variants have functional consequences including in response to therapy.…”