Posttranscriptional Regulation Controls Calretinin Expression in Malignant Pleural Mesothelioma

Our plain-English summary of Posttranscriptional Regulation Controls Calretinin Expression in Malignant Pleural Mesothelioma, by J. Kresoja-Rakic, M. Sulemani, M.B. Kirschner, et al (Frontiers in Genetics, 2017).

The study

• Authors: J. Kresoja-Rakic, M. Sulemani, M.B. Kirschner, M. Ronner, G. Reid, S. Kao, B. Schwaller, W. Weder, R.A. Stahel, E. Felley-Bosco
• Journal: Frontiers in Genetics
• Published: 2017;8:70
• Cited by: 11 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

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.

In plain English

This laboratory study examined how mesothelioma cells control their levels of calretinin, a protein used as a marker for malignant pleural mesothelioma. It did not study asbestos exposure. The authors attached the end section of the calretinin gene's message (its 3′UTR) to a light-producing reporter gene and put it into mesothelioma cell lines. Adding this section lowered reporter activity by 40% compared with the reporter alone. Altering one stabilizing site in it (an ARE) lowered activity by a further 20%, and this site bound a protein called HuR. Altering two binding sites for small RNAs of the miR-30 family brought activity back to the reporter-alone level. Adding miR-30e-5p lowered reporter activity by about 60% in ONE58 cells and about 50% in ACC-MESO-4 cells, and it also lowered calretinin protein. Blocking miR-30e-5p raised both. In 60 mesothelioma tumor samples from patients in Sydney, miR-30e-5p and calretinin levels showed a weak but significant negative correlation. The authors concluded that AU-binding proteins help stabilize calretinin and that miR-30e-5p reduces calretinin expression through the 3′UTR.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (cell-culture) molecular study, using reporter-gene, mutation, binding and miRNA mimic/inhibitor experiments, plus a re-analysis (data mining) of earlier tumor-sample measurements
• Who was studied: Four mesothelioma cell lines (ACC-MESO-4, ZL55, SPC111, ONE58), plus a subset of 60 tumor samples from mesothelioma patients who underwent extrapleural pneumonectomy between 1994 and 2009 at Royal Prince Alfred and Strathfield Private Hospital, Sydney, Australia
• Exposure: No asbestos or fiber exposure was studied. The experimental factors were the calretinin (CALB2) 3′UTR, normal or with mutated ARE and miR-30 sites, inserted after a luciferase reporter, and miRNA mimics (miR-30b-5p, miR-30c-5p, miR-9-5p, miR-30e-5p at 1 or 5 nM) or anti-miR-30e-5p (10 or 30 nM)
• Main results:
  • In ONE58 cells, inserting the CALB2-3′UTR decreased firefly luciferase expression by 40% (p = 0.0012) compared with the empty vector
  • Mutating the predicted ARE motif caused a further significant decrease of 20% (p = 0.0055), which the authors interpreted as the ARE site binding a stabilizing factor
  • Mutating both miR-30 binding sites restored luciferase reporter expression to the level of the empty vector
  • A 25-nucleotide RNA containing the ARE motif bound a protein from mesothelioma cell extracts and pulled down HuR. HuR was expressed in all tested cell lines and was 5- to 50-fold enriched compared with TTP
  • In ONE58 cells stably carrying the reporter, 5 nM miR-30e-5p mimic led to about 60% downregulation of reporter expression (p = 0.0039) compared with negative control mimics, and calretinin protein also decreased
  • In ACC-MESO-4 cells, 5 nM miR-30e-5p mimic gave about a 50% decrease (p = 0.0039) in reporter expression and also lowered calretinin protein
  • Blocking miR-30e-5p with anti-miR caused a moderate but significant rise in reporter expression and an increase in calretinin protein
  • miR-9 mimics did not lower reporter or calretinin expression
  • In 60 mesothelioma tumor samples, calretinin expression (by immunohistochemistry) and miR-30e-5p expression showed a weak but significant negative correlation
  • In ONE58 cells stably overexpressing the CALB2-3′UTR (about 580-fold above the cells' own CALB2 mRNA), miR-30e-5p mimics did not lower calretinin. Calretinin fell only in empty-vector cells, which the authors suggested reflects the extra 3′UTR soaking up the mimics
• The authors' conclusion: The authors concluded that their data show a role for AU-binding proteins in stabilizing calretinin and for miR-30e-5p in negatively regulating calretinin expression after transcription, through its 3′UTR. They also suggested a possible physiological role for calretinin's alternatively spliced transcripts.

Topics

Medical terms in this study, explained in our Lexicon: Calretinin, Pleural Mesothelioma.

Related studies

• Regulation of calretinin in malignant mesothelioma is mediated by septin 7 binding to the CALB2 promoter — W. Blum, L. Pecze, et al. (2018)
• Tumor Suppressor microRNAs Contribute to the Regulation of PD-L1 Expression in Malignant Pleural Mesothelioma — S.C. Kao, Y.Y. Cheng, et al. (2017)
• Low calretinin expression and high neutrophil-to-lymphocyte ratio are poor prognostic factors in patients with malignant mesothelioma undergoing extrapleural pneumonectomy — S.C. Kao, S. Klebe, et al. (2011)
• SV40-induced expression of calretinin protects mesothelial cells from asbestos cytotoxicity and may be a key factor contributing to mesothelioma pathogenesis — T. Henzi, W.V. Blum, et al. (2009)
• Modulation of Calretinin Expression in Human Mesothelioma Cells Reveals the Implication of the FAK and Wnt Signaling Pathways in Conferring Chemoresistance towards Cisplatin — J. Wörthmüller, V. Salicio, et al. (2019)
• Double-Stranded RNA Structural Elements Holding the Key to Translational Regulation in Cancer: The Case of Editing in RNA-Binding Motif Protein 8A — A. Abukar, M. Wipplinger, et al. (2021)

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