Post-transcriptional regulation of PIAS3 expression by miR-18a in malignant mesothelioma

Our plain-English summary of Post-transcriptional regulation of PIAS3 expression by miR-18a in malignant mesothelioma, by T. He, K. McColl, N. Sakre, et al (Molecular Oncology, 2018).

The study

• Authors: T. He, K. McColl, N. Sakre, Y. Chen, G. Wildey, A. Dowlati
• Journal: Molecular Oncology
• Published: 2018;12(12):2124-2135
• Cited by: 13 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Protein inhibitor of activated STAT3 (PIAS3) is an endogenous suppressor of signal transducer and activator of transcription 3 (STAT3) signaling. By directly interacting with phosphorylated STAT3, PIAS3 can block the downstream transcriptional activity of STAT3, which is hyper-activated in various cancers. We previously reported that in malignant mesothelioma (MM), low PIAS3 expression is associated with increased STAT3 activation and correlates with poor patient survival, yet the regulatory mechanism(s) governing PIAS3 expression in MM remain unclear. Here, we demonstrate that PIAS3 protein expression does not correlate with its mRNA level in MM cell lines, indicating that PIAS3 expression is regulated at a post-transcriptional level. Inhibition of proteasomal degradation with MG132 (10 μm) or bortezomib (1 μm), alone and in combination, did not increase PIAS3 protein levels; furthermore, inhibition of protein synthesis by cycloheximide treatment did not decrease PIAS3 levels within 48 h, suggesting that PIAS3 expression is not actively regulated at a post-translational level. To determine whether miRNA (miRs) can translationally regulate PIAS3 expression, we combined miR microarray analysis with bioinformatic screening to identify candidate miRs, in MM cell lines with low PIAS3 expression, followed by luciferase reporter assays to validate miR regulation of the PIAS3 3'UTR. We identified miR-18a as a suppressor of PIAS3 expression that is upregulated in MM cells and whose inhibition can increase PIAS3 expression and suppress STAT3 activity. Moreover, we showed that miR-18a inhibition can decrease MM cell viability and that its expression is negatively correlated with MM patient survival. Taken together, these results suggest that targeting miR-18a may have therapeutic benefit in MM.

© 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

In plain English

This was a laboratory study of human mesothelioma cell lines grown in culture, plus an analysis of public TCGA database records for 87 malignant mesothelioma patients. It did not study asbestos exposure. The authors asked why some mesothelioma cells make little PIAS3, a protein that dampens STAT3 signaling. Three cell lines (211H, H2052, H2452) had low PIAS3 protein compared with the H28 line, yet PIAS3 messenger RNA was similar in all five cell lines tested. Blocking protein breakdown with MG-132 or bortezomib did not raise PIAS3. A screen of small regulatory RNAs (microRNAs) narrowed 687 candidates to 19. Of four tested, only miR-18a significantly lowered a PIAS3 reporter signal compared with a control (p=0.0012). Blocking miR-18a in H2452 cells raised PIAS3 and reduced cell viability at 72 hours (p=0.027) and 100 hours (p=0.0092). In H2052 cells it lowered a STAT3 reporter signal (p=0.021). In the patient data, higher miR-18a was associated with poorer survival (p=0.0341). The authors concluded that miR-18a "may represent a promising therapeutic target" in malignant mesothelioma.

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 (in vitro) study of human cell lines, combined with a survival analysis of publicly available TCGA gene expression and clinical data
• Who was studied: Six mesothelioma cell lines (H28, 211H, H2052, H2452 from ATCC; HAY and YOU from the National Cancer Institute), one non-small cell lung cancer cell line (A549), HEK293T cells for reporter assays, and TCGA data for 87 malignant mesothelioma patients (43 with miR-18a above the median, 44 below)
• Exposure: No asbestos exposure was studied. The laboratory treatments were proteasome inhibitors MG-132 (10µM) and bortezomib (1µM), alone or together, for 5 hours; the protein synthesis inhibitor cycloheximide (100µg/ml); miR mimics; and miR-18a inhibitors (100nM). In the patient data, the factor examined was miR-18a expression level.
• Main results:
  • PIAS3 protein was markedly lower in mesothelioma cell lines 211H, H2052 and H2452 than in H28 mesothelioma cells and A549 lung cancer cells. PIAS3 mRNA was at a similar level in all five cell lines.
  • In the three low-PIAS3 mesothelioma cell lines, the proteasome inhibitors (alone or combined, 5 hours) did not increase PIAS3. Cycloheximide treatment of H28 cells showed little PIAS3 degradation.
  • The microarray screen identified 687 miR candidates. Prediction of binding sites in the PIAS3 3'UTR reduced these to 19 candidates.
  • In HEK293T reporter assays, only miR-18a, of the four candidates tested (miR-335, miR-543, miR-31, miR-18a), significantly decreased PIAS3 3'UTR luciferase activity compared with a scramble control (p=0.0012).
  • Inhibiting miR-18a in H2452 mesothelioma cells increased PIAS3 mRNA and protein within 72 hours.
  • In H2052 mesothelioma cells, STAT3 luciferase reporter activity was significantly lower after miR-18a inhibition than in the control group (p=0.021).
  • In H2452 mesothelioma cells, miR-18a inhibition significantly decreased cell viability compared with mock control at 72 hours (p=0.027) and 100 hours (p=0.0092).
  • In the TCGA data for 87 mesothelioma patients, higher miR-18a expression was significantly correlated with poorer survival (p=0.0341). None of the other miRs in the same miR-17-92 cluster showed a similar significant correlation.
• The authors' conclusion: The authors concluded that protein degradation played no regulatory role in PIAS3 expression in mesothelioma cells. They concluded that miR-18a may help lower PIAS3 by binding its 3'UTR, and that miR-18a may represent a promising therapeutic target in malignant mesothelioma.
• Limitations the authors state:
  • Adding miR-18a mimics to H28 cells did not produce a reduction in natural PIAS3 protein that could be shown after 48 hours. The authors attributed this to the high stability of PIAS3 protein.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Lung cancer, Mesothelioma.

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

Related studies

• Post-transcriptional regulation of plasminogen activator inhibitor type-1 expression in human pleural mesothelial cells — S. Shetty, T. Velusamy, et al. (2010)
• Transcriptional over-expression of chloride intracellular channels 3 and 4 in malignant pleural mesothelioma — V. Tasiopoulou, D. Magouliotis, et al. (2015)
• Tumor Suppressor microRNAs Contribute to the Regulation of PD-L1 Expression in Malignant Pleural Mesothelioma — S.C. Kao, Y.Y. Cheng, et al. (2017)
• Differential microRNA expression profiling of mesothelioma and expression analysis of miR-1 and miR-214 in mesothelioma — V.J. Amatya, A.S. Mawas, et al. (2016)
• miR-193a-3p is a potential tumor suppressor in malignant pleural mesothelioma — M. Williams, M.B. Kirschner, et al. (2015)
• Transcriptional up-regulation of MMP12 and MMP13 by asbestos occurs via a PKCdelta-dependent pathway in murine lung — A. Shukla, T.F. Barrett, et al. (2006)

Browse more research in Molecular Oncology, or return to medical research.