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01.12.2017 | Research | Ausgabe 1/2017 Open Access

Journal of Hematology & Oncology 1/2017

miR-380-5p-mediated repression of TEP1 and TSPYL5 interferes with telomerase activity and favours the emergence of an “ALT-like” phenotype in diffuse malignant peritoneal mesothelioma cells

Zeitschrift:
Journal of Hematology & Oncology > Ausgabe 1/2017
Autoren:
Graziella Cimino-Reale, Paolo Gandellini, Francesca Santambrogio, Marta Recagni, Nadia Zaffaroni, Marco Folini
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​s13045-017-0510-3) contains supplementary material, which is available to authorized users.

Abstract

Background

Understanding the molecular/cellular underpinnings of diffuse malignant peritoneal mesothelioma (DMPM), a fatal malignancy with limited therapeutic options, is of utmost importance for the fruitful management of the disease. In this context, we previously found that telomerase activity (TA), which accounts for the limitless proliferative potential of cancer cells, is prognostic for disease relapse and cancer-related death in DMPM patients. Consequently, the identification of factors involved in telomerase activation/regulation may pave the way towards the development of novel therapeutic interventions for the disease. Here, the capability of miR-380-5p, a microRNA negligibly expressed in telomerase-positive DMPM clinical specimens, to interfere with telomerase-mediated telomere maintenance and, hence, with cancer cell growth was assessed on preclinical models of DMPM.

Methods

DMPM cells were transfected with a miR-380-5p synthetic precursor, and the effects of miRNA replacement were evaluated in terms of growing capability, induction of apoptosis and interference with TA. Reiterated weekly transfections were also performed in order to analyse the phenotype arising upon prolonged miR-380-5p reconstitution in DMPM cells.

Results

The ectopic expression of miR-380-5p elicited a remarkable inhibition of TA and resulted in DMPM cell growth impairment and apoptosis induction. In particular, we demonstrated for the first time that these effects were the result of a molecular circuitry converging on telomerase associated protein 1 (TEP1), where the miRNA was able to target the gene both directly in unconventional targeting modality and indirectly via p53 accumulation consequent to miRNA-mediated downregulation of testis-specific protein, Y-encoded-like 5 gene. Moreover, miR-380-5p did not cause telomere attrition and cell growth arrest in long-term DMPM transfectants, which in turn showed slightly elongated telomeres and molecular features (e.g. c-circle DNA and reduced expression levels of chromatin remodeler ATRX) resembling an alternative lengthening of telomeres (ALT) phenotype.

Conclusions

miR-380-5p interferes with TA in DMPM cells by targeting TEP1. Notably, in the long-term setting, miR-380-5p-mediated impairment of TA did not result in telomere attrition. Instead, a phenotype reminiscent of ALT emerged in DMPM cells as possible compensatory pathway that safeguards DMPM cell growth, an event that may be regarded as a potential resistance mechanism to anticancer therapies based on telomerase inhibitors.

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Zusatzmaterial
Additional file 3: Figure S3. In silico prediction analysis of putative miR-380-5p target genes by miRWalk 2.0. Description of data: (A) By the predicted target module of miRWalk 2.0—a comprehensive database that provides indications on predicted and validated binding sites on miRNA target genes [14]—we obtained a combined information on putative miR-380-5p binding sites within the 3′UTRs of human RefSeq mRNAs in terms of union of the predictions generated by five distinct algorithms (i.e. miRWalk 2.0; miRanda-rel2010; miRMap; RNA22v2 and Targetscan6.2). (B) Representative western immunoblotting showing the amounts of protein encoded by predicted miR-380-5p target genes in STO cells transfected with preNeg or miR-380-5p. Target proteins have been selected among those known to play a role in TMM and reported in panel A. Cropped images of selected proteins are shown. (TIF 432 kb)
Additional file 4: Figure S4. Effects of miR-380-5p reconstitution on A549 lung adenocarcinoma cells. Description of data: (A) Assessment of miR-380-5p expression levels in preNeg and miR-380-5p-transfected cells (Log10(RQ) vs. NT cells; mean values ± s.d.). (B) Growth curves of NT, preNeg- and miR-380-5p-transfected cells (number of growing cells; mean values ± s.d.); **P < 0.02. (C) Assessment of TEP1 (black bars) and TSPYL5 (white bars) mRNA expression levels in p53 proficient (siCTR) and p53-depleted (sip53) cells ectopically expressing miR-380-5p (Log10(RQ) vs. NT cells; mean values ± s.d.); *P < 0.05 vs. siCTR-transfected cells. (D) Representative immunoblotting showing TSPYL5, TEP1 and p53 protein amounts in NT, preNeg- and miR-380-5p-transfected A549 cells. Cropped images of selected proteins are shown. (E) Assessment of TSPYL5 mRNA expression levels in preNeg- and miR-380-5p-transfected U-2 Os cells (RQ vs. NT cells; mean values ± s.d.). (F) Representative immunoblotting showing TSPYL5 and p53 protein amounts in NT, preNeg- and miR-380-5p-transfected U-2 Os cells. Cropped images of selected proteins are shown. (G) Representative immunoblotting showing p53, TEP1 and TSPYL5 protein levels in p53 proficient (siCTR) and p53-depleted (sip53) cells ectopically expressing miR-380-5p. Cropped images of selected proteins are shown. The graph on the right shows the quantification of TEP1 (black bars) and TSPYL5 (white bars) protein amounts as a function of the different transfected oligomers (relative quantity vs. NT cells; mean values ± s.d.); *P < 0.05 vs. siCTR-transfected cells. (H) Representative immunobloting showing TSPYL5, p53 and TEP1 amounts in preNeg- and miR-380-5p-transfected cells ± target protector (TSPYL5 TP). Cropped images of selected proteins are shown. (I) Quantification of TSPYL5 (white bars), TEP1 (black bars) and p53 (grey bars) protein amounts in preNeg- and miR-380-5p-transfected cells ± TSPYL5 TP (relative amounts with respect to preNeg-transfected cells; mean values ± s.d.); **P < 0.01 vs. miR-380-5p-transfected cells. (TIF 1515 kb)
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