MicroRNA-mediated loss of ADAR1 in metastatic melanoma promotes tumor growth
Yael Nemlich, … , Gideon Rechavi, Gal Markel
Yael Nemlich, … , Gideon Rechavi, Gal Markel
Published May 24, 2013
Citation Information: J Clin Invest. 2013;123(6):2703-2718. https://doi.org/10.1172/JCI62980.
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Research Article Oncology

MicroRNA-mediated loss of ADAR1 in metastatic melanoma promotes tumor growth

Abstract

Some solid tumors have reduced posttranscriptional RNA editing by adenosine deaminase acting on RNA (ADAR) enzymes, but the functional significance of this alteration has been unclear. Here, we found the primary RNA-editing enzyme ADAR1 is frequently reduced in metastatic melanomas. In situ analysis of melanoma samples using progression tissue microarrays indicated a substantial downregulation of ADAR1 during the metastatic transition. Further, ADAR1 knockdown altered cell morphology, promoted in vitro proliferation, and markedly enhanced the tumorigenicity in vivo. A comparative whole genome expression microarray analysis revealed that ADAR1 controls the expression of more than 100 microRNAs (miRNAs) that regulate many genes associated with the observed phenotypes. Importantly, we discovered that ADAR1 fundamentally regulates miRNA processing in an RNA binding–dependent, yet RNA editing–independent manner by regulating Dicer expression at the translational level via let-7. In addition, ADAR1 formed a complex with DGCR8 that was mutually exclusive with the DGCR8-Drosha complex that processes pri-miRNAs in the nucleus. We found that cancer cells silence ADAR1 by overexpressing miR-17 and miR-432, which both directly target the ADAR1 transcript. We further demonstrated that the genes encoding miR-17 and miR-432 are frequently amplified in melanoma and that aberrant hypomethylation of the imprinted DLK1-DIO3 region in chromosome 14 can also drive miR-432 overexpression.

Authors

Yael Nemlich, Eyal Greenberg, Rona Ortenberg, Michal J. Besser, Iris Barshack, Jasmine Jacob-Hirsch, Elad Jacoby, Eran Eyal, Ludmila Rivkin, Victor G. Prieto, Nitin Chakravarti, Lyn M. Duncan, David M. Kallenberg, Eitan Galun, Dorothy C. Bennett, Ninette Amariglio, Menashe Bar-Eli, Jacob Schachter, Gideon Rechavi, Gal Markel

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Figure 6

ADAR1 regulates miRNAs processing by affecting the biogenesis proteins.

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ADAR1 regulates miRNAs processing by affecting the biogenesis proteins. ...
Expression of Dicer, Drosha, and DGCR8 at the (A) mRNA and (B) protein level as determined by qPCR and Western blot, respectively, in the transfectants indicated. Densitometry quantification of the immunoblots was performed using ImageJ software. Band intensities for Dicer, Drosha, and DGCR8 were determined relative to those of the endogenous control β-actin. All values were normalized to control cells. Data represent the mean ± SEM of 3 independent cell systems. *P < 0.05 (2-tailed t test). (C) Comparison between the expression level (fold change) of let-7 miRNA family members in ADAR1-KD and control cells. (D) Western blot of Dicer, Drosha and DGCR8, ADAR1, and CEACAM1 following pulldown of ADAR1 (right) or CEACAM1 (left). S1–S3 represents lysate samples from 3 independent transfected cell systems extracted both before (total cell lysate) and after IP procedure. (E) Western blotting for ADAR1 (left) or CEACAM1 (right) from total cell lysate or after the respective protein pulldown. Fold values in the total lysate samples represent the fold overexpression of each pri-miRNA over endogenous expression, after normalization to GAPDH. The presence (+) or absence (–) of pri-miRNAs in the pulldown is marked.