Analysis of Argonaute Complex Bound mRNAs in DU145 Prostate Carcinoma Cells Reveals New miRNA Target Genes.

Jaroslaw Szczyrba,Michaela Beitzinger,Volker Jung,Bernd Wullich,Friedrich A Grässer,Helge Taubert,Martin Hart,Gunther Wennemuth,Gunter Meister,Sven Wach,Elke Nolte,Norbert Eichner,Marc Wiesehöfer,Thomas Stempfl,Sandra Sapich

doi:10.1155/2017/4893921

Abstract

Posttranscriptional gene regulation by microRNAs (miRNAs) contributes to the induction and maintenance of prostate carcinoma (PCa). To identify mRNAs enriched or removed from Ago2-containing RISC complexes, these complexes were immunoprecipitated from normal prostate fibroblasts (PNFs) and the PCa line DU145 and the bound mRNAs were quantified by microarray. The analysis of Ago complexes derived from PNFs or DU145 confirmed the enrichment or depletion of a variety of mRNAs already known from the literature to be deregulated. Novel potential targets were analyzed by luciferase assays with miRNAs known to be deregulated in PCa. We demonstrate that the mRNAs of the death effector domain-containing protein (DEDD), the tumor necrosis factor receptor superfamily, member 10b protein (TNFRSF10B), the tumor protein p53 inducible nuclear protein 1 (TP53INP1), and the secreted protein, acidic, cysteine-rich (SPARC; osteonectin) are regulated by miRNAs miR-148a, miR-20a, miR-24, and miR-29a/b, respectively. Therefore, these miRNAs represent potential targets for therapy. Surprisingly, overexpression of miR-24 induced focus formation and proliferation of DU145 cells, while miR-29b reduced proliferation. The study confirms genes deregulated in PCa by virtue of their presence/absence in the Ago2-complex. In conjunction with the already published miRNA profiles of PCa, the data can be used to identify miRNA-regulated mRNAs.

Highlights

Prostate carcinoma (PCa) is a leading cause of cancer morbidity and mortality in men worldwide [1]
To identify mRNAs differentially enriched or excluded from the RISC complex, Ago-2 was immunoprecipitated from extracts derived from normal prostate fibroblasts (PNF-08) and from the prostate carcinoma (PCa) cell line DU145 using the previously described Ago-2 antibody 11A9 and an appropriate isotype control [15, 16]
In addition to the laminin alpha 3 (LAMA3) mRNA, the highly RISC-enriched adenylate cyclase 3 (ADCY3) mRNA is a potential target for the upregulated miRNAs miR-25 and miR-27a/b

Summary

Introduction

Prostate carcinoma (PCa) is a leading cause of cancer morbidity and mortality in men worldwide [1]. The etiology of PCa is complex and numerous genetic [2] and epigenetic alterations [3] have previously been reported. MicroRNAs (miRNAs) are short noncoding RNAs of 18–23 nt length that posttranscriptionally regulate gene expression by binding to mRNA targets to inhibit protein synthesis (reviewed in [4]). In the context of tumorigenesis, miRNAs can function both as tumor suppressors and as oncogenes [5]. They are bound to their target mRNAs in the RISC complex, which contains an Argonaute (Ago) protein that is tethered to the mRNA by the miRNA. Binding of the miRNA to the target initially inhibits protein synthesis by reducing translation but eventually leads to degradation of the mRNA target [6]

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