DSCAM-AS1-Driven Proliferation of Breast Cancer Cells Involves Regulation of Alternative Exon Splicing and 3'-End Usage.

Jamal Elhasnaoui,Aline S C Fabricio,Anna Sapino,Elena Doria,Giulio Ferrero,Isabella Castellano,Valentina Miano,Laura Annaratone,Antonette E Leon,Michele De Bortoli

doi:10.3390/cancers12061453

Abstract

DSCAM-AS1 is a cancer-related long noncoding RNA with higher expression levels in Luminal A, B, and HER2-positive Breast Carcinoma (BC), where its expression is strongly dependent on Estrogen Receptor Alpha (ERα). DSCAM-AS1 expression is analyzed in 30 public datasets and, additionally, by qRT-PCR in tumors from 93 BC patients, to uncover correlations with clinical data. Moreover, the effect of DSCAM-AS1 knockdown on gene expression and alternative splicing is studied by RNA-Seq in MCF-7 cells. We confirm DSCAM-AS1 overexpression in high grade Luminal A, B, and HER2+ BCs and find a significant correlation with disease relapse. In total, 908 genes are regulated by DSCAM-AS1-silencing, primarily involved in the cell cycle and inflammatory response. Noteworthily, the analysis of alternative splicing and isoform regulation reveals 2085 splicing events regulated by DSCAM-AS1, enriched in alternative polyadenylation sites, 3′UTR (untranslated region) shortening and exon skipping events. Finally, the DSCAM-AS1-interacting splicing factor heterogeneous nuclear ribonucleoprotein L (hnRNPL) is predicted as the most enriched RBP for exon skipping and 3′UTR events. The relevance of DSCAM-AS1 overexpression in BC is confirmed by clinical data and further enhanced by its possible involvement in the regulation of RNA processing, which is emerging as one of the most important dysfunctions in cancer.

Highlights

Non-coding RNAs are an established layer of regulation in the molecular pathophysiology of complex diseases, including cancer [1]
In addition to these microarray and RNA-seq public data, we quantified, by Quantitative Real Time PCR (qRT-PCR), the DSCAM-AS1 expression in RNA samples derived from primary cancer tissues from two Breast Carcinoma (BC) cohorts composed, respectively, of 42 (Cohort_1) and 51 (Cohort_2) subjects
In addition to heterogeneous nuclear ribonucleoprotein L (hnRNPL), we successfully identified a set of RNA-binding proteins (RBPs) previously known to regulate Alternative PolyA (APA) site selection in the case of 30 UTR-shortening/lengthening events, including muscleblind-like proteins 1, 2 and 3 (MBNL1, MBNL2 and MBNL3), which are known to bind preferentially to 30 UTR regions [41], Ras guanosine triphosphate (GTP)-ase-activating protein-binding protein 1 (G3BP1), an essential splicing factor for normal stress granule assembly and, the preservation of polyadenylated mRNAs [42], Splicing Factor proline/glutamine rich (SFPQ) known to facilitate miRNA-target binding [42,43], as well as the RNA-binding motif 47 protein (RBM47), which was previously reported to inhibit the proliferation of different BC cell lines and whose binding was predominantly at 30 UTRs [44]

Summary

Introduction

Non-coding RNAs are an established layer of regulation in the molecular pathophysiology of complex diseases, including cancer [1]. In the context of Breast Carcinoma (BC), lncRNA expression and molecular activity were related to different stages of the disease as well as to the different BC subtypes [4]. Among these subtypes, the Estrogen Receptor alpha (ERα)-positive BC (Luminal A and B subtypes) represents the most frequent breast neoplasm with over 270,000 estimated new cases in the US population for 2020 [5]. Among the lncRNA genes related to the onset and progression of luminal BC, HOTAIR, MIAT, and DSCAM-AS1 were described in multiple studies [7]

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