Unraveling Interaction between Estrogen Receptor alpha and long non coding RNA, DSCAM-AS1

Estrogen receptor alpha (ERα) is a master transcriptional factor in estrogen receptor-positive (ER+) breast cancer, involved in the expression of genes essential for tumor growth. Recent evidence highlights the binding of transcription factors with long non-coding RNAs indicating the function as RNA binding proteins, not only association with DNA acting on genomic level. This study focused on the interaction between ERα and long non-coding DSCAM-AS1, highly expressed in breast cancer, elucidating its molecular basis and implications in breast cancer progression. The analysis of CLIP-Seq against ERα showed a signal on DSCAM-AS1 mRNA suggesting a possible interaction with ERα. In order to demonstrate the interaction between DSCAM-AS1 and ERα, we designed a probe for RNA pull-down assay. The probe is an oligonucleotide with a sequence complementary to the position on DSCAM-AS1 based on the parameters of A, B and C. The biotinylated probe was incubated with lysates of MCF7 breast cancer cells. After Western Blot analysis, we found an enrichment of ERα protein in samples with DSCAM-AS1-specific probe respect to unrelated probe. RNA immunoprecipitation assay was performed using ERα antibodies followed by Real Time PCR for DSCAM-AS1. The subcellular localization of the complex might suggest the function, therefore we performed RNA Pull Down in cytosolic and nuclear fractions. We found an increased enrichment in the cytosol fraction, suggesting that ERα-DSCAM-AS1 may act in non-genomic pathway. Altogether, these data indicate that ERα interacts with DSCAM-AS1 and the complex is enriched in the cytosol. Further experiments will be done to understand the regulation of this complex and its biological function. In the TF-RNA interaction, RNA acts as a guide, scaffold, or decoy, regulating TF occupancy on the promoter and transcription output. In this work, the lncRNA DSCAM-AS1 was considered in its modulating function of the transcription factor Estrogen Receptor-alpha. ERα is normally a ligand-responsive transcription factor activated by the ligand Estrogen E2, a steroid hormone involved in numerous pathways in the human physiology of the reproductive, growth, and developmental systems. Like other TFs, canonically ERα binds a specific double-stranded estrogen response element (ERE) sequence via its DBD. ERα has been shown to be a key factor for more than 70% of breast cancers studied for years in the role of transcription factor. For this reason, an attempt has been made to inhibit ERα to try to stop cancer progression with anticancer drugs such as Tamoxifen, an estrogen antagonist, often used as a therapeutic agent of first resort as it blocks the activity of ERα. Unfortunately, however, it turned out to be an extenuating circumstance as many patients developed a relapse, becoming insensitive to this antagonist, and in others, ERα remained active. These results have led to the investigation and characterization of the estrogen receptor α, leading to the discovery that ERα represents a strong non-canonical RNA-binding protein. One of these RNAs involved with ERα binding turns out to be the lncRNA DSCAM-AS1 (a variant of the DSCAM gene) protagonist of breast cancer progression. In this research, we verified the association of DSCAM-AS1 with the ERα protein in the MCF-7 breast cancer cell line with the aim of understanding the role of the ERα-DSCAM complex, its cytosolic and nuclear localization, and the presence or absence of this complex in MCF-7 stimulated with estrogen E2. The research project began by studying ERα interactions in various public scientific databases to search among the most relevant lncRNAs that interact with the transcription factor in question. DSCAM-AS1 was our lncRNA resulting from the research because it also had a strong interaction with the estrogen receptor α (given a specific RNA-protein interaction analysis).