The role of the long non coding RNA HAS2-AS1 in breast cancer cells

Extracellular matrix (ECM) is a network made by proteins and proteoglycans, whose structure is essential to maintain tissue architecture and to provide molecules diffusion and cellular communications. A deregulated synthesis of ECM components is often associated to a pathological status. Among various glycosaminoglycans, hyaluronan (HA) is a ubiquitous ECM component with a remarkable structural importance. It is able to modulate cell adhesion, motility, growth and inflammation after the binding with cellular receptors (CD44 and RHAMM) and the activation of different cellular pathways. In tumour microenvironment, the up-regulation of HAS2 and the overproduction of HA are often associated with tumour progression and metastasis. This also applies to breast cancer, where the accumulation of HA and the overexpression of hyaluronan synthases (HASes) in stromal and tumoral cells correlate with tumor malignancy and patients survival. The study of the regulation of HAS2, the main enzyme in the production of HA, is very important to understand the development and the progression of breast cancer. Recently, it has been discovered that the lncRNA HAS2-AS1 can modulate the expression of HAS2 and the production of hyaluronan in aortic smooth muscle cells via epigenetic modifications [1]. Although the role of HA and HAS2 in breast cancer is widely described, little is known about HAS2-AS1. Given this considerations, the aim of this project is to study the role of HAS2-AS1 in breast cancer. In particular, we compared the behaviour of MDA-MB-231 and MCF-7 cells after the modulation of HAS2-AS1 expression with functional assays evaluating cell proliferation, migration and invasion. In the same conditions, we analysed the expression of HA related genes and receptors in MDA-MB-231 cells. This analysis revealed that HAS2-AS1 knockdown stimulated the presence of a malignant phenotype, as its abrogation increased cell motility and invasion, as well as the expression of several HA related genes and the receptor CD44. These evidences suggested that HAS2-AS1 plays an important role breast tumor progression through alteration of HA metabolism. Further analysis were conducted to understand the molecular mechanisms at the basis of the changes observed. LncRNAs can orchestrate gene expression through a variety of mechanisms, regulating transcription and translation, chromatin remodelling and the interaction with other RNA species, i.e. miRNAs. HAS2-AS1 transcript contains a putative binding site for miRNA 186, a negative regulator of the pro-apoptotic receptor P2X7 [2]. In our results we demonstrated that the overexpression of HAS2-AS1 decreased the abundance of miR-186, while the transcript of P2X7 and other targets of miRNA 186 (involved in cell cycle and autophagy) raised. All together, these data suggest that the “sponge effect” of HAS2-AS1 is able to antagonise the function of miRNA 186 on its downstream targets and could explain the presence of a malignant phenotype after HAS2-AS1 silencing in MDA-MB-231. 1. Vigetti D, Deleonibus S, Moretto P, Bowen T, Fischer JW, Grandoch M, et al. Natural antisense transcript for hyaluronan synthase 2 (HAS2-AS1) induces transcription of HAS2 via protein O-GlcNAcylation. J. Biol. Chem. 2014;289:28816–26. 2. Zhou L, Qi X, Potashkin JA, Abdul-Karim FW, Gorodeski GI. MicroRNAs miR-186 and miR-150 down-regulate expression of the pro-apoptotic purinergic P2X7 receptor by activation of instability sites at the 3ʹ-untranslated region of the gene that decrease steady-state levels of the transcript. J. Biol. Chem. 2008;283:28274–86.