Long noncoding RNA H19 promotes the acquisition of a mesenchymal-like invasive phenotype in mesothelial primary cells through an HDAC1-mediated WT1/Sp1 switch

Abstract

: Peritoneal fibrosis is a pathological alteration of the peritoneal membrane occurring in pro-inflammatory conditions, including peritoneal dialysis (PD), a renal replacement therapy. Characteristic of this process is the acquisition of invasive/pro-fibrotic abilities by mesothelial cells (MCs) through induction of mesothelial to mesenchymal transition (MMT), a cell-specific form of EMT. Long noncoding (lnc) RNAs act as major players in physiologic regulatory circuitries of the cell. While LncRNA-H19 (lncH19), one of the first lncRNAs identified, has been broadly studied in tumorigenesis, its role in peritoneum fibrotic diseases has been scarcely addressed so far. Aim of this study was to investigate the role of H19 in the acquisition of a mesenchymal-like phenotype in primary fibrotic MCs from PD patients, and to elucidate epigenetic mechanisms controlling its expression. Genetic silencing/ectopic expression experiments revealed that H19 promoted the expression of MMT markers while downregulating the epithelial marker E-Cadherin, and favored MC directed migration and invasion on a collagen matrix. Silencing of three main H19 isoforms revealed a synergistic activity in the induction of a mesenchymal phenotype. Treatment with MS-275, an HDAC1-3 specific inhibitor previously known to promote MMT reversal, as well as HDAC1 genetic silencing, downregulated lncRNA H19 expression. Bioinformatic analysis revealed a binding sequence of Wilm's Tumor Protein 1 (WT1), the master gene of mesothelial differentiation, on the H19 promoter at an area with multiple acetylation peaks partially overlapping the binding site of Specificity protein 1 (Sp1), another transcription factor active in cellular plasticity regulation. Genetic silencing and Chromatin Immunoprecipitation (ChIP) experiments demonstrated that HDAC1 inhibition promotes a switch between WT1 and Sp1 in H19 promoter occupancy, favoring an inhibitory effect of WT1 on H19 expression and the reversal towards an epithelial-like phenotype. Overall, we discovered an HDAC1-WT1/Sp1-H19 axis potentially relevant to the design of new therapies aimed at counteracting peritoneal fibrosis.