Long non-coding RNA H19 regulates matrisome signature and impacts cell behavior on MSC-engineered extracellular matrices

Abstract

AbstractBackgroundThe vast and promising class of long non-coding RNAs (lncRNAs) has been under investigation for distinct therapeutic applications. Nevertheless, their role as molecular drivers of bone regeneration remains poorly studied. The lncRNAH19mediates osteogenic differentiation of Mesenchymal Stem/Stromal Cells (MSCs) through the control of intracellular pathways. However, the effect ofH19on the extracellular matrix (ECM) components is still largely unknown. This research study was designed to decode theH19-mediated ECM regulatory network, and to reveal how the decellularized siH19-engineered matrices influence MSC proliferation and fate. This is particularly relevant for diseases in which the ECM regulation and remodeling processes are disrupted, such as osteoporosis.MethodsMass spectrometry-based quantitative proteomics analysis was used to identify ECM components, after oligonucleotides delivery to osteoporosis-derived hMSCs. Moreover, qRT-PCR, immunofluorescence and proliferation, differentiation and apoptosis assays were performed. Engineered matrices were decellularized, characterized by atomic force microscopy and repopulated with hMSC and pre-adipocytes. Clinical bone samples were characterized by histomorphometry analysis.ResultsOur study provides an in-depth proteome-wide and matrisome-specific analysis of the ECM proteins controlled by the lncRNAH19. Using bone marrow-isolated MSC from patients with osteoporosis, we identified fibrillin-1 (FBN1), vitronectin (VTN) and collagen triple helix repeat containing 1 (CTHRC1), among others, as having different pattern levels followingH19silencing. Decellularized siH19-engineered matrices are less dense and have a decreased collagen content compared with control matrices. Repopulation with naïve MSCs promotes a shift towards the adipogenic lineage in detriment of the osteogenic lineage and inhibits proliferation. In pre-adipocytes, these siH19-matrices enhance lipid droplets formation. Mechanistically,H19is targeted by miR-29c, whose expression is decreased in osteoporotic bone clinical samples. Accordingly, miR-29c impacts MSC proliferation and collagen production, but does not influence ALP staining or mineralization, revealing thatH19silencing and miR-29c mimics have complementary but not overlapping functions.ConclusionOur data suggestH19as a therapeutic target to engineer the bone ECM and to control cell behavior.