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

Abstract

Background The 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 lncRNA H19 mediates osteogenic differentiation of Mesenchymal Stem/Stromal Cells (MSCs) through the control of intracellular pathways. However, the effect of H19 on the extracellular matrix (ECM) components is still largely unknown. This research study was designed to decode the H19-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. Methods Mass 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. Results Our study provides an in-depth proteome-wide and matrisome-specific analysis of the ECM proteins controlled by the lncRNA H19. 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 following H19 silencing. 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, H19 is 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 that H19 silencing and miR-29c mimics have complementary but not overlapping functions. Conclusion Our data suggest H19 as a therapeutic target to engineer the bone ECM and to control cell behavior.

Mechanical strain regulates osteogenesis via Antxr1/LncRNA H19/Wnt/β‐catenin axis

“…Wnt/β-catenin signaling plays an important role in the regulation of bone metabolism. Wnt proteins induce the differentiation of osteoblast Moura et al, 2023). The lack of lncRNA H19…”

“…Consistently, other research showed that in stem cells from apical papilla, the deficiency of lncRNA H19 inhibited osteo/odontogenesis, while overexpression of of lncRNA H19 induced the opposite effect (Li et al, 2019). In addition, lncRNA H19 activation regulates BMSCs extracellular matrix components, and accelerates healing of calvarial bone defect (Kieu Nguyen et al, 2023; Moura et al, 2023). The lack of lncRNA H19 overexpression is a shortcoming in this study, which will be further explored in future experiments.…”

Long non‐coding RNA H19 mediates osteogenic differentiation of bone marrow mesenchymal stem cells through the miR‐29b‐3p/DKK1 axis

“…H19 is reduced in BMSCs of ovariectomized mice and is associated with Foxc2 synergistically promoted osteogenic differentiation of BMSCs via Wnt/β‐catenin pathway 29 . Knockout of H19 impairs the proliferation and osteogenic differentiation of BMSCs, while enhancing lipid droplet formation in preadipocytes 30 . Currently, research on osteoporosis after SCI is still limited 23,31 .…”

“… 29 Knockout of H19 impairs the proliferation and osteogenic differentiation of BMSCs, while enhancing lipid droplet formation in preadipocytes. 30 Currently, research on osteoporosis after SCI is still limited. 23 , 31 Consistent with the results of previous studies, we found that H19 expression was significantly upregulated in the SCI group.…”

Stage-Specific Modulation of Multinucleation, Fusion and Resorption by the Long Non-coding RNA DLEU1 and miR-16 in Human Primary Osteoclasts

“…For the mass-spectrometry analysis, 40 μg of protein lysates from transfected OCs were used. Protein identification and label-free quantitation were performed by nanoLC-MS/MS, composed by an Ultimate 3000 liquid chromatography system coupled to a Q-Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Scientific), as previously reported [41,50,54]. Raw data were processed using Proteome Discoverer 2.3.0.523 software (Thermo Scientific).…”