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ADSCs Promote Tenocyte Proliferation by Reducing the Methylation Level of lncRNA Morf4l1 in Tendon Injury.

ABSTRACT: Objective: Tendons are the special connective tissue that connects bones to muscles and governs joint movement in response to loads passed by muscles. The healing of tendon injuries is still a challenge. In recent years, adipose-derived mesenchymal stem cells (ADSCs) have been increasingly used for tissue regeneration, but the underlying mechanism of tendon injury still remains unclear. Methods: High-throughput sequencing was used to identify a novel lncRNA, whose expression was significantly decreased in injured tendon compared with normal tendon. Furthermore, pyrosequencing, nuclear-cytoplasmic separation, FISH assay and qRT-PCR analysis were used to verify the level of lncRNA methylation in the injured tenocytes. lncRNA was confirmed to promote the proliferation of tenocytes by flow cytometry, wound healing assay, qRT-PCR, and western blot, and the target gene of lncRNA was predicted and verified. To confirm that ADSCs could repair injured tendons, ADSCs and injured tenocytes were co-cultured in vitro, and ADSCs were injected into injured tendons in vitro, respectively. Results: The lncRNA Morf4l1 promoter methylation in injured tendons led to down-regulation of its expression and inhibition of tenocyte proliferation. LncRNA Morf4l1 promoted the expression of TGF-β2 by targeting 3'U of miR-145-5p. After co-cultured ADSCs and injured tenocytes, the expression of lncRNA Morf4l1 was up-regulated, and the proliferation of injured tenocytes in vitro was promoted. The ADSCs were injected into the injured tendon to repair the injured tendon in vivo. Conclusion: This study confirmed that ADSCs promoted tendon wound healing by reducing the methylation level of lncRNA Morf4l1.

SUBMITTER: Zhao H

PROVIDER: S-EPMC9290323 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundTendon derived stem cells (TDSCs) have proven to be effective in tendon repair by secreting paracrine factors, which modulate the function of resident cells and inflammatory process. Exosomes, which are secreted from cells to mediate intercellular communication, may be used to treat tendon injuries. Here, we aimed to determine the effects of exosomes from TDSCs (TDSC-Exos) on tendon repair and to explore the underlying mechanism by investigating the role of microRNAs (miRNAs).MethodsTDSC-Exos were isolated from TDSC conditioned medium. In vitro studies were performed to investigate the effects of TDSC-Exos on the proliferation, migration, cytoprotection, collagen production and tendon-specific markers expression in tenocytes. In order to determine the therapeutic effects of TDSC-Exos in vivo, we used a scaffold of photopolymerizable hyaluronic acid (p-HA) loaded with TDSC-Exos (pHA-TDSC-Exos) to treat tendon defects in the rat model. Subsequently, RNA sequencing and bioinformatic analyses were used to screen for enriched miRNAs in TDSC-Exos and predict target genes. The miRNA-target transcript interaction was confirmed by a dual-luciferase reporter assay system. In order to determine the role of candidate miRNA and its target gene in TDSC-Exos-regulated tendon repair, miRNA mimic and inhibitor were transfected into tenocytes to evaluate cell proliferation and migration.ResultsTreatment with TDSC-Exos promoted proliferation, migration, type I collagen production and tendon-specific markers expression in tenocytes, and also protected tenocytes from oxidative stress and serum deprivation. The scaffold of pHA-TDSC-Exos could sever as a sustained release system to treat the rat model of tendon defects. In vivo study showed that TDSC-Exos promoted early healing of injured tendons. Rats treated with TDSC-Exos had better fiber arrangement and histological scores at the injury site. Besides, the injured tendons treated with TDSC-Exos had better performance in the biomechanical testing. Therefore, the pHA-TDSC-Exos scaffold proved to facilitate tendon repair in the rat model. miR-144-3p was enriched in TDSC-Exos and promoted tenocyte proliferation and migration via targeting AT-rich interactive domain 1A (ARID1A).ConclusionsTDSC-Exos enhanced tenon repair through miR-144-3p-regulated tenocyte proliferation and migration. These results suggest that TDSC-Exos can serve as a promising strategy to treat tendon injuries.

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ADSCs Promote Tenocyte Proliferation by Reducing the Methylation Level of lncRNA Morf4l1 in Tendon Injury.

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