A total of 664 were identified in bone marrow mesenchymal stem cell-derived exosomes, with 113 being novel circRNAs.
Novel_circ_0001686 was significantly downregulated in exosomes from patients with steroid-induced osteonecrosis of the femoral head (SONFH).
This circRNA exhibited features typical of circular RNAs, including a stable structure and localization in the cytoplasm.
Functional assays demonstrated that novel_circ_0001686 enhanced the proliferation, migration, and anti-apoptotic activity of human umbilical vein endothelial cells (HUVECs).
Mechanistically, novel_circ_0001686 functions as a competing endogenous RNA, sponging miR-34a-5p and mitigating its inhibition of Piezo1.
Rescue experiments showed that the pro-angiogenic effects of novel_circ_0001686 were negated when miR-34a-5p mimic or Piezo1 knockdown was applied.
Ex vivo and in vivo assays confirmed that novel_circ_0001686 facilitates microvessel sprouting and capillary formation.
Simplified
BACKGROUND: Steroid-induced osteonecrosis of the femoral head (SONFH) is characterized by impaired angiogenesis and bone remodeling, ultimately leading to joint dysfunction. Exosomes derived from bone marrow mesenchymal stem cells (BMSCs) carry bioactive molecules, including circular RNAs (), which play crucial roles in regulating angiogenesis and tissue repair. This study investigated the role of a novel BMSC-derived exosomal circRNA (novel_circ_0001686) in promoting angiogenesis through a competing endogenous RNA () mechanism, targeting the miR-34a-5p/Piezo1 axis.
METHODS: This study employed a comprehensive approach to determine the roles of circRNAs in BMSC-derived exosomes from patients with SONFH. High-throughput sequencing identified circRNA profiles, with a focus on novel_circ_0001686. Its structure and expression were confirmed via Sanger sequencing, qRT-PCR, agarose gel electrophoresis, and ribonuclease R (RNase R) treatment, while its subcellular localization was determined through fluorescence in situ hybridization (FISH) and nuclear/cytoplasmic fractionation. The biological effects of novel_circ_0001686 were explored using human umbilical vein endothelial cells (HUVECs) with lentivirus-mediated overexpression and knockdown to assess its impact on cell proliferation, migration, apoptosis, and angiogenesis. Exosome functionality was investigated using co-culture systems, exosome uptake assays, and GW4869 treatment to evaluate the role of exosome-mediated transfer in these processes. Mechanistic studies involved miRNA prediction tools, dual-luciferase reporter assays, RNA immunoprecipitation (RIP), RNA antisense purification (RAP), and rescue experiments. Angiogenic effects were further evaluated using ex vivo aortic ring and in vivo chicken chorioallantoic membrane (CAM) assays.
RESULTS: Sequencing identified 664 circRNAs in BMSC-derived exosomes, including 113 novel circRNAs. Novel_circ_0001686, which was significantly downregulated in SONFH exosomes, exhibited hallmark features of circRNAs, including a stable circular structure and cytoplasmic localization. Functional studies showed that it significantly enhanced HUVEC proliferation, migration, anti-apoptotic activity, and tube formation. Mechanistic analyses revealed that novel_circ_0001686 acts as a ceRNA, sponging miR-34a-5p and reducing its suppressive effect on Piezo1. Rescue experiments confirmed that miR-34a-5p mimic or Piezo1 knockdown abolished the pro-angiogenic effects of novel_circ_0001686. Ex vivo aortic ring assays and the in vivo CAM model substantiated the ability of exosomal novel_circ_0001686 to enhance microvessel sprouting and capillary formation.
CONCLUSIONS: This study provides the first reported circRNA profile of BMSC-derived exosomes in SONFH, identifying novel_circ_0001686 as a key regulator of angiogenesis. By acting through the novel_circ_0001686/miR-34a-5p/Piezo1 axis, it enhances endothelial function and vascular remodeling. These findings imply that novel_circ_0001686 is a promising therapeutic target, underscoring the potential of exosome-based strategies for restoring vascularization and bone homeostasis in SONFH.
Key numbers
40-fold
Significant Downregulation
Expression of in after overexpression.
90%
Enhanced Proliferation
Reduction in after knockdown of .
12
Microvessel Sprouting
Count of microvessels sprouted in the aortic ring assay with OE-exo treatment.
Full Text
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Declarations. Ethics approval and consent to participate: Experiments involving human cells and animals were approved by the Ethics Committee of The Affiliated Hospital of Qingdao University (No: AHQU-MAL20240329ZYX; Date of approval: March 29, 2024); The original source (Procell) has confirmed that there was initial ethical approval for collection of human cells, and that the donors had signed informed consent. Consent for publication: The manuscript was read and approved for publication by all authors. Competing interests: The authors declare that they have no competing interests.
PubMed
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