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Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow
Published on: July 15, 2016
Hsa_circ_0066523 promotes the proliferation and osteogenic differentiation of bone mesenchymal stem cells by
Wei Xin1, Shuai Yuan1, Bo Wang1
1Department of Orthopedics, Changzheng Hospital, Shanghai, China.
Aims:
Circular RNAs (circRNAs) are a novel type of non-coding RNA that plays major roles in the development of diverse diseases including osteonecrosis of the femoral head (ONFH). Here, we explored the impact of hsa_circ_0066523 derived from forkhead box P1 (FOXP1) (also called circFOXP1) on bone mesenchymal stem cells (BMSCs), which is important for ONFH development.
Methods:
RNA or protein expression in BMSCs was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot, respectively. Cell Counting Kit 8 (CCK8) and 5-ethynyl-2'-deoxyuridine (EdU) were used to analyze cell proliferation. Alkaline phosphatase (ALP) activity, ALP staining, and Alizarin Red S staining were employed to evaluate the osteoblastic differentiation. Chromatin immunoprecipitation (ChIP), luciferase reporter, RNA pull down, and RNA immunoprecipitation (RIP) assays were combined for exploring molecular associations.
Results:
Circ_0066523 was upregulated in osteogenic induction process of BMSCs. Silencing circ_0066523 restrained the proliferation and osteogenic differentiation of BMSCs. Mechanistically, circ_0066523 activated phosphatidylinositol-4,5-bisphosphate 3-kinase / AKT serine/threonine kinase 1 (PI3K/AKT) pathway via recruiting lysine demethylase 5B (KDM5B) to epigenetically repress the transcription of phosphatase and tensin homolog (PTEN). Functionally, AKT signalling pathway agonist or PTEN knockdown counteracted the effects of silenced circ_0066523 on BMSC proliferation and differentiation.
Conclusion:
Circ_0066523 promotes the proliferation and differentiation of BMSCs by epigenetically repressing PTEN and therefore activating AKT pathway. This finding might open new avenues for the identification of therapeutic targets for osteoblast differentiation related diseases such as ONFH. Cite this article: Bone Joint Res 2021;10(8):526-535.
Insights
Circular RNA FOXP1 (circFOXP1) promotes bone mesenchymal stem cell proliferation and osteogenic differentiation. It achieves this by epigenetically repressing PTEN, thereby activating the AKT pathway, offering potential therapeutic targets for osteonecrosis of the femoral head (ONFH).
Area of Science:
- Molecular Biology
- Stem Cell Biology
- RNA Biology
Background:
- Circular RNAs (circRNAs) are crucial regulators in various diseases, including osteonecrosis of the femoral head (ONFH).
- Bone mesenchymal stem cells (BMSCs) play a vital role in ONFH pathogenesis.
- Understanding the role of specific circRNAs in BMSC function is essential for developing ONFH therapies.
Purpose of the Study:
- To investigate the role of hsa_circ_0066523 (circFOXP1) in regulating the proliferation and osteogenic differentiation of BMSCs.
- To elucidate the underlying molecular mechanisms by which circFOXP1 affects BMSC function.
- To explore the potential of circFOXP1 as a therapeutic target for ONFH.
Main Methods:
- Quantitative real-time PCR (qRT-PCR) and Western blot to assess RNA and protein expression.
- Cell Counting Kit-8 (CCK8) and 5-ethynyl-2'-deoxyuridine (EdU) assays for cell proliferation analysis.
- Alkaline phosphatase (ALP) activity, ALP staining, and Alizarin Red S staining for osteogenic differentiation assessment.
- Chromatin immunoprecipitation (ChIP), luciferase reporter, RNA pull-down, and RNA immunoprecipitation (RIP) assays to determine molecular interactions.
Main Results:
- CircFOXP1 expression was upregulated during the osteogenic induction of BMSCs.
- Silencing circFOXP1 inhibited BMSC proliferation and osteogenic differentiation.
- CircFOXP1 epigenetically repressed PTEN transcription by recruiting KDM5B, leading to PI3K/AKT pathway activation.
- AKT pathway activation or PTEN knockdown rescued the inhibitory effects of circFOXP1 silencing on BMSC function.
Conclusions:
- CircFOXP1 promotes BMSC proliferation and differentiation by epigenetically inhibiting PTEN and activating the AKT pathway.
- These findings highlight circFOXP1 as a key regulator in BMSC biology relevant to ONFH.
- CircFOXP1 represents a potential therapeutic target for diseases involving abnormal osteoblast differentiation, such as ONFH.
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