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CircHIPK3 Regulates Vascular Smooth Muscle Cell Calcification Via the miR-106a-5p/MFN2 Axis

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Abstract

Atherosclerosis is the most common arterial disease and is closely related to vascular calcification. CircHIPK3 has been implicated in atherosclerosis development, but the possible downstream regulatory mechanisms remain unclear. The levels of circHIPK3, miR-106a and MFN2 in tissues and blood samples of patients with atherosclerosis were detected by RT-qPCR. The levels of circHIPK3, miR-106a and MFN2 were detected by RT-qPCR and the expression levels of MFN2, osteogenic and cartilage differentiation marker proteins were detected by western blot in vitro. ALP staining, Alizarin Red staining, and calcium content detection evaluated the degree of osteogenic differentiation of cells. Alcian blue staining detected the level of cell cartilage differentiation. Luciferase detected the targeting relationship between circHIPK3 and miR-106a-5p, as well as miR-106a-5p and MFN2. CircHIPK3 and MFN2 were low expressed and miR-106a-5p was highly expressed in tissues and blood samples of patients with atherosclerosis, as well as vascular smooth muscle cell (VSMC) with osteogenic and cartilage differentiation. Overexpression of circHIPK3 reduced the cell mineralization and calcium content. Overexpression of circHIPK3 inhibited osteogenic differentiation by decreasing ALP activity, RUNX2, and OPG expression, and increasing SM22α and SMA level. What’s more, overexpression of circHIPK3 decreased the chondrogenic differentiation by inhibiting the protein level of SOX9, aggrecan, and collagen II. CircHIPK3 targeted miR-106a-5p and miR-106a-5p targeted MFN2. MiR-106a-5p overexpression or MFN2 depletion repressed the effect of circHIPK3 overexpression on VSMC calcification. CircHIPK3 regulated osteogenic and cartilage differentiation of VSMC via miR-106a-5p/MFN2 axis, indicating a target for treating vascular calcification.

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Data Availability

All data generated or analyzed during this study are included in this article. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

ALP:

Alkaline phosphatase

CircRNA:

Circular RNA

FBS:

Fetal bovine serum

MiRNA:

MicroRNA

MFN2:

Mitochondrial fusion protein 2

PBS:

Phosphate-buffered solution

RIP:

RNA binding protein immunoprecipitation

RT-qPCR:

Real-time fluorescence quantitative PCR

VSMC:

Vascular smooth muscle cells

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Acknowledgements

This project was supported by Hainan Province Clinical Medical Center

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Authors and Affiliations

  1. Department of Vascular Surgery, Hainan General Hospital, Hainan Affiliated Hospital of Hainan Medical University, No.19, **uhua Road, Haikou, 570311, Hainan Province, People’s Republic of China

    Wen-Bo Zhang, You-Fei Qi, Zhan-**ang **ao, Hao Chen, Sa-Hua Liu, Zhen-Zhen Li, Zhao-Fan Zeng & Hong-Fei Wu

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  1. Wen-Bo Zhang
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Contributions

W. B. Z.: Conceptualization.

Y. F. Q.: Funding acquisition.

Z. X. X.: Writing—original draft.

H. C.: Data curation; resources.

S. H. L.: Methodology; formal analysis.

Z. Z. L.: Investigation; software; visualization.

Z. F. Z.: Project administration; supervision.

H. F. W.: Validation; writing—review and editing.

All authors have read and approved the final version of this manuscript to be published.

Corresponding author

Correspondence to Wen-Bo Zhang.

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Ethical Approval

All participating subjects gave informed consent. This study was approved by the Hainan General Hospital, Hainan Affiliated Hospital of Hainan Medical University Ethics Committee. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

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Informed consent was obtained from all individual participants included in the study.

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The authors declare no competing interests.

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Associate Editor Adrian Chester oversaw the review of this article

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ESM 1

Supplementary Fig. 1 The expression of HIPK3 in atherosclerosis (A) The expression of HIPK3 in normal tissues (n=12) and atherosclerotic tissues (n=9) were detected by RT-qPCR. (B) RT-qPCR detected the expression of HIPK3 in blood samples of patients with atherosclerosis (n=20) and normal volunteers (n=20). *P < 0.05, **P < 0.01, ***P < 0.001. (PNG 26 kb)

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Zhang, WB., Qi, YF., **ao, ZX. et al. CircHIPK3 Regulates Vascular Smooth Muscle Cell Calcification Via the miR-106a-5p/MFN2 Axis. J. of Cardiovasc. Trans. Res. 15, 1315–1326 (2022). https://doi.org/10.1007/s12265-022-10247-8

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