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HNGF6A Inhibits Oxidative Stress-Induced MC3T3-E1 Cell Apoptosis and Osteoblast Phenotype Inhibition by Targeting Circ_0001843/miR-214 Pathway

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Abstract

Humanin (HN), a mitochondrial derived peptide, plays cyto-protective role under various stress. In this study, we aimed to investigate the effects of HNGF6A, an analogue of HN, on osteoblast apoptosis and differentiation and the underlying mechanisms. Cell proliferation of murine osteoblastic cell line MC3TC-E1 was examined by CCK8 assay and Edu staining. Cell apoptosis was detected by Annexin V assay under H2O2 treatment. The differentiation of osteoblast was determined by Alizarin red S staining. We also tested the expression of osteoblast phenotype related protein by real-time PCR and Western blot. The interaction between Circ_0001843 and miR-214, miR-214 and TAFA5 was examined by luciferase report assay. Circ_0001843 was inhibited by siRNA and miR-214 was suppressed by miR-214 inhibitor to determine the effects of Circ_0001843 and miR-214 on cell proliferation, apoptosis, and differentiation. HNGF6A, an analogue of HN, exerted cyto-protection and osteogenesis-promotion in MC3T3-E1 cells. The expression of osteoblast phenotype related protein was significantly induced by HNGF6A. Additionally, HNGF6A treatment decreased Circ_0001843 and increased miR-214 levels, as well as inhibited the phosphorylation of p38 and JNK. We further found that Circ_0001843 directly bound with miR-214, which in turn inhibited the phosphorylation of p38 and JNK. Furthermore, both Circ_0001843 overexpression and miR-214 knockdown significantly decreased the cyto-protection and osteogenic promotion of HNGF6A. In summary, our data showed that HNGF6A protected osteoblasts from oxidative stress-induced apoptosis and osteoblast phenotype inhibition by targeting Circ_0001843/miR-214 pathway and the downstream kinases, p38 and JNK.

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Abbreviations

HN:

Humanin

MAPK:

Mitogen-activated protein kinase

JNK:

C-Jun N-terminal kinase

RT-PCR:

Reverse transcription polymerase chain reaction

CCK-8:

Cell counting Kit-8

EdU:

5-Ethynyl-2′-deoxyuridine

ALP:

Alkaline phosphatase

OCN:

Osteocalcin

BMP-2:

Bone morphogenetic protein 2

RUNX2:

Runt-related transcription factor 2

GSH:

Glutathione

ROS:

Reactive oxygen species

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Funding

This work was supported by National Natural Science Foundation of China (No. 81400849), The Natural Science Foundation of Guangdong Province (China, No. 2014A030310490), The Science and Technology Planning Project of Guangdong Province (China, No. 2017A020215189), Guangdong Provincial Key Laboratory of Bone and Joint Degeneration Disease, Hunan Provincial Natural Science Foundation (China, No. 2017JJ2154), Scientific Research Project of Hunan Health Commission Grant (China, No. B2019067), International Training Plan for Outstanding Young Scientific Research Talents in Universities of Guangdong Province and The Science and Technology Planning Project of Tianhe District (Guangdong, China, No. 2013kw004).

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Authors and Affiliations

  1. Department of Endocrinology, The Third Affiliated Hospital of Southern Medical University, No. 183 West Zhongshan Road, Tianhe District, Guangzhou, 510630, Guangdong, China

    Xiao Zhu, Jicheng Lv, Daoyan Pan & Jie Shen

  2. Department of Joint Surgery, Guangdong Provincial Key Laboratory of Bone and Joint Degeneration Diseases, The Third Affiliated Hospital of Southern Medical University, No.183 West Zhongshan Road, Tianhe District, Guangzhou, 510630, Guangdong, China

    Ziping Zhao & Daozhang Cai

  3. Department of Foot and Ankle Surgery, The Third Affiliated Hospital of Southern Medical University, Guangzhou, 510630, Guangdong, China

    Canjun Zeng

  4. Department of Endocrinology, Guangdong Second Provincial General Hospital, Guangzhou, 510317, Guangdong, China

    Bo Chen

  5. Department of Internal Medicine, the Eastern Hospital of the First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, 510700, Guangdong, China

    Haifeng Huang

  6. Department of Clinical Laboratory, The Third Affiliated Hospital of Southern Medical University, Guangzhou, 510630, Guangdong, China

    Youming Chen & Jing Zhang

  7. Department of Medical Image, The Third Affiliated Hospital of Southern Medical University, Guangzhou, 510630, Guangdong, China

    Quan Zhou

  8. Department of Endocrinology, People’s Hospital of Hunan Province, Changsha, 410011, Hunan, China

    Li Yang

  9. Department of Medicine, Western Health, The University of Melbourne, St Albans, Victoria, 3021, Australia

    Gustavo Duque

  10. Australian Institute for Musculoskeletal Science (AIMSS), The University of Melbourne and Western Health, St Albans, Victoria, 3021, Australia

    Gustavo Duque

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  1. Xiao Zhu
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Contributions

Study design/planning: XZ; Data collection/entry: HH, YC, LY and JZ; Data analysis/statistics: ZZ, HH, YC, QZ, JZ and DP; Data interpretation: XZ, ZZ, BC, BC, JL, DC and JS; Preparation of manuscript: XZ; Literature analysis/search: XZ, ZZ, BC, YC, QZ, LY, JL, DP, DC and JS; Manuscript revise: XZ, CZ and GD; Funds collection: XZ, LY and DC. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Jie Shen, Gustavo Duque or Daozhang Cai.

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Xiao Zhu, Ziping Zhao, Canjun Zeng, Bo Chen, Haifeng Huang, Youming Chen, Quan Zhou, Li Yang, Jicheng Lv, Jing Zhang, Daoyan Pan, Jie Shen, Gustavo Duque and Daozhang Cai declare that they have no conflict of interest.

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223_2020_660_MOESM1_ESM.tif

Supplementary file1 (TIF 4930 kb) Supplementary figure 1. Effects of HNGF6A and Circ_0001843 on oxidation in MC3T3-E1 cells. MC3T3-E1 cells were transfected with Circ_0001843 and exposed to H2O2 (400 μM) for 4 h. Then the cells were exposed to control or HNGF6A for 3 days and collected to determine the levels of GSH (A) and ROS (B). *, p<0.05, **, p<0.01.

223_2020_660_MOESM2_ESM.tif

Supplementary file2 (TIF 915 kb) Supplementary figure 2. Effects of miR-214, p38 and JNK on the cyto-protection of HNGF6A. MC3T3-E1 cells were transfected with miR-214 and exposed to H2O2 (400 μM) for 4 h. Cells were exposed to HNGF6A alone or in combination with SB203580 (10 μΜ) or SP600125 (10 μΜ). After treatment, the cells were collected to determine the expression of TAFA5 by RT-PCR (A) and western blot (B). *p<0.05, **p<0.01 as compared with the parent MC3T3-E1 cells treated with NC or control.

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Zhu, X., Zhao, Z., Zeng, C. et al. HNGF6A Inhibits Oxidative Stress-Induced MC3T3-E1 Cell Apoptosis and Osteoblast Phenotype Inhibition by Targeting Circ_0001843/miR-214 Pathway. Calcif Tissue Int 106, 518–532 (2020). https://doi.org/10.1007/s00223-020-00660-z

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