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lncRNA SERPINB9P1 Regulates SIRT6 Mediated Osteogenic Differentiation of BMSCs via miR-545-3p

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Abstract

Evidence has shown that the altered osteogenic differentiation of human bone marrow stromal cells (BMSCs) under pathological conditions, such as osteoporosis, lead to the imbalance of bone tissue generation and destruction. Recent studies have indicated that long noncoding RNAs may play a role in regulating BMSCs osteogenic differentiation. This contributed to our impetus to move forward with the investigation of the function of lncRNA SERPINB9P1 in osteogenic differentiation of BMSCs and the potential mechanisms involved. Osteogenic differentiation of BMSCs was induced by osteogenic medium. Relative expression of lncRNA SERPINB9P1 and miR-545-3p were tested by qRT-PCR. Osteogenic mineralization was examined by Alizarin S Red staining, ALP staining, and ALP activity assay. Expression of osteoblastic markers were detected by Western blot. RNA-binding protein immunoprecipitation and dual-luciferase reporter assays were performed to test the interaction between lncRNA SERPINB9P1 and miR-545-3p. BMSCs osteogenic differentiation resulted in LncRNA SERPINB9P1 overexpression while miR-545-3p inhibition. Functional assays suggest that knockdown of lncRNA SERPINB9P1 or overexpression of miR-545-3p both inhibit BMSC osteogenic differentiation. lncRNA SERPINB9P1 was proven to regulate the osteogenic differentiation of BMSCs by altering SIRT6 expression through its suppressive effects on miR-545-3p. lncRNA SERPINB9P1 promotes osteogenic differentiation of BMSCs through the miR-545-3p/SIRT6 pathway.

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Abbreviations

lncRNA:

Long non-coding RNA

MiR:

MicroRNA

MSC:

Marrow stem cell

BMSC:

Bone marrow stem cell

OP:

Osteoporosis

OB:

Osteoblasts

OM:

Osteogenic medium

ALP:

Alkaline phosphatase

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Acknowledgements

We would like to give our sincere gratitude to the reviewers for their constructive comments.

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Authors and Affiliations

  1. Department of Orthopedics, The Affiliated Children’s Hospital of Nanchang University, Nanchang, 330006, People’s Republic of China

    Min Wu & Qunqun Zeng

  2. Department of Orthopedics, Jiangxi Provincial Children’s Hospital, Nanchang, 330006, People’s Republic of China

    Min Wu & Qunqun Zeng

  3. Department of Orthopedics, The First Affiliated Hospital of Nanchang University, Nanchang, 330006, People’s Republic of China

    Min Dai & Xuqiang Liu

  4. Department of Plastic Surgery, The First Affiliated Hospital of Nanchang University, No. 17, Yongwai Zheng Street, Nanchang, 330006, People’s Republic of China

    Yingjie Lu

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  1. Min Wu
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Correspondence to Yingjie Lu.

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Conflict of interest

Min Wu, Min Dai, Xuqiang Liu, Qunqun Zeng, and Yingjie Lu declare that they have no conflict of interest.

Ethics approval and consent to participate

Human bone marrow was collected in orthopaedic surgery. All people have consented to the study, and the procedures were approved by the Ethics Committee of the Affiliated Children's Hospital of Nanchang University (JXSETYY-YXKY-20220183).

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Supplementary Information

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223_2022_1034_MOESM1_ESM.tif

Supplemental Figure 1. lncRNA SERPINB9P1 was elevated and miR-545-3p was decreased during BMSC osteogenic differentiation. (A) The phenotype of BMSCs were tested by flow cytometry. (B) Relative expression of ALP, Runx2, and OCN were detected on days 0, 1, 5, 10, and 15. GAPDH as the loading control. (C) Relative expression of lncRNA SERPINB9P1 and miR-545-3p during BMSC osteogenic differentiation on days 0, 1, 5, 10, and 15. Data indicate mean ± SD, n = 3. *p < 0.05, **p < 0.01, and ***p < 0.001. Supplementary file1 (TIF 9702 KB)

223_2022_1034_MOESM2_ESM.tif

Supplemental Figure 2 lncRNA SERPINB9P1 overexpression and miR-545-3p inhibition decreased the expression of RANKL and increased the expression of OPG. (A and B) Western blot was performed to test the protein expression of RANKL and OPG in BMSCs treated with lncRNA SERPINB9P1 overexpression plasmid (oe-SERPINB9P1) or miR-545-3p inhibitor. Supplementary file2 (TIF 832 KB)

Supplemental Figure 3 UCSC homology prediction. Supplementary file3 (PNG 23 KB)

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Wu, M., Dai, M., Liu, X. et al. lncRNA SERPINB9P1 Regulates SIRT6 Mediated Osteogenic Differentiation of BMSCs via miR-545-3p. Calcif Tissue Int 112, 92–102 (2023). https://doi.org/10.1007/s00223-022-01034-3

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