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LncRNA RAD51-AS1 Regulates Human Bone Marrow Mesenchymal Stem Cells via Interaction with YBX1 to Ameliorate Osteoporosis

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Abstract

Long noncoding RNA (lncRNA) is a new key regulatory molecule in the occurrence of osteoporosis, but its research is still in the primary stage. In order to study the role and mechanism of lncRNA in the occurrence of osteoporosis, we reannotated the GSE35956 datasets, compared and analyzed the differential expression profiles of lncRNAs between bone marrow mesenchymal stem cells (hBMSCs) from healthy and osteoporotic patients, and then screened a lncRNA RAD51-AS1 with low expression in hBMSCs from osteoporotic patients, and its role in the occurrence of osteoporosis has not been studied. We confirmed that the expression level of lncRNA RAD51-AS1 in hBMSCs from patients with osteoporosis was significantly lower than those from healthy donors. A nuclear cytoplasmic separation experiment and RNA fluorescence in situ hybridization showed that RAD51-AS1 was mainly located in the nucleus. RAD51-AS1 knockdown significantly inhibited the proliferation and osteogenic differentiation of hBMSCs and significantly increased their apoptosis, while RAD51-AS1 overexpression significantly promoted the proliferation, osteogenic differentiation, and ectopic bone formation of hBMSCs. Mechanistically, we found that RAD51-AS1 banded to YBX1 and then activated the TGF-β signal pathway by binding to Smad7 and Smurf2 mRNA to inhibit their translation and transcription up-regulated PCNA and SIVA1 by binding to their promoter regions. In conclusion, RAD51-AS1 promoted the proliferation and osteogenic differentiation of hBMSCs by binding YBX1, inhibiting the translation of Smad7 and Smurf2, and transcriptionally up-regulated PCNA and SIVA1.

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Data Availability

GSE35956 raw data were downloaded from The Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE35956).

Abbreviations

BMSCs:

Bone marrow mesenchymal stem cells

hBMSCs:

Human Bone marrow mesenchymal stem cells

LncRNAs:

Long noncoding RNAs

RBP:

RNA-binding protein

ALP:

Alkaline phosphatase

FISH:

Fluorescent in situ hybridization

IF:

Immunofluorescence

CHIRP:

Chromatin isolation by RNA purification

RIP:

RNA immunoprecipitation

CUT&Tag:

Cleavage under targets and tagmentation

SD:

Standard deviation

ANOVA:

Analysis of variance

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Acknowledgements

We thank Prof. Deng-Shun Miao for the research platform and our partners in A426 lab for their help. We also thank HaploX for providing bioinformatics analysis.

Funding

This work was supported by the National Natural Science Foundation of China (Grant No. 81730066) and the Nanjing Health Science and Technology Development Special Funds (Grant No. ZKX19025).

Author information

Authors and Affiliations

  1. Department of Orthopedics, Nanjing First Hospital, Nanjing Medical University, Nanjing, 210006, China

    Beichen Li, Qinjue Wang, Quan Liu, Guantong Wang & Qiang Sun

  2. State Key Laboratory of Reproductive Medicine, Department of Anatomy, Histology and Embryology, The Research Center for Bone and Stem Cells, Nanjing Medical University, Nanjing, 211100, China

    Jing Wang, Fangrong Xu & Dengshun Miao

  3. Department of Plastic Surgery, The Affiliated Friendship Plastic Surgery Hospital of Nanjing Medical University, Nanjing, 211161, China

    Dengshun Miao

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  1. Beichen Li
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Contributions

Qiang Sun, Dengshun Miao, and Beichen Li were involved in the conception and design of this study. Beichen Li performed the experiments and wrote the manuscript. Jing Wang and Fangrong Xu provided suggestions and performed the analysis. Qinjue Wang provided suggestions for revisions and embellished the article. Liu Quan and Wang Guan Tong insightfully discussed and revised the manuscript. All authors read and approved the final submitted manuscript.

Corresponding authors

Correspondence to Dengshun Miao or Qiang Sun.

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Ethics Approval and Consent to Participate

This study was approved by the ethics committee of the Nanjing First Hospital Medical University (approval number: 00001501). Written informed consent was obtained from all participants included in the study.

This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Nanjing Medical University. All procedures involving animals were approved by the Animal Use and Care Committee of the Nanjing Medical University (approval number: IACUC-2203058).

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The datasets supporting the conclusions of this article are included within the article.

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The authors declare no competing interests.

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Supplementary Information

Supplementary Table S1

The forward and reverse primers of qPCR for each gene are listed in this table. (XLS 32 kb)

Supplementary Table S2

The used probes in CHIRP assays were listed in this table. (XLSX 9.21 kb)

Supplementary Table S3

The identified 103 DE lncRNAs (68 upregulated and 35 downregulated) in the osteoporosis group compared with the normal group. (XLS 219 kb)

Supplementary Table S4

The detailed information of all human cell donors. (XLS 42 kb)

Supplementary Image S5

The full (uncropped and unadjusted) images of Western Blot. (PNG 547 kb)

High resolution image (TIF 3485 kb)

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Li, B., Wang, J., Xu, F. et al. LncRNA RAD51-AS1 Regulates Human Bone Marrow Mesenchymal Stem Cells via Interaction with YBX1 to Ameliorate Osteoporosis. Stem Cell Rev and Rep 19, 170–187 (2023). https://doi.org/10.1007/s12015-022-10408-x

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