Mir155 regulates osteogenesis and bone mass phenotype via targeting S1pr1 gene

  1. Zhichao Zheng
  2. Lihong Wu
  3. Zhicong Li
  4. Ruoshu Tang
  5. Hongtao Li
  6. Yinyin Huang
  7. Tianqi Wang
  8. Shaofen Xu
  9. Haoyu Cheng
  10. Zhitong Ye
  11. Dong Xiao
  12. Xiaolin Lin
  13. Gang Wu
  14. Richard T Jaspers
  15. Janak L Pathak

  • Curated by eLife

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    Evaluation Summary:

    Caner/cancer metastasis-induced bone loss-mediated fracture is a serious clinical problem. In this regard the authors have done an interesting study to show how miR155 exhibits a catabolic effect on osteogenesis and bone mass phenotype via interaction with the Sphingosine 1-phosphate receptor-1 (S1PR1) gene. The study suggests that inhibition of miR155 could be a potential strategy for bone regeneration and bone defect healing.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

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Abstract

MicroRNA-155 (miR155) is overexpressed in various inflammatory diseases and cancer, in which bone resorption and osteolysis are frequently observed. However, the role of miR155 on osteogenesis and bone mass phenotype is still unknown. Here, we report a low bone mass phenotype in the long bone of Mir155 -Tg mice compared with wild-type mice. In contrast, Mir155 -KO mice showed a high bone mass phenotype and protective effect against inflammation-induced bone loss. Mir155 -KO mice showed robust bone regeneration in the ectopic and orthotopic model, but Mir155 -Tg mice showed compromised bone regeneration compared with the wild-type mice. Similarly, the osteogenic differentiation potential of bone marrow stromal stem cells (BMSCs) from Mir155 -KO mice was robust and Mir155 -Tg was compromised compared with that of wild-type mice. Moreover, Mir155 knockdown in BMSCs from wild-type mice showed higher osteogenic differentiation potential, supporting the results from Mir155 -KO mice. TargetScan analysis predicted sphingosine 1-phosphate receptor-1 ( S1pr1 ) as a target gene of Mir155 , which was further confirmed by luciferase assay and Mir155 knockdown. S1pr1 overexpression in BMSCs robustly promoted osteogenic differentiation without affecting cell viability and proliferation. Furthermore, osteoclastogenic differentiation of Mir155 -Tg bone marrow-derived macrophages was inhibited compared with that of wild-type mice. Thus, Mir155 showed a catabolic effect on osteogenesis and bone mass phenotype via interaction with the S1pr1 gene, suggesting inhibition of Mir155 as a potential strategy for bone regeneration and bone defect healing.

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  1. Version published to 10.7554/elife.77742 on eLife
  2. eLife

    Evaluation Summary:

    Caner/cancer metastasis-induced bone loss-mediated fracture is a serious clinical problem. In this regard the authors have done an interesting study to show how miR155 exhibits a catabolic effect on osteogenesis and bone mass phenotype via interaction with the Sphingosine 1-phosphate receptor-1 (S1PR1) gene. The study suggests that inhibition of miR155 could be a potential strategy for bone regeneration and bone defect healing.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    The authors have investigated MicroRNA-155 (miR155) that is overexpressed in various inflammatory diseases and cancer. Both these conditions present with bone resorption and osteolysis. Cancer/cancer metastasis-induced bone loss-mediated fracture is a serious clinical problem. In the studies performed by the authors miR155 showed a catabolic effect on osteogenesis and bone mass phenotype via interaction with the Sphingosine 1-phosphate receptor-1 (S1PR1) gene, suggesting inhibition of miR155 as a potential strategy for bone regeneration and bone defect healing.
    The study highlights the importance that miR155 inhibitors could be potential therapeutics to promote bone regeneration even in inflammatory conditions.
    The study delivers a partial mechanistic study. The study is simple and understandable, and the results are quite new.

  4. eLife

    Reviewer #2 (Public Review):

    In this manuscript, Zheng et al. investigated the regulation of S1PR1 gene by MicroRNA-155 and determined the inhibitory role of this miRNA in osteogenic differentiation of bone marrow mesenchymal stem cells. They observed two opposite phenotypes, low bone mass in mice overexpressing miR-155 and high bone mass in miR-155 null mice. They showed miR-155 sponge (inhibitor of miR-155) increases S1PR1 protein levels. Furthermore, they found that miR-155 inhibits osteoblast differentiation markers alkaline phosphatase and Runx-2 in invitro and invivo experiments. Although several components of the miR-155 and S1PR1 cascade have been previously reported in other cellular systems, the connection with osteogenesis is novel and very interesting. However, several concerns should be addressed:

    1. Are the similar effects observed in female mice? Authors did experiments in 8 week old male mice. Does this high bone mass phenotype in miR-155 KO or miR-155 Tg mice change over the period of time as mice age? It would be interesting in the bone field if these mice are resistant/prone to age related bone loss or inflamaging.

    2. To check miRNA expression levels from serum, plasm, tissue or cells TaqMan-based pre-miRNA assay is a better detection method for its specificity and accuracy than SYBR green detection method. For small RNAs like miRNAs, U6 and 5S are the two commonly used normalizers for miRNA qRT-PCR. Please comment and revise.

    3. What are the expression levels of miR-155 in bone tissue from miR-155 Tg and miR-155 KO mice? It is important to show miR-155 levels are reduced or knocked down in miR-155 KO bones and increased in miR-155 Tg bones. Authors should mention and explain these effects in the results and discussion.

    4. What are the effects at cellular levels in miR-155 TG and miR-155 KO mice? Static histomorphometry data using Goldner's Trichome and TRAP staining will be important to study osteoblast and osteoclast numbers. Authors should mention and explain these effects in the results and discussion.

    5. What are the effects on Dynamic histomorphometry in miR-155 TG and miR-155 KO mice? Calcein/Alizarin Red injections followed by histomorphentry will be important to study Bone formation and mineral apposition rates. Authors should mention and explain these effects in the results and discussion.

    6. What are the effects on serum levels of bone formation and resorption markers in miR-155 TG and miR-155 KO mice? PINP or Osteoclacin and CTX-1 eliza histomorphentry will be important to study how these mice lose or gain bone. Authors should mention and explain these effects in the results and discussion.

    7. Fig.5F : Authors observed no change or possibly reduction in Runx2 levels in miR-155 KO BMSCs. However, they observed increase in Runx2 levels in BMSCs treated with miR-155 sponge. The authors should mention and explain why they did not observe increased Runx2 levels in miR-155 KO BMSCs, in the result and discussion.

    8. Fig 7A, Authors showed binding site of miR-155-5p in the 3'UTR of S1PR1 gene but did not mention whether it is conserved among different species like mouse, human or rat etc. Authors should comment on these points in the result and discussion.

  5. eLife

    Reviewer #3 (Public Review):

    This manuscript will be of broad interest to orthopedists, tissue engineers, and particularly to those who are involved in developing osteoinductive scaffolds. Anti-miR155- could be used as an anabolic tool with the potential of improving bone density phenotypes, as well as being used directly as an additive to tissue engineering components. Having characterized the effects of silencing mir-155 on various pathways and stem cells, its use in a clinical setting may be possible, as said in the paper. The author might add some additional experiments to make it more robust when it comes to validating the finding and taking things forward for further clinical application.

    As miR155 is a potent regulator of thousands of mRNA targets, the author could include some experiments looking at effects on a couple of different pathways.

  6. Version published to 10.1101/2022.02.18.480982v1 on bioRxiv