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MicroRNA-378 Suppressed Osteogenesis of MSCs and Impaired Bone Formation via Inactivating Wnt/β-Catenin Signaling

Feng, Lu, Zhang, Jin-fang, Shi, Liu, Yang, Zheng-meng, Wu, Tian-yi, Wang, Hai-xing, Lin, Wei-ping, Lu, Ying-fei, Lo, Jessica Hiu Tung, Zhu, Da-hai, Li, Gang·2020·Molecular Therapy Nucleic Acids
DOI 10.1016/j.omtn.2020.07.018PMC PMC7452050

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Figure 1 Abnormal Bone Tissues and Impaired Bone Quality Were Observed in miR-378 TG Mice (A and B) Bone phenotype of femurs (A) and tibia (B) of miR-378 TG mice and their wild-type (WT) mice were examined by digital radiography. (C–F) Representative microarchitecture 3D images of cortical (C and E) and trabecular (D and F) bone of femur (C and D), as well as tibia (E and F). BV/TV of the cortical and trabecular bone all showed that the bone mass was significantly decreased in the miR-378 TG mice (age: 3 months; n = 10; ∗p < 0.05 versus WT group). (G) H&E staining of transverse and coronal plane of femurs revealed thicker cortices, smoother edge of cortical bone, and more paralleled alignment of osteocytes in the WT mice than those in the miR-378 TG group. Scale bars, 400 μm. (H) H&E staining of the sagittal plane of the metaphyseal region of femurs revealed more disrupted bone microarchitecture and homeostasis in cancellous bone of the miR-378 TG mice femur. Scale bars, 100 μm.
Figure 1 Abnormal Bone Tissues and Impaired Bone Quality Were Observed in miR-378 TG Mice (A and B) Bone phenotype of femurs (A) and tibia (B) of miR-378 TG mice and their wild-type (WT) mice were examined by digital radiography. (C–F) Representative microarchitecture 3D images of cortical (C and E) and trabecular (D and F) bone of femur (C and D), as well as tibia (E and F). BV/TV of the cortical and trabecular bone all showed that the bone mass was significantly decreased in the miR-378 TG mice (age: 3 months; n = 10; ∗p < 0.05 versus WT group). (G) H&E staining of transverse and coronal plane of femurs revealed thicker cortices, smoother edge of cortical bone, and more paralleled alignment of osteocytes in the WT mice than those in the miR-378 TG group. Scale bars, 400 μm. (H) H&E staining of the sagittal plane of the metaphyseal region of femurs revealed more disrupted bone microarchitecture and homeostasis in cancellous bone of the miR-378 TG mice femur. Scale bars, 100 μm.CC BY · PubMed Central
Figure 2 Bone Fracture Healing Was Impaired in the miR-378 TG Group (A) Representative images of X-ray radiography showed that less callus formation was found in the miR-378 TG group at week 4, and the gap in the fracture sites almost disappeared in the WT group. (B) Three-dimensional micro-CT images of the mouse femur fracture zone were taken 4 weeks after surgery, and the result confirmed that less continuous callus was found in the miR-378 TG group. (C) Micro-CT analysis was performed, and highly mineralized and newly formed calluses were reconstructed based on a different threshold. The results showed that BV/TV of total mineralized bone (threshold 158–1,000), highly mineralized bone (threshold 211–1,000), as well as new callus (threshold 158–211) in the WT group was much higher than that in the miR-378 TG groups. (D) Three-point bending mechanical testing in the miR-378 TG group showed a significant decrease of ultimate load, E-modulus, and energy to failure compared to the WT group. (E, a and b) H&E staining revealed that the miR-378 TG group has fewer calluses covered on the femur fracture site at week 4 postsurgery, and the bone remodeling was less vigorous than the WT group. Scale bar, 800 μm. (E, c–g) Immunohistochemistry staining and semiquantitative analysis revealed less OCN and OPN expression in the miR-378 TG group. Scale bars, 100 μm. (E, g) Age: 3 months; n = 10; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus WT group.
Figure 2 Bone Fracture Healing Was Impaired in the miR-378 TG Group (A) Representative images of X-ray radiography showed that less callus formation was found in the miR-378 TG group at week 4, and the gap in the fracture sites almost disappeared in the WT group. (B) Three-dimensional micro-CT images of the mouse femur fracture zone were taken 4 weeks after surgery, and the result confirmed that less continuous callus was found in the miR-378 TG group. (C) Micro-CT analysis was performed, and highly mineralized and newly formed calluses were reconstructed based on a different threshold. The results showed that BV/TV of total mineralized bone (threshold 158–1,000), highly mineralized bone (threshold 211–1,000), as well as new callus (threshold 158–211) in the WT group was much higher than that in the miR-378 TG groups. (D) Three-point bending mechanical testing in the miR-378 TG group showed a significant decrease of ultimate load, E-modulus, and energy to failure compared to the WT group. (E, a and b) H&E staining revealed that the miR-378 TG group has fewer calluses covered on the femur fracture site at week 4 postsurgery, and the bone remodeling was less vigorous than the WT group. Scale bar, 800 μm. (E, c–g) Immunohistochemistry staining and semiquantitative analysis revealed less OCN and OPN expression in the miR-378 TG group. Scale bars, 100 μm. (E, g) Age: 3 months; n = 10; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus WT group.CC BY · PubMed Central
Figure 3 miR-378 Suppressed Osteogenesis of MSCs (A–C) Under osteogenic induction, ALP activity (A), calcified nodules (B), and osteogenic marker gene expression (C) were all suppressed in MSCs derived from miR-378 TG mice (n = 3; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus WT group). (D–F) ALP activity (D), calcified nodule formation (E), and osteogenic marker genes expression (F) were all decreased by miR-378-3p or miR-378-5p mimics (n = 3; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus miR-NC group). (G–I) ALP activity (G), calcified nodule formation (H), and osteogenesis marker gene expression (I) were promoted by anti-miR-378-3p and anti-miR-378-5p (n = 3; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus anti-miR-NC group).
Figure 3 miR-378 Suppressed Osteogenesis of MSCs (A–C) Under osteogenic induction, ALP activity (A), calcified nodules (B), and osteogenic marker gene expression (C) were all suppressed in MSCs derived from miR-378 TG mice (n = 3; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus WT group). (D–F) ALP activity (D), calcified nodule formation (E), and osteogenic marker genes expression (F) were all decreased by miR-378-3p or miR-378-5p mimics (n = 3; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus miR-NC group). (G–I) ALP activity (G), calcified nodule formation (H), and osteogenesis marker gene expression (I) were promoted by anti-miR-378-3p and anti-miR-378-5p (n = 3; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus anti-miR-NC group).CC BY · PubMed Central
Figure 4 Wnt10a and Wnt6 Were Bona Fide Targets for miR-378-3p and miR-378-5p, Respectively (A and B) Schematic diagrams of the interaction between miR-378-3p (A) and miR-378-5p (B) transcripts and their direct target genes Wnt10a (A) and Wnt6 (B). (C and D) miR-378-3p (C) or miR-378-5p (D) was cotransfected with the Wnt10a (C) or Wnt6 (D) luciferase reporter into HEK293 cells, and the luciferase activity was measured (n = 3; ∗p < 0.05 versus miR-NC group). (E and F) Expression of Wnt10a was suppressed by miR-378-3p mimics (E), whereas promoted by its antagonist (F) at mRNA and protein levels (n = 3; ∗p < 0.05 versus miR-NC or anti-miR-NC group, respectively). (G and H) Expression of Wnt6 was reduced by miR-378-5p mimics (G), whereas enhanced by its antagonist (H) at mRNA and protein levels (n = 3; ∗p < 0.05 versus miR-NC or anti-miR-NC group, respectively).
Figure 4 Wnt10a and Wnt6 Were Bona Fide Targets for miR-378-3p and miR-378-5p, Respectively (A and B) Schematic diagrams of the interaction between miR-378-3p (A) and miR-378-5p (B) transcripts and their direct target genes Wnt10a (A) and Wnt6 (B). (C and D) miR-378-3p (C) or miR-378-5p (D) was cotransfected with the Wnt10a (C) or Wnt6 (D) luciferase reporter into HEK293 cells, and the luciferase activity was measured (n = 3; ∗p < 0.05 versus miR-NC group). (E and F) Expression of Wnt10a was suppressed by miR-378-3p mimics (E), whereas promoted by its antagonist (F) at mRNA and protein levels (n = 3; ∗p < 0.05 versus miR-NC or anti-miR-NC group, respectively). (G and H) Expression of Wnt6 was reduced by miR-378-5p mimics (G), whereas enhanced by its antagonist (H) at mRNA and protein levels (n = 3; ∗p < 0.05 versus miR-NC or anti-miR-NC group, respectively).CC BY · PubMed Central
Figure 5 Wnt/β-Catenin Signaling Was Suppressed in miR-378-Mediated Osteogenesis (A and B) miR-378 mimics suppressed (A), whereas their antagonist promoted (B) the TOPflash luciferase activity (n = 3; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus miR-NC or anti-miR-NC group, respectively). (C–F) Expression of β-catenin was evaluated with miR-378 mimics or the antagonists’ treatment at mRNA (C and D) and protein (E and F) levels (n = 3; ∗p < 0.05, ∗∗p < 0.01 versus miR-NC or anti-miR-NC group, respectively). (G and H) miR-378 mimics (G) suppressed, whereas their antagonists (H) activated several downstream target genes of β-catenin (n = 3; ∗p < 0.05, ∗∗p < 0.01 versus miR-NC or anti-miR-NC group, respectively).
Figure 5 Wnt/β-Catenin Signaling Was Suppressed in miR-378-Mediated Osteogenesis (A and B) miR-378 mimics suppressed (A), whereas their antagonist promoted (B) the TOPflash luciferase activity (n = 3; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus miR-NC or anti-miR-NC group, respectively). (C–F) Expression of β-catenin was evaluated with miR-378 mimics or the antagonists’ treatment at mRNA (C and D) and protein (E and F) levels (n = 3; ∗p < 0.05, ∗∗p < 0.01 versus miR-NC or anti-miR-NC group, respectively). (G and H) miR-378 mimics (G) suppressed, whereas their antagonists (H) activated several downstream target genes of β-catenin (n = 3; ∗p < 0.05, ∗∗p < 0.01 versus miR-NC or anti-miR-NC group, respectively).CC BY · PubMed Central
Figure 6 Wnt/β-Catenin Signaling Was Suppressed in miR-378 TG Mice (A) Expression of Wnt10a, Wnt6, and β-catenin was decreased in the MSCs derived from miR-378 TG mice at the mRNA level (n = 3; ∗p < 0.05 versus WT group). (B and C) Western blot (B) and semiquantitative analysis (C) revealed that Wnt10a, Wnt6, and β-catenin protein expression was suppressed in the MSCs isolated from miR-378 TG mice. (D) Several downstream target genes of β-catenin were downregulated in the MSCs derived from miR-378 TG mice (n = 3; ∗p < 0.05 versus WT group).
Figure 6 Wnt/β-Catenin Signaling Was Suppressed in miR-378 TG Mice (A) Expression of Wnt10a, Wnt6, and β-catenin was decreased in the MSCs derived from miR-378 TG mice at the mRNA level (n = 3; ∗p < 0.05 versus WT group). (B and C) Western blot (B) and semiquantitative analysis (C) revealed that Wnt10a, Wnt6, and β-catenin protein expression was suppressed in the MSCs isolated from miR-378 TG mice. (D) Several downstream target genes of β-catenin were downregulated in the MSCs derived from miR-378 TG mice (n = 3; ∗p < 0.05 versus WT group).CC BY · PubMed Central
Figure 7 sh-miR-378-Mediated MSCs Accelerated Bone-Fracture Healing in Mice (A) X-ray radiography was taken during the course of fracture healing. Representative images showed that the fractured site was covered by more calluses, and bone remodeling was more vigorous in the sh-miR-378 group. (B) Three-dimensional micro-CT images showed a better fracture-healing effect condition and more calluses in the sh-miR-378 group. (C) Statistical diagram of BV/TV calculated from micro-CT results was displayed (age: 3 months; n = 10; ∗p < 0.05 versus sh-NC group). (D) Three-point bending mechanical testing at week 4 postsurgery was performed, and the statistical data were displayed (n = 10; ∗p < 0.05 versus sh-NC group). (E, a and b) H&E staining revealed more callus covered in the femur fracture site of the sh-miR-378 group at week 4. Scale bar, 800 μm. (E, c–g) Immunohistochemistry staining and semiquantitative analysis revealed more OCN and OPN expressed in the new bone zone in the sh-miR-378 treatment group by immunohistochemistry staining. Scale bars, 100 μm.
Figure 7 sh-miR-378-Mediated MSCs Accelerated Bone-Fracture Healing in Mice (A) X-ray radiography was taken during the course of fracture healing. Representative images showed that the fractured site was covered by more calluses, and bone remodeling was more vigorous in the sh-miR-378 group. (B) Three-dimensional micro-CT images showed a better fracture-healing effect condition and more calluses in the sh-miR-378 group. (C) Statistical diagram of BV/TV calculated from micro-CT results was displayed (age: 3 months; n = 10; ∗p < 0.05 versus sh-NC group). (D) Three-point bending mechanical testing at week 4 postsurgery was performed, and the statistical data were displayed (n = 10; ∗p < 0.05 versus sh-NC group). (E, a and b) H&E staining revealed more callus covered in the femur fracture site of the sh-miR-378 group at week 4. Scale bar, 800 μm. (E, c–g) Immunohistochemistry staining and semiquantitative analysis revealed more OCN and OPN expressed in the new bone zone in the sh-miR-378 treatment group by immunohistochemistry staining. Scale bars, 100 μm.CC BY · PubMed Central

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Metadata

Authors
Feng, L., Zhang, J.-F., Shi, L., Yang, Z.-M., Wu, T.-Y., Wang, H.-X., Lin, W.-P., Lu, Y.-F., Lo, J. H. T., Zhu, D.-H., & Li, G.
Venue
Molecular Therapy Nucleic Acids
Publisher
Elsevier BV
Published
2020-09
Volume
21
Pages
1017-1028
ISSN
2162-2531
DOI
10.1016/j.omtn.2020.07.018
PMC
PMC7452050
Cited by
46
References
2
Language
en
Licence
Funders
  • National Natural Science Foundation of China (81772322)
  • Chinese University of Hong Kong
  • Innovation and Technology Commission - Hong Kong (ITS/448/18)
  • General Research Fund of Shanghai Normal University (14120118, 14160917, 9054014 N_CityU102/15, C7030-18G, T13-402/17-N)

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