MicroRNA-15b shuttled by bone marrow mesenchymal stem cell-derived extracellular vesicles binds to WWP1 and promotes osteogenic differentiation
Authors: Shuanke Wang
Kai Feng
Guangjie Li
Wenjia Du
Haijun Feng
Jing Wang
Yanhong Li
Yanchao Ma
Journal: Arthritis research & therapy (2020)
Discipline: CLINICAL MEDICINE
Keywords: Bone marrow mesenchymal stem cell; Extracellular vesicles; KLF2; NF-κB signaling pathway; Osteogenic differentiation
Affiliations: Department of Orthopaedics, Lanzhou University Second Hospital, No. 82, Cuiyingmen, Chengguan District, Lanzhou, 730030, Gansu Province, People's Republic of China., Department of Orthopaedics, Lanzhou University Second Hospital, No. 82, Cuiyingmen, Chengguan District, Lanzhou, 730030, Gansu Province, People's Republic of China. .

Abstract:  Background: Osteogenic differentiation is an essential process for bone regeneration involving bone marrow mesenchymal stem cells (BMSCs). BMSC-secreted extracellular vesicles (EVs) enriched with microRNAs (miRs) have vital roles to play in mediating osteogenic differentiation. Therefore, this study aimed to explore the effect of BMSC-derived EVs loaded with miR-15b on osteogenic differentiation. Methods: Human BMSCs (hBMSCs) were cultured and treated with plasmids overexpressing or knocking down KLF2, WWP1, and miR-15b to define the role of derived EVs in osteogenic differentiation in vitro. The expression of osteogenic differentiation-related marker was measured by Western blot analysis. The interaction among miR-15b, WWP1, and ubiquitination of KLF2 was investigated by dual-luciferase reporter, immunoprecipitation, and GST pull-down assays. Moreover, EVs from hBMSCs transfected with miR-15b inhibitor (EV-miR-15b inhibitor) were injected into ovariectomized rats to verify the effect of miR-15b on bone loss in vivo. Results: WWP1 was downregulated, and KLF2 was upregulated during osteogenic differentiation. After co-culture with EVs, miR-15b expression was elevated and WWP1 expression was reduced in hBMSCs. Upregulation of miR-15b or KLF2 or downregulation of WWP1 or NF-κB increased ALP activity and cell mineralization, as well as osteogenic differentiation-related marker expression in hBMSCs. Mechanistically, miR-15b targeted and inhibited WWP1, thus attenuating KLF2 degradation and inhibiting NF-κB activity. Co-culture of EVs increased the bone volume and trabecular number, but decreased bone loss in ovariectomized rats, which could be reversed after treatment with EV-miR-15b inhibitor. Conclusion: Collectively, BMSC-derived EVs loaded with miR-15b promoted osteogenic differentiation by impairing WWP1-mediated KLF2 ubiquitination and inactivating the NF-κB signaling pathway. 

Retracted: No

PubPeer comments:
 Two images in Figure 7B have edges which are quite similar. Could the authors explain how this might happen? *** ![file] , 
 #1 Here I have black white reversed one of the panels... ![file] ... and superimposed them, so that similar areas cancel out. ![file] The area of cancellation (where the two reconstructed meshes are the same) is surprisingly large. , 
 There also appears to be some image overlap between Figures 4 and 5. Possible change in dimension and rotation and scale bars may be off. *** ![file] , 
 - *[above]* Fig 6A. - *[below]* Fig 5A from  Proper mechanical stress promotes femoral head recovery from steroid-induced osteonecrosis in rats through the OPG RANK RANKL system ([Fu et al 2020] ). ![file] , 
 - *[left]* Fig 4F. - *[right]* Fig 2C from  microRNA-130b downregulation potentiates chondrogenic differentiation of bone marrow mesenchymal stem cells by targeting SOX9 ([Zhang et al 2021] ). ![file] , 
 - *[above]* Fig 7F from  LncRNA nuclearu2010enriched abundant transcript 1 shuttled by prostate cancer cellsu2010secreted exosomes initiates osteoblastic phenotypes in the bone metastatic microenvironment via miRu2010205u20105p runtu2010related transcription factor 2 splicing factor prolineu2010 and glutamineu2010rich polypyrimidine tractu2010binding protein 2 axis ([Mo et al 2021] ). - *[below]* **Fig 7B** again. ![file] Images are constructed from CT scans of the tibia of mice and rats respectively, but the overlap suggests that a CT scan was shared. ![file] , 
 Fig 3. All the WB-like images in this paper display a stylised  row-of-bowties appearance, often found in the output of a prolific papermill. ![file] , 
 #6 Wait, another sighting! - *[left]* A panel from Fig 7F from Mo et al (2021). - *[right]* A panel from **Fig 7B**. - *[below]* A panel from Fig 2B of  Upu2010regulated CST5 inhibits bone resorption and activation of osteoclasts in rat models of osteoporosis via suppression of the NFu2010u03baB pathway ([Wang et al 2019] ). ![file] The context. - *[above]* Fig 7B. - *[below]* Fig 2B from Wang et al (2019). ![file] And another pair of panels. ![file] , 
 The journal contacted me in September to inform me that a correction was imminent. I have notified the journal of the additional concerns. ,