Huang et al. Experimental & Molecular Medicine (2021) 53:973–985 https://doi.org/10.1038/s12276-021-00631-w Experimental & Molecular Medicine ARTICLE Open Access miR-19b enhances osteogenic differentiation of mesenchymal stem cells and promotes fracture healing through the WWP1/Smurf2-mediated KLF5/β-catenin signaling pathway Yan Huang1,2, Yongqiang Xu 1, Siyin Feng1,PanHe1, Bing Sheng1 and Jiangdong Ni2 Abstract Bone marrow mesenchymal stem cell (BMSC)-derived exosomes have been found to enhance fracture healing. In addition, microRNAs contributing to the healing of various bone fractures have attracted widespread attention in recent years, but knowledge of the mechanisms by which they act is still very limited. In this study, we clarified the function of altered microRNA-19b (miR-19b) expression in BMSCs in fracture healing. We modulated miR-19b expression via mimics/inhibitors in BMSCs and via agomirs in mice to explore the effects of these changes on osteogenic factors, bone cell mineralization and the healing status of modeled fractures. Through gain- and loss-of function assays, the binding affinity between miR-19b and WWP1/Smurf2 was identified and characterized to explain the underlying mechanism involving the KLF5/β-catenin signaling pathway. miR-19b promoted the differentiation of human BMSCs into osteoblasts by targeting WWP1 and Smurf2. Overexpression of WWP1 or Smurf2 degraded the target protein KLF5 in BMSCs through ubiquitination to inhibit fracture healing. KLF5 knockdown delayed fracture β 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; healing by modulating the Wnt/ -catenin signaling pathway. Furthermore, miR-19b enhanced fracture healing via the KLF5/β-catenin signaling pathway by targeting WWP1 or Smurf2. Moreover, miR-19b was found to be enriched in BMSC-derived exosomes, and treatment with exosomes promoted fracture healing in vivo. Collectively, these results indicate that mesenchymal stem cell-derived exosomal miR-19b represses the expression of WWP1 or Smurf2 and elevates KLF5 expression through the Wnt/β-catenin signaling pathway, thereby facilitating fracture healing. Introduction and biomineralization3. Fracture sites are commonly Bone fractures are the most frequently occurring type of subjected to increasing oxidative stress after injuries, large-organ, traumatic damage in humans1. The delayed which impairs osteoblast function and impedes the pro- healing process of bone fractures is a serious clinical and cess of fracture healing and remodeling4. The repair of economic problem for both patients and health services2. fracture wounds is critically influenced by mechanical The healing process of bone fractures is widely accepted loading as well as the geometric configuration of the as a complicated physiological course of events driven by fractured fragments5. The recent available evidence on the early inflammatory reactions and is accompanied by var- molecular mechanism indicates that bone fracture healing ious biological activities, such as osteogenic differentiation involves the complex coordination of various cell types, proteins and genes to restore structural integrity6. A limited amount of data have documented the paracrine Correspondence: Yongqiang Xu ([email protected]) mechanisms associated with the orchestration of 1 ’ Department of Orthopaedics, Hunan Provincial People s Hospital, Changsha, mesenchymal stem cell (MSC) transplantation in bone China 2Department of Orthopaedics, The Second Xiangya Hospital of Central South fractures, and exosomes are pivotal elements of the University, Changsha, China © The Author(s) 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Korean Society for Biochemistry and Molecular Biology Huang et al. Experimental & Molecular Medicine (2021) 53:973–985 974 paracrine effects7. MSC-derived exosome-mediated delivery exosomes. BMSCs were cultured in exosome-free med- of microRNAs (miRNAs), such as miR-126, has shown ium containing 10% FBS, and BMSCs at passages 4-6 were potential in accelerating fracture healing8.Ofinterest, selected for exosome collection. A total of 2 × 106 MSCs upregulation of miR-19b could cause a significant increase were cultured in 100 mm culture dishes under normal/ in the transcription and translation of osteogenic factor hypoxic conditions for 72 h, and 10 ml of the supernatant genes during the process of osteogenic differentiation9.We was taken. Exosomes were then isolated from the super- used a bioinformatic prediction program to predict the natant after centrifugation16. Then, exosomes were potential target genes of miR-19b, and WWP1 and Smurf2 resuspended in PBS. were predicted as the candidate target genes of miR-19b. For identification of exosomes, samples were evaluated Notably, the PY motif-containing transcription factor under a JEM-2100 transmission electron microscope Kruppel like factor 5 (KLF5) is frequently degraded through (TEM; JEOL, Tokyo, Japan). Images were acquired using ubiquitination by E3 ubiquitin ligases such as WWP110.In the PARTICLEMEIRIX system. Nanoparticle tracking addition, prior evidence has suggested that SMAD ubiqui- analysis (NTA) was performed using the NanoSight tination regulatory factor 2 (Smurf2), an E3 ubiquitin ligase, NS300 system (Malvern Instruments, Malvern, UK). The is an interacting protein of KLF5 and diminishes the protein Brownian motion of exosomes in PBS was recorded and stability of KLF511. Furthermore, KLF5 has been revealed to tracked, and the size distribution was analyzed using the activate β-catenin signaling in breast cancer12.Inturn, Stokes-Einstein equation. The exosome characteristics Wnt/β-catenin signaling pathway activation has been pro- were identified by detecting the expression of the posed to enhance the production of osteogenic factors and exosome-specific surface markers with rabbit anti-CD63 thus expedite tibial fracture healing13.Inthisstudy,we (1:2000, ab216130, Abcam, UK), rabbit anti-TSG101 sought to explain the regulatory mechanism of exosomal (1:10,000, ab125011, Abcam), rabbit anti-CD81 (1:10,000, miR-19b derived from bone marrow mesenchymal stem ab109201, Abcam) and rabbit anti-Calnexin (1:100,000, cells (BMSCs) in the process of fracture healing, which may ab92573, Abcam) antibodies by Western blot analysis. involve the WWP1/Smurf2/KLF5/β-catenin axis. Experimental protocols Materials and methods When the cell density was 90% and the cells were in in Sample collection logarithmic growth phase, trypsin digestion and pipetting The bone marrow of healthy volunteers at The Second were performed to prepare a 2.5 × 104 cell/ml cell sus- Xiangya Hospital of Central South University was col- pension, which was added to a 6-well plate at 2 ml per lected, and informed consent was obtained from the par- well. When the cells were in logarithmic growth phase ticipants. This study was approved by the Ethics and were 30% confluent, the corresponding lentiviruses Committee of The Second Xiangya Hospital of Central (2 × 106 TU) and 5 μg of polybrene were added to 1 ml of South University. After surgery, portions of the fresh tissue serum/antibacterial drug-free medium. Forty-eight hours samples were stored in liquid nitrogen within 30 min and after transduction, 1 μg/ml puromycin was added to each were then transferred to a −80 °C freezer for further use. well to select stably transduced cells. After successful lentiviral infection, cells at a confluence of 70% to 80% Isolation and culture of human BMSCs were subjected to transient transduction of the miR-19b Human BMSCs were isolated from healthy volunteers mimic/inhibitor, WWP1 overexpression plasmid (oe- and amplified according to previously reported meth- WWP1), Smurf2 overexpression plasmid (oe-Smurf2), ods14. BMSCs below passage 4 were used in the following KLF5 overexpression plasmid (oe-KLF5), β-catenin over- experiments. Maintenance medium comprising dulbec- expression plasmid (oe-β-catenin), shRNA targeting KLF5 co’s modified Eagle’s medium (DMEM) (Invitrogen), 10% (sh-KLF5) or the corresponding negative controls (NCs) FBS and 100 U/ml penicillin/streptomycin (Invitrogen) according to the instructions for LipofectamineTM 2000 was employed to incubate BMSCs at 37 °C in 5% CO2. (Invitrogen). Cells were collected 48 h after transduction, The osteogenic differentiation induction medium was and the transduction efficiency was determined for sub- composed of 10% FBS, 50 mg/ml L-ascorbic acid, 10 mM sequent experiments. glycerophosphate, and 100 nM dexamethasone and anti- biotics (complete alpha-MEM) (Sigma, St. Louis, MO). Western blot analysis Detection was performed on the 7th day after osteogenic The transduced cells and fracture healing tissues har- differentiation was induced15. vested at 2 and