Exosomal miR-186 derived from BMSCs promote osteogenesis through hippo signaling pathway in postmenopausal osteoporosis

Abstract

Background: Postmenopausal osteoporosis (PMO) that results from estrogen withdrawal is the most common primary osteoporosis among older women. However, little is known about the mechanism of PMO, and effective treatment of PMO is limited.

Methods: We used real-time polymerase chain reaction (qPCR), Western blotting, and RNA pull down to investigate the relationship between miR-186 and MOB Kinase Activator 1A (Mob1). Also, we investigated the effect of exosome in osteogenesis using alkaline phosphatase (ALP) staining. And hematoxylin eosin (HE) staining was used to verify the osteogenesis in PMO model.

Results: Exosomal miR-186 plays an important role in bone formation. The results of miRNA-seq and q-PCR showed that miR-186 was upregulated in a PMO + Exo treatment group. Results of RNA-pull down and luciferase reporter assays verified interactions between miR-186 and Mob1. We also verified the Hippo signaling pathway plays an important role in osteogenesis.

Conclusions: We concluded that exosomes derived from human bone marrow mesenchymal stem cells (hBMSCs) can transfer miR-186 to promote osteogenesis in ovariectomy (OVX) rats through the Hippo signaling pathway.

Keywords: Exosomes; Osteogenesis; Postmenopausal osteoporosis; miRNA.

Fig. 1

a The morphology of exosomes derived from hBMSCs were detected using transmission electron microscopy. Phosphotungstic acid staining was used before exosome identification. b The size distribution of exosomes was identified using DLS. c The surface biomarkers of exosomes, including Alixs, CD81, and CD63 were detected using Western blot. d The exosomes were labeled with PKH26, and the labeled exosomes were observed using confocal microscopy

Fig. 2

a CCK-8 assay was conducted to detect the proliferation rates of BMSCs extracted from OVX rats. b The CCK-8 results were calculated using the SPSS software. c Bone metabolism markers, including BMP2 and RANKL, were detected using Western blot. d The expressions of BMP2 were calculated using ImageJ. e The alizarin red staining was performed to detect the osteogenesis effect of exosomes

Fig. 3

a The differentially expressed miRNAs were detected between BMSCs extracted in OVX rats (PMO) and BMSCs extracted in OVX rats + exosomes groups (PMO + Exos) using miRNA-seq. b q-PCR was performed to investigate the expression of miRNA between PMO and PMO + exosome group. c RNA in tibia of rats were extracted and the expression of miR-186 in tibia of rats were verified using q-PCR. d microT, miRanda, Target Scan, and PITA were used to predict the downstream targets of miR-186. e The binding sites between Mob1 and miR-186 were shown, and mutant or wild-type nucleotides were labeled in red. f The relationship between Mob1 and miR-186 was verified using the luciferase reporter assay. g RNA-pull down assays were conducted to further verify the interaction between Mob1 and miR-186. h q-PCR was performed to detect the expression of Mob1. i The expression of Mob1 was detected between the PMO and PMO + Exos groups using q-PCR

Fig. 4

a The expression of YAP was detected in the control group, PMO group, PMO + Exo group, PMO + Exo + miR-186 inhibitor group, and PMO + miR-186 mimics group. b YAP expression was calculated using ImageJ. c ALP staining and oil red staining was performed to detect osteogenesis and adipogenesis between different groups. d Quantitative calculation of ALP-positive areas. e HE staining of tibia in rats receiving different treatments was performed to evaluate the osteogenesis of exosomal miR-186. f The number of osteocytes were calculated. g The number of osteoblasts was calculated. h The number of osteoclasts was calculated

Fig. 5

a The microCT was used to evaluate the bone mass between different groups. b The bone parameters were calculated between different groups