Exosomal miR-365a-5p derived from HUC-MSCs regulates osteogenesis in GIONFH through the Hippo signaling pathway

Abstract

The pathogenesis of glucocorticoid (GC)-induced osteonecrosis of the femoral head (GIONFH) is still disputed, and abnormal bone metabolism caused by GCs may be an important factor. In vitro, Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) staining were used to evaluate cellular proliferation, and western blotting was used to investigate osteogenesis. In vivo, we used micro-computed tomography (micro-CT), H&E staining, Masson staining, and immunohistochemistry (IHC) analysis to evaluate the impact of exosomes. In addition, the mechanism by which exosomes regulate osteogenesis through the miR-365a-5p/Hippo signaling pathway was investigated using RNA sequencing (RNA-seq), luciferase reporter assays, fluorescence in situ hybridization (FISH), and western blotting. The results of western blotting verified that the relevant genes in osteogenesis, including BMP2, Sp7, and Runx2, were upregulated. RNA-seq and qPCR of the exosome and Dex-treated exosome groups showed that miR-365a-5p was upregulated in the exosome group. Furthermore, we verified that miR-365a-5p promoted osteogenesis by targeting SAV1. Additional in vivo experiments revealed that exosomes prevented GIONFH in a rat model, as shown by micro-CT scanning and histological and IHC analysis. We concluded that exosomal miR-365a-5p was effective in promoting osteogenesis and preventing the development of GIONFH via activation of the Hippo signaling pathway in rats.

Keywords: GIONFH; HUC-MSCs; Hippo signaling pathway; exosomes; osteogenesis.

Graphical abstract

Figure 1

Exosomes extracted from HUC-MSCs were characterized using TEM, NanoSight analysis, and western blotting (A) Morphology of HUC-MSC-Exos identified by transmission electron microscopy (TEM). Scale bar, 100 nm. (B) Western blot analysis of the surface biomarkers CD9, CD63, CD81, and TSG101. (C) Particle size distribution and concentration of HUC-MSC-Exos measured by NanoSight analysis. (D) The enrichment of exosomes was calculated using NanoSight analysis. (E) HUC-MSC-Exos were labeled with PKH26, and their endocytosis was observed via confocal microscopy.

Figure 2

HUC-MSC-Exos promoted cell proliferation and osteogenesis as determined by CCK-8 assay, alizarin red staining, and western blotting (A) BMSCs were cultured in conditioned medium containing Dex with or without exosomes, and proliferation was determined by a CCK-8 assay. (B) Statistical analysis was performed to investigate the proliferation rate. (C) Alizarin red staining was used to detect the osteogenesis of BMSCs treated with Dex with or without exosomes. Scale bars, 200 μm. (D) Western blotting was performed to detect the expression of Runx2, BMP2, and Sp7 in BMSCs treated with Dex with or without exosomes. (E–G) The relative levels of Runx2 (E), Sp7 (F), and BMP2 (G) were verified using ImageJ. ∗p < 0.05.The error bars stand for standard deviation. The scale bars stand for 220 μm.

Figure 3

RNA-seq was performed to identify differentially expressed exosomal miRNAs (A) miRNA expression in the control group is shown with a Venn diagram. (B) miRNA expression in the experimental group is shown with a Venn diagram. (C) Differential miRNA expression between the control and experimental groups is shown with a Venn diagram. (D) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed to evaluate the related signaling pathways. (E) Cluster analysis was performed in each group, and heatmaps were used to show the differential expression of miRNAs. (F) Differentially expressed genes were classified into biological pathways, and the metabolic pathways involved were divided into six groups as follows: cellular processes, environmental information processing, genetic information processing, human disease, metabolism, and organismal systems. (G) The candidate gene number term was calculated using the Q value. (H) Gene Ontology analysis was used for the functional classification of differentially expressed genes.

Figure 4

The interaction between miR-365a-5p and SAV1 was verified using qPCR, luciferase reporter assay, RNA pull-down, and RNA FISH (A) The related miRNAs, such as miR-21, miR-155, miR-125b, miR-122 and miR-221-3p, participating in regulating bone metabolism and osteogenesis that are contained in exosomes. (B) RT-PCR was performed to detect the level of miR-365a-5p in Dex-treated exosomes and the negative control. (C) RT-PCR was performed to detect the level of miR-365a-5p in BMSCs cocultured with PBS or exosomes. (D) The miR-365a-5p-SAV1 binding site. (E and F) The transfection efficiency of the miR-365a-5p agomir (E) and miR-365a-5p antagomir (F) was evaluated using RT-PCR. (G) SAV1 expression was evaluated after transfection with miR-365a-5p agomir. (H) SAV1 expression was evaluated after transfection with miR-365a-5p antagomir. (I) A luciferase reporter assay was performed to verify the interaction between miR-365a-5p and SAV1. (J) RNA FISH was performed to detect the cellular location of miR-365a-5p. (K) The interaction between miR-365a-5p and SAV1 was investigated using RNA pull-down. ∗p < 0.05. The error bars stand for standard deviation.

Figure 5

miR-365a-5p regulated osteogenesis through the Hippo signaling pathway (A) Western blotting was used to detect the expression of related genes, including SAV1, p-LATS, total (t-)YAP, and p-YAP, in the Hippo signaling pathway. (B–D) ImageJ was used to calculate the expression of SAV1 (B), p-LATS (C), and p-YAP/t-YAP (D). (E) Immunofluorescence staining was performed to evaluate the nuclear and cytoplasmic location of YAP. (F) Western blotting was performed to evaluate the nuclear expression of YAP. (G) Western blotting was performed to evaluate the cytoplasmic expression of YAP. (H) Alizarin red staining and ALP staining were used to detect osteogenesis in BMSCs in the control, agomir, Dex+agomir, and Dex+antagomir+YAP small interfering RNA (si-YAP) groups. (I) Calculation of the alizarin red-positive area. (J) EdU staining was used to detect proliferation in the control, agomir, Dex+agomir, and Dex+antagomir+si-YAP groups. (K) Calculation of the percentage of EdU-positive cells using ImageJ. (L) Calculation of the ALP-positive area. ∗p < 0.05. The error bars stand for standard deviation. The scale bars stand for 220μm.

Figure 6

miR-365a-5p regulated osteogenesis in a GIONFH rat model (A) Schematic of the MPS-induced GIONFH rat model and the different treatment groups. (B) H&E and Masson staining of the femoral heads of rats receiving different treatments. IHC staining was used to detect the level of YAP. (C) Micro-CT scans were performed to assess femoral head bone volume (BV) in the different treatment groups. (D) Quantitative analyses of BV per tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular number (Tb.N) in the different treatment groups. ∗p < 0.05. The error bars stand for standard deviation.