Abstract：Objective: To explore the role and mechanism of circular RNA (circRNA) YAP1 in the regulation of microvascular endothelial cell (MVEC) angiogenesis in steroid-induced osteonecrosis of the femoral head (SONFH) rats. Methods: Adult male SD rats were injected with methylprednisolone (MPS, 20 mg/kg/d) for 3 weeks to induce the SONFH model (n=30). The control group (n=30) was injected with an equal amount of saline. Micro-CT scanning and HE staining were performed to evaluate whether the model was successfully established. MVECs were isolated from the femoral heads of the rats using CD31 as a marker and identified by immunofluorescence staining. The MVECs were divided into three groups: the pcDNA-YAP1 group (overexpression of circRNA YAP1), the pcDNA-NC group (negative control), and the pcDNA-YAP1+dickkopf-1 group (treated with Wnt1/β-catenin pathway inhibitor dickkopf-1). The expression of circRNA YAP1 was detected using qPCR, while the expressions of Wnt1, β-catenin, VEGF-A, and vimentin were detected using Western blotting. Cell proliferation was detected using the CCK-8 assay, while cell migration was detected using the Transwell assay. Tube formation assays were performed to detect the angiogenic ability of the cells. Results: The HE staining results showed that the control group was healthy, with no bone necrosis, while the SONFH group showed severe femoral head necrosis and deterioration. The results of micro-CT scanning showed that the control group had dense and regular bone trabeculae, with normal numbers and morphology of bone trabeculae. The SONFH group was accompanied by an empty bone pit, a decrease in the number of bone trabeculae, and shortened bone trabeculae. Compared with the control group (1.00±0.08; 1.00±0.12; 1.00±0.15), the expressions of circRNA YAP1 (0.27±0.04), Wnt1 (0.49±0.03), and β-catenin (0.33±0.03) in the SONFH group were significantly reduced (t=12.158, P=0.009; t=10.596, P=0.012; t=10.080, P=0.014). In addition, MVECs from the femoral heads of the Ctrl and SONFH groups were successfully isolated. Compared with the control group (1.00±0.00; 1.00±0.02; 1.00±0.01), the expressions of circRNA YAP1 (0.15±0.01), Wnt1 (0.28±0.05), and β-catenin (0.31±0.02) in the MVECs from the SONFH group were significantly reduced (t=17.625, P=0.004; t=16.154, P=0.005; t=13.596, P=0.007).In MVECs, the expressions of circRNA YAP1, Wnt1, β-catenin, VEGF-A and vimentin were up-regulated in pcDNA-YAP1 group compared with pcDNA-NC group. The cell proliferation rate, cell migration rate and tubule formation rate were significantly increased (all P<0.05). Compared with pcDNA-YAP1 group, the expression of circRNA YAP1 in pcDNA-YAP1+dickkopf-1 group had no significant change (P>0.05), while the expression of Wnt1, β-catenin, VEGF-A and vimentin were down-regulated. The cell proliferation rate, cell migration rate and tubule formation rate were significantly inhibited (all P<0.05). Conclusion: CircRNA YAP1 promotes the angiogenesis of bone endovascular endothelial cells in SONFH rats by activating Wnt1/β-catenin pathway.
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2023, Vol. 29 
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