Establishment of a juvenile rabbit Perthes disease model
We established an avascular necrosis model of the femoral head in juvenile rabbits using femoral neck ligation (Fig. 1A). Micro-CT scans were performed on days 0, 14, and 28 to assess the progression of femoral head necrosis (Fig. 1B, 1D). The results indicated that necrosis progressively worsened with longer ligation periods. Comparing the femoral heads at days 0, 14, and 28, we observed that the femoral heads at day 28 were larger, flatter, and had a paler overall color compared to those at days 0 and 14 (Fig. 1C). After Western blot analysis, we observed a decrease in osteogenic markers such as Runt-related transcription factor (RUNX), Osteopontin (OPN), and Osteocalcin (OC), and an increase in adipogenic markers such as Peroxisome Proliferator-Activated Receptor Alpha (PPARα) (Fig. 1E). Concurrently, the expression of B-cell lymphoma-2 (Bcl-2) was downregulated, while Caspase-3 (CASP-3) and Bcl-2-associated X protein (Bax) were upregulated (Fig. 1F).
The expression of miR-223-5p is downregulated during the establishment of the juvenile rabbit Perthes disease model and decreased in BMSCs under hypoxic conditions
Research indicates that a hypoxic environment is generated in the region of femoral head apoptosis, and transfection of mesenchymal stem cells alone is not sufficient to effectively repair the necrotic femoral head[25, 26]. Studies have shown that miRNAs can regulate gene expression through various mechanisms and play significant roles in the regulation of apoptosis[9,27]. Therefore, to investigate miRNA changes in the femoral head under hypoxic conditions, we ligated the femoral head and maintained it for 28 days, followed by microarray analysis to obtain miRNA profiles of the femoral head under normal or hypoxic conditions. Our results indicated that 19 miRNAs were upregulated and 7 miRNAs were downregulated under hypoxic conditions, with 7 specific miRNAs being downregulated by more than 2-fold after hypoxia, as depicted in the heatmap (Fig. 2A, 2B). qPCR testing of normal and necrotic femoral heads revealed that miR-223-5p exhibited the most significant changes (Fig. 2C). To further verify the changes in miRNA expression in BMSCs under hypoxic conditions in vitro (Fig. 2D), we found that the expression of miR-223-5p in the BMSCs of the model (hypoxia) group was significantly downregulated (Fig. 2E).
MiR-223-5p can inhibit hypoxia-induced apoptosis of BMSCs and activate the β-catenin signaling pathway in vitro
To further investigate the effects of miR-223-5p on hypoxia-induced apoptosis of BMSCs, we transfected BMSCs with miR-223-5p mimics, mimic NC, inhibitor, and inhibitor NC using Lipofectamine 3000. After transfecting miR-223-5p into BMSCs, the expression of miR-223-5p increased significantly (Fig. 3A). As shown in Fig. 3B, oligo transfection significantly impacted BMSC proliferation, as detected by the CCK-8 assay. Then, BMSCs were exposed to hypoxia for 48 hours. The results showed that under hypoxia, β-catenin and Bcl-2 expression levels were downregulated, while Bax and Cleaved CASP-3 expression levels were upregulated (Fig. 3C, 3D), with the BMSC apoptotic rate exceeding 70% (Fig. 3E, 3F). However, overexpression of miR-223-5p reversed these effects, significantly reducing the apoptotic rate of BMSCs and promoting their survival under hypoxia while inhibiting the β-catenin signaling pathway (Fig. 3G, 3H). Notably, β-catenin expression was clearly upregulated by miR-223-5p overexpression. These findings suggest that miR-223-5p inhibits hypoxia-induced apoptosis of BMSCs and activate the β-catenin signaling pathway.
CHAC2 mRNAs are direct targets of miR-223-5p
To further explore the interaction between miRNA and mRNA, we ligated the femoral head and maintained it for 28 days. We then used microarray analysis to obtain RNA profiles of the femoral head under normal or hypoxic conditions. Our results indicated that 6218 mRNAs were upregulated and 5872 mRNAs were downregulated under hypoxic conditions, with 232 potential miRNAs being upregulated by more than 2-fold after hypoxia, as shown in the heatmap (Fig. 4A, 4B). To investigate the potential mechanism of miR-223-5p in rabbit BMSCs, we speculated the promising targets of miR-223-5p using miRanda, PITA, TargetScan, and RNAhybrid. A total of 391 genes were found to have common intersections(Fig. 4C). By intersecting the database-predicted genes with the upregulated genes identified through microarray analysis, we identified three genes: CCDC102B, GRIN2B, and CHAC2 (Fig. 4D). Current research suggests that CCDC102B is primarily involved in the development of myopic macular degeneration and tumorigenesis[28, 29], while GRIN2B is mainly involved in neurotransmitter transmission[30], and CHAC2 acts as a primary enzyme for GSH degradation[31]. We, therefore, hypothesize that the regulatory relationship between miR-223-5p and CHAC2 affects the apoptosis of BMSCs under hypoxic conditions. Furthermore, miR-223-5p can suppress the expression of CHAC2 at the protein level. Conversely, inhibition of miR-223-5p leads to an upregulation of CHAC2 expression (Fig. 4E-F). Additionally, dual luciferase assays demonstrated that miR-223-5p reduced the luciferase activity of wild-type CHAC2 constructs compared to mutant groups. The positive control confirmed the validity of the method (Fig. 4G-H).
MiR-223-5p inhibits hypoxia-induced apoptosis of BMSCs by regulating CHAC2 and activate the β-catenin signaling pathway
We first constructed a lentivirus for CHAC2 overexpression in BMSCs and transfected BMSCs with CHAC2 siRNA. It was found that CHAC2 levels in BMSCs exhibited significant changes(Fig. 5A). We next determined whether miR-223-5p inhibited hypoxia-induced apoptosis of BMSCs by regulating CHAC2. We inhibited CHAC2 by downregulating CHAC2 expression using gene-specific small interfering (si)RNAs, and then subjected BMSCs to hypoxia for 48 h. After silencing with siRNA, it was observed that, compared to the hypoxia group, β-catenin and Bcl-2 gene expression was downregulated, while CHAC2, Bax and Cleaved CASP-3 gene expression was upregulated (Fig. 5B, 5C). Simultaneously, the apoptosis rate of BMSCs decreased. In the subsequent rescue experiment, an increase in CHAC2 gene expression led to a rise in the apoptosis rate of BMSCs, suggesting that the CHAC2 gene can induce apoptosis in BMSCs(Fig. 5D-F). Furthermore, co-transfection of BMSCs overexpressing the CHAC2 gene with a mimic led to an even higher apoptosis rate compared to BMSCs transfected with the mimic alone (Fig. 3D-H). This indicates that miR-223-5p can regulate BMSC apoptosis through CHAC2 and activate the β-catenin signaling pathway.
Repair effects of transplantation of miR-223-5p-overexpressed BMSCs into the perthes model
The effect of miR-223-5p-overexpressed BMSC transplantation was analyzed in the juvenile rabbit Perthes disease model. First, the juvenile rabbit Perthes disease model was successfully generated, as shown in Fig. 1A. Micro-CT scanning results showed that the NC group exhibited only slight improvement compared to the model group. By contrast, miR-223-5p-overexpressed BMSC transplantation significantly attenuated the pathological changes (Fig. 6A). Furthermore, femoral neck ligation seriously deteriorated trabecular parameters, such as BV/TV, Tb.N, Tb.Th, and Tb.Sp. However, miR-223-5p-overexpressed BMSC transplantation significantly improved these parameters, while they were only slightly restored in the NC group (Fig. 6B). When examined histologically, miR-223-5p-overexpressed BMSC transplantation clearly enhanced osteogenesis in the juvenile rabbit Perthes disease model. Bone histomorphometry results showed more trabecular bone structure and fewer empty lacunae in the femoral head of the miR-223-5p-overexpressed group compared to the model or NC groups (Fig. 6C, 6D). WB showed that Runx2, OCN, and OC were downregulated, and PPARα was upregulated in the model group. The expression of Runx2, OCN, and OC was significantly restored by miR-223-5p-overexpressed BMSC transplantation (Fig. 6F).