MiR-106a-5p Targets SFRP2 To Regulate Apoptosis And Osteogenic Differentiation Of Osteoblast Precursors In Perthes Disease

Objective:RNA sequencing（RNA-seq）was employed to identify the differentially expressed genes（DEGs）in the femoral head of an animal model with Perthes disease in this study.The regulatory mechanism which miR-106a-5p targets SFRP2 to regulate proliferation,apoptosis,and osteogenic differentiation of osteoblast precursors via the Wnt/β-catenin pathway was investigated both in vitro and in vivo.The aim of this study was to elucidate the pathogenesis of Perthes disease and identify effective treatment options and potential therapeutic targets.Methods:1.To create animal models of Perthes disease,the femoral neck of immature rabbits was ligated.After 8 weeks,the animals were euthanized,and the success of developing the Perthes disease animal model was assessed through gross morphology and X-ray imaging.RNA was extracted from the femoral head,and RNA-seq analysis was used to screen for DEGs,which were further analyzed using the GO database and the KEGG database.Through literature research,candidate genes were identified and confirmed using quantitative polymerase chain reaction（q PCR）and western blot（Wb）assays in damaged femoral heads.2.Multiple experiments were performed to investigate the function of SFRP2 and miR-106a-5p in chondrocytes and osteoblast progenitor cells.（1）Mouse chondrocyte line ATDC5 cells were cultured and induced with chondrocyte induction medium to investigate the expression of the SFRP2 gene at 0,7,14,and 28 days.Additionally,the expression of the SFRP2 gene was examined at 7 and 28 days under a hypoxic condition induced by Co Cl₂using q PCR.（2）Immature rabbit BMSCs and mouse embryonic osteoblast progenitor cell line MC3T3-E1 cells were cultured,identified,and subjected to SFRP2 gene silencing or overexpression using a lentivirus vector.The cells were divided into four groups,including control,sh-SFRP2,oe-SFRP2,and oe-NC.Cell proliferation and apoptosis were detected using MTT,Ed U,and flow cytometry assays.Cell osteogenic differentiation was determined using ALP staining and alizarin red staining,and the expression of osteogenesis-related molecules and Wnt/β-catenin pathway were examined using q PCR and Wb.（3）BMSCs were transfected with overexpressed or silenced genes of miR-106a-5p and then categorized into three groups:control group,miR-106a mimics group,and miR-106a inhibitor group.The expression of miR-106a-5p and SFRP2 during osteogenic differentiation was investigated using q PCR and Wb.（4）BMSCs that were transfected with miR-106a-5p mimics or over-expressed SFRP2 were classified into four groups:control group,miR-106a group（miR-106a-5p mimics）,SFRP2 group（SFRP2 over-expressed gene）,and miR-106a+SFRP2 group（miR-106a-5p mimics+SFRP2 over-expressed gene）.The expression of cell proliferative,apoptotic,osteogenesis-related,and Wnt/β-catenin pathway molecules was measured using q PCR and Wb.The study aimed to determine whether the biological effects regulated by miR-106a-5p can be rescued by SFRP2.（5）The SFRP2 m RNA-3’UTR mutant and wild gene vector based on the predicted base binding sites from the targetscan website were constructed.The transfected 293T cells were divided into four groups:miR-NC+SFRP2-WTgroup,miR-106a-5p+SFRP2-WT group,miR-NC+SFRP2-MUT group,and miR-106a-5p+SFRP2-MUT group.The binding sites were verified using a dual-luciferase reporter gene assay.3.The animal models of Perthes disease in immature rabbits were established,and p AAV-zs Green-SFRP2 or miR-106a-5p agomir were injected locally into the hip postoperatively for two weeks.The animals were classified into five different groups,including the Control group,Model group,miR-106a group,Ad-SFRP2 group,and Ad-SFRP2+miR-106a group.These animals were euthanized postoperatively for 8weeks,and the morphology of the femoral head was observed through micro CT,HE staining,ALP staining,and immunohistochemical staining.The bone structure,osteogenic differentiation,and SFRP2 expression were also analyzed to determine the bone repair function on the damaged femoral head and the improvement of femoral head deformity.Results:1.The animal model of Perthes disease in immature rabbits was successfully established by ligating the femoral neck.The DEGs were screened in the damaged femoral heads by RNA-seq technique,which included 128 m RNAs（123up-expressions and 5 down-expressions）and 19 micro RNAs（3 up-regulations and 16down-regulations）.The differential expression of SFRP2 and miR-106a-5p in the damaged femoral heads was confirmed by q PCR,which was consistent with the results of the RNA-seq analysis.2.（1）The expression of SFRP2 gene was gradually up-regulated during chondrocyte differentiation in ATDC5 cells,and its expression was further increased under hypoxia induced by Co Cl₂（P<0.05）.（2）It was observed that the oe-SFRP2 group in BMSCs or MC3T3-E1 cells during osteogenic differentiation led to enhanced cell proliferation and osteogenic differentiation abilities,and inhibited apoptosis.The expression of PCNA,Bcl-2,ALP,Col1a1,Runx1,Osterix,Wnt3a,β-catenin,LRP5,and LRP6 were up-regulated（P<0.05）;however,the expressions of Bax and Caspase3were down-regulated（P<0.05）.Conversely,the biological effect of the sh-SFRP2group was the opposite of that of the oe-SFRP2 group.（3）The expression of miR-106a-5p was significantly up-regulated,whereas the expression of SFRP2 was significantly down-regulated in the miR-106a-5p mimic group（P<0.05）.However,the expression of miR-106a-5p and SFRP2 had completely different patterns in the miR-106a inhibitor group compared to the miR-106a-5p mimic group.（4）During the late stage of osteogenic differentiation of BMSCs,the expressions of PCNA,Bcl-2,ALP,Col1a1,Runx1,Osterix,Wnt3a,β-catenin,LRP5,and LRP6 were significantly down-regulated（P<0.05）,while the expressions of Bax and Caspase3 were significantly up-regulated in the miR-106a group（P<0.05）.Conversely,the expression of these molecules in the SFRP2 group was opposite compared to that in the miR-106a group（P<0.05）,and the findings in the SFRP2+miR-106a group suggested that SFRP2 could rescue the biological effect of miR-106a-5p on BMSCs.（5）The luciferase activity had significantly decreased in the miR-106a-5p+SFRP2-WT group（P<0.01）,while there was no significant difference observed in the other groups（P>0.05）.3.The gross morphology and micro CT images of the femoral head in the Model group were found to be highly similar to those observed in Perthes disease.The damaged femoral head may still be in the early stages of repair postoperatively for 8weeks.Compared to the Model group,the Ad-SFRP2 group showed a closer-to-normal gross morphology of the damaged femoral head,reduced the number of empty bone lacunae,deepened ALP staining,increased SFRP2 expression,alleviated femoral head deformity,and increased trabecular density and bone mass.Conversely,the miR-106a group showed the opposite performance to that of the Ad-SFRP2 group.The study results indicated that SFRP2 could reverse the inhibitory function of miR-106a-5p on the repair of the damaged femoral head.Conclusion:1.Ligating the femoral neck is a reliable method to establish an animal model of Perthes disease in immature rabbits.2.The expression of miR-106a-5p is significantly down-regulated during the repair of the damaged femoral head,the up-regulation of SFRP2 expression may be related to the epiphyseal chondrocytes adjacent to the lesion area.3.MiR-106a-5p targets SFRP2 to inhibit proliferation and osteogenic differentiation of osteoblast precursor cells,promotes apoptosis via Wnt/β-catenin pathway.4.SFRP2 promotes the bone repair of the damaged femoral head,which can lead to a reduction in the deformity of the femoral head.Conversely,miR-106a-5p is observed to inhibit the repair of the damaged femoral head.5.SFRP2and miR-106a-5p are both potential therapeutic targets for the treatment of Perthes disease...