| Objective: The repair of large bone defects has always been a difficult clinical problem.Although autologous bone transplantation is commonly used,the damage to the donor site is relatively large.The concept of bone tissue engineering was proposed in 1995,that is,the application of engineering technology,first to prepare a scaffold material with good performance,then load cells on the scaffold,and finally implant the cell-loaded scaffold material into the bone defect to repair the bone defect.In recent years,many studies have focused on the osteogenic differentiation mechanism of bone marrow mesenchymal stem cells(BMSCs).However,the content of BMSCs in the bone marrow is very small,and it takes a long time to expand in vitro.Although the source and quantity of BMSCs are not limited,there is immune rejection after implantation.Therefore,it is difficult for BMSCs to meet the needs of clinical treatment when applied to bone tissue engineering.Human adipose-derived stem cells(h ADSCs)exist in adipose tissue and have the potential to maintain self-renewal and differentiate into a variety of cells,including osteoblasts,chondrocytes,and adipocytes.Quiescent h ADSCs account for 70% of the total number of cells with stem cell properties in the body.The osteogenic differentiation process of h ADSCs is regulated by genetic and epigenetic mechanisms,but the specific molecular mechanism remains unclear.Numerous studies have shown that micro RNAs(miRNAs)play a key role in the osteogenic differentiation of h ADSCs.According to current studies,at least 27 miRNAs are involved in the osteogenic differentiation of h ADSCs.miR-146 a and miR-26 a can inhibit the BMP signaling pathway by inhibiting the expression of Smad1 and Smad4 proteins,thereby inhibiting the osteogenic differentiation of h ADSCs.miR-23 a can target RUNX2,and miR-31 can target Osterix,which in turn negatively regulates the osteogenic differentiation of h ADSCs.Studies have reported that miR-204-5p can promote the adipogenic differentiation of h ADSCs and inhibit the osteogenic differentiation of BMSCs,and the Target Scan website predicts that GDF7 may be the target gene of miR-204-5p,and the JASPAR website shows that the transcription factor FOXC1 can bind to the promoter of miR-204-5p and GDF7.Therefore,this study intends to explore the mechanism of FOXC1/miR-204-5p/GDF7 regulatory axis in the osteogenic differentiation of h ADSCs,and to provide a theoretical basis for the repair of clinical bone defects.Methods:1.Extract h ADSCs from adipose tissue after liposuction,and verify their multi-directional differentiation ability of adipogenic,osteogenic,and chondrogenic,and analyze their surface markers CD44,CD45,CD90,CD105 and HLA-DR by flow cytometry expression.2.The h ADSCs were cultured for osteogenic induction,and the expression levels of miR-204-5p,osteogenesis-related genes RUNX2,ALP,and OCN in the cells were detected by q PCR at 0,3,5,7,10,and 14 days of osteogenic induction.3.The h ADSCs were transfected with miR-204-5p mimics or inhibitor,and cultured in proliferation medium or osteogenic induction medium,respectively.After 14 days of culture,the expression levels of miR-204-5p,RUNX2,ALP,and OCN were detected by q PCR.ALP and alizarin red staining were used to detect the osteogenic differentiation ability and calcium ion deposition level of cells.Western blot is to detect the expression levels of RUNX2,ALP,OCN,FOXC1 and GDF7 in cells,and immunofluorescence staining is to detect the expression of OCN of cells.4.In order to verify the targeting relationship between miR-204-5p and GDF7,a complementary experiment was performed.The experimental groups were as follows:(1)OM+mimics+vector,(2)OM+mimics+GDF7,(3)OM+inhibitor+NC,(4)OM+inhibitor+si-GDF7.After 14 days of cell culture in each group,the expression levels of miR-204-5p,GDF7,RUNX2,ALP and OCN were detected by q PCR,the osteogenic differentiation ability and calcium ion deposition level of the cells were detected by ALP and alizarin red staining,and GDF7,RUNX2,ALP,OCN,p-AKT,AKT,p-P38,P38 protein expression levels were detected by Western blot.The dual-luciferase reporter gene vector was constructed after predicting the binding site of miR-204-5p and GDF7,and the dual-luciferase activity was verified.5.To verify the role of FOXC1 in the regulation of miR-204-5p in the osteogenic differentiation of h ADSCs,the experimental groups were as follows:(1)OM+mimics+vector,(2)OM+mimics+FOXC1,(3)OM+inhibitor+NC,(4)OM+inhibitor+si-FOXC1.After 14 days of cell culture in each group,the expression levels of miR-204-5p,FOXC1,GDF7,RUNX2,ALP and OCN were detected by q PCR,the osteogenic differentiation ability and calcium ion deposition level of cells were detected by ALP and alizarin red staining,and FOXC1,GDF7 and the expression levels of osteogenesis-related proteins were detected by Western blot.The relationship of transcription factor FOXC1 on miR-204-5p and GDF7 promoters was verified by dual luciferase activity assay.6.Ch IP experiment was used to detect the relationship between FOXC1 on the promoter region of miR-204-5p and GDF7 and the relationship among HDAC2,H3K9 AC and miR-204-5p gene promoter.Results:1.The extracted h ADSCs have multi-directional differentiation ability.The results showed that the cells were positive for CD44,CD90,and CD105 and negative for CD45 and HLADR,which suggested that the primary cultured cells were h ADSCs.2.The expression level of miR-204-5p decreased during the osteogenic differentiation of h ADSCs.Overexpression of miR-204-5p decreased the expression levels of RUNX2,ALP and OCN,while silencing miR-204-5p could increase their expression levels.Alizarin red staining and ALP staining showed that overexpression of miR-204-5p inhibited the osteogenic differentiation of h ADSCs,and silencing of miR-204-5p enhanced the osteogenic differentiation of h ADSCs.3.The expression level of GDF7 increased during the osteogenic differentiation of h ADSCs,and miR-204-5p mimics decreased the expression level of GDF7.Overexpression of GDF7 could inhibit the expression level of miR-204-5p and increase the expression levels of RUNX2,ALP,and OCN.Silencing GDF7 had the opposite result.Alizarin red and ALP staining showed that overexpression of GDF7 could reduce the inhibitory effect of miR-204-5p on the osteogenic differentiation of h ADSCs.Meanwhile,overexpression of GDF7 could promote the phosphorylation of AKT and P38 under miR-204-5p mimics treatment.The dual-luciferase reporter gene results indicated that GDF7 was the target of miR-204-5p.4.Overexpression of FOXC1 can inhibit the expression level of miR-204-5p,and promote the expression level of GDF7,RUNX2,ALP,and OCN.On the contrary,the results of silencing FOXC1 are opposite.Alizarin red and ALP staining results suggest that overexpression of FOXC1 can reduce the inhibitory effect of miR-204-5p on the osteogenic differentiation of h ADSCs.5.The dual luciferase reporter gene results showed that FOXC1 combined with the promoters of miR-204-5p and GDF7,and regulated the promoter activities of miR-204-5p and GDF7.The results of Ch IP experiments further confirmed that FOXC1 regulates the transcription of GDF7 and miR-204-5p through direct binding.At the same time,the results of Ch IP experiments also showed that FOXC1 repressed the transcription of miR-204-5p by affecting the interaction of histone deacetylase 2 and H3K9 AC with the miR-204-5p promoter.Conclusions:1.The expression level of miR-204-5p gradually decreased during the osteogenic differentiation of h ADSCs,silencing miR-204-5p promoted the osteogenic differentiation of h ADSCs,and its overexpression inhibited the osteogenic differentiation.2.miR-204-5p targets GDF7 to inhibit the osteogenic differentiation of h ADSCs.GDF7 attenuates the inhibitory effect of miR-204-5p on the osteogenic differentiation of h ADSCs by promoting the phosphorylation of AKT and P38.3.FOXC1 combined with the promoters of miR-204-5p and GDF7 to promote the deacetylation of the promoter of miR-204-5p and the transcription of GDF7,and attenuate the inhibitory effect of miR-204-5p on the osteogenic differentiation of hADSCs. |
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