Study On The Interaction Between Circle＿0001421 And MiR-1253 To Regulate The Role And Mechanism Of SMAD4 In Osteogenic Differentiation Of Adipose Stem Cells

Objective: Human Adipose-Derived Stem Cells(hADSCs)are mesenchymal-derived stem cells that exist in adipose tissue and have multiple differentiation potentials.Compared with other tissues containing stem cells,adipose tissue is rich in content and easy to obtain,is an ideal adult stem cell in tissue engineering.hADSCs can differentiate into various types of cells under appropriate induction conditions,and can be used as seed cells for tissue engineering.Can differentiate into adipocytes,chondrocytes,osteoblasts and other forms.,and can secrete a variety of pro-angiogenic and anticoagulant factors.Many studies have proposed to apply the multi-lineage differentiation potential of hADSCs combined with tissue engineering technology to solve clinical diseases.However,its ability and efficiency of differentiation will directly affect its clinical application prospects.In recent years,scholars at home and abroad have confirmed the osteogenic differentiation potential of hADSCs,and determined that hADSCs are seed cells that can be used in osteogenic differentiation tissue engineering,but the mechanism of osteogenic differentiation has not been fully elucidated.Circular RNA(circular RNA,circRNA)is a kind of non-coding RNA(non-coding RNA,ncRNA),which was first discovered in 1976 and widely exists in eukaryotic transcriptome.Research hotspots.It was originally considered to be a by-product of abnormal splicing.Unlike long non-coding RNA(lncRNA),micro RNA(micro RNA,miRNA)and traditional linear RNA(linear RNA),circRNA molecules are closed circular The structure is not affected by RNA exonuclease,relatively stable,and the expression has certain tissue specificity,which can be a potential marker for disease diagnosis and treatment.Studies have shown that circRNA can participate in the process of osteogenic differentiation and play a certain regulatory role in the osteogenic differentiation signaling pathway.The purpose of this study was to analyze the differential expression of circ＿0001421 in uninduced and induced osteogenic states,and to explore the effect of circ＿0001421 on the biological functions of downstream miRNAs and the interaction between miRNAs and m RNAs to regulate the osteogenic differentiation of hADSCs.To explore the molecular mechanism of circ＿0001421 in hADSCs osteogenic differentiation.Methods: 1.The extracted hADSCs have multi-directional differentiation potential.Flow cytometry results: positive expression of CD29,CD44,CD73,CD90,CD105,etc.,and almost no expression of CD14,CD31,CD34,CD45,CD117;2.It has been verified that the expression trend of circ＿0001421 is consistent with the chip results.The expression of circ＿0001421 was significantly increased during osteogenic differentiation.After silencing circ＿0001421,the differentiation ability of hADSCs decreased in OM medium,and the m RNA and protein expression levels of osteogenesis-related factors decreased.3.The expression level of circ＿0001421 was increased during the osteogenic differentiation of hADSCs.Overexpression of circ＿0001421 increased the expression levels of RUNX2,ALP,and OCN,while silencing circ＿0001421 could inhibit the expression levels of the three.Alizarin red staining and ALP staining results showed that overexpression of circ＿0001421 promoted the osteogenic differentiation of hADSCs,and silencing circ＿0001421 could weaken the osteogenic differentiation ability of hADSCs.Ectopic osteogenesis experiments in mice also confirmed that silencing circ＿0001421 inhibited the osteogenic differentiation of hADSCs.4.The results of dual luciferase experiments confirmed that circ＿0001421 can bind to miR-1253,and miR-1253 can also target SMAD4,and the three form the regulatory axis of circ＿0001421-miR-1253-SMAD4.5.Complementation experiments confirmed that circ＿0001421 can regulate the interaction between miR-1253 and SMAD4.Overexpression of miR-1253 can inhibit the expression level of SMAD4,while overexpression of circ＿0001421 can negatively regulate the inhibitory effect of miR-1253 on SMAD4,thereby promoting SMAD4 The expression of hADSCs can improve the osteogenic differentiation ability of hADSCs.Result:1.The expression level of circ＿0001421 gradually increased during the osteogenic differentiation of hADSCs,silencing circ＿0001421 inhibited the osteogenic differentiation of hADSCs,and its overexpression promoted osteogenic differentiation;2.miR-1253 binds to SMAD4,overexpressing miR-1253 can inhibit the expression of SMAD4,and knocking down miR-1253 can promote the expression of SMAD4.3.circ＿0001421 can bind miR-1253,negatively regulate the inhibitory effect of miR-1253 on SMAD4,promote the expression of SMAD4,and promote the osteogenic differentiation of hADSCs.Conclusion:1.The expression level of circ＿0001421 gradually increased during the osteogenic differentiation of hADSCs,silencing circ＿0001421 inhibited the osteogenic differentiation of hADSCs,and its overexpression promoted osteogenic differentiation;2.miR-1253 binds to SMAD4,overexpressing miR-1253 can inhibit the expression of SMAD4,and knocking down miR-1253 can promote the expression of SMAD4.3.circ＿0001421 can bind miR-1253,which can negatively regulate the inhibitory effect of miR-1253 on SMAD4,promote the expression of SMAD4,and promote the osteogenic differentiation of hADSCs.