The Role And Mechanism Of CircSEC31A During Osteogenic Differentiation In Human Adipose-derived Stem Cells

Objective: Adipose Stem Cells(ADSCs)are a type of mesenchymal stem cells,which are derived from adipose tissue and have the characteristics of self-renewal and the potential for multidirectional differentiation.Compared with bone marrow stem cells,adipose stem cells are easier to obtain,have more abundant sources,more content,and fewer ethical restrictions.Many studies have confirmed the osteogenic differentiation potential of ADSCs,and believe that ADSCs are good seed cells for bone formation and regeneration.Adipose stem cells have thus become the most valuable seed cells in the field of tissue engineering repair and reconstruction.Circ RNAs(circ RNAs)are noncoding RNAs that are widely present in eukaryotic transcripts.Unlike traditional linear RNAs,circ RNAs form a closed loop structure through covalent bonds and are not affected by RNA exonuclease.The expression of Circ RNAs is more stable,not easily degraded and has new characteristics.They have the ability to express specifically in developmental stages or tissues.They are ideal biomarkers for disease diagnosis and treatment and play an important regulatory role in many life activities..In terms of function,studies have shown that circ RNAs are rich in binding sites for micro RNAs(mi RNAs)and enter cells as mi RNA sponges,thereby releasing mi RNAs from shearing and degrading their messenger RNA(m RNA)or inhibiting their transcription The role of translation,thereby increasing the expression level of target genes,this mechanism of action is called the competitive endogenous RNA(ce RNA)mechanism.Since the discovery of circ RNAs,a large number of studies have shown that circ RNAs are also widely involved in tissue regeneration and stem cell differentiation processes,and initiate roles in various osteogenic-related signal pathways.However,we are still unclear about the specific role and mechanism of circ RNAs in the osteogenic differentiation of human ADSCs.Therefore,in order to explore the role and mechanism of circ RNAs in the osteogenesis of ADSCs,we carried out the following research.Methods: 1.In this study,the adipose tissues of five healthy young women who underwent liposuction were obtained.The primary ADSCs were extracted from them by collagenase digestion,and the cell surface markers CD34,CD44,CD45,CD73,CD90were analyzed by flow cytometry.The expression of CD105 was tested.In addition,we have verified the differentiation potential of ADSCs through adipogenic,osteogenic,chondrogenic differentiation induction medium culture and oil red O(oli red O),alizarin red(ARS),and Alcian blue(Alcian)staining.Under the guidance of gene chip data results,circ SEC31 A was selected as the main research target of this study.2.Use q PCR to detect the expression level of circ SEC31 A in non-induced and induced differentiated ADSCs cell samples.After knockdown or overexpression of circ SEC31 A,m RNA changes of osteogenic indicators were detected and ALP and alizarin red staining were performed after differentiation culture.Intracellular localization of circ SEC31 A was performed using fluorescence in situ hybridization.3.The mi RNA-m RNA and mi RNAcirc RNA interactions were predicted using Target Scan and mi Randa databases,and the predicted results were plotted into a network diagram using Cytoscape.According to the predicted network diagram,mi RNAs that might bind to circ SEC31 A were selected,and their targeted binding was verified by dual luciferase assay.The function of mi RNA was verified,and its downstream target genes were predicted and verified.Results: After verification,the expression trend of circ SEC31 A is consistent with the microarray results.The expression of circ SEC31 A was significantly increased during osteogenic differentiation.The osteogenic differentiation ability of ADSCs in osteogenic induction medium was decreased after circ SEC31 A silencing,while the osteogenic differentiation ability of adipose stem cells in osteogenic induction medium was increased after circ SEC31 A overexpression.ALP staining,alizarin red staining,m RNA and protein expressions of osteogenic factors were consistent with the above trend.Circ SEC31 A is formed by circularization of the 24 th,25th,and 26 th exons of the SEC31 A gene,with a length of 406 nt,and is located in the cytoplasm of ADSCs.Circ SEC31 A is positively correlated with the expression of CLDN11 in cells,and plays a role in promoting osteogenesis in ADSCs by binding with mi R-612.Conclusion: Circ SEC31 A plays a role in promoting the osteogenic differentiation of ADSCs and is involved in the regulation of their osteogenic differentiation ability.It can affect the expression of CLDN11 through competitive combination of mi R-612,thereby participating in the osteogenic differentiation process of ADSCs.The effect of circ SEC31 A on promoting osteogenic differentiation provides a theoretical basis for tissue engineering to repair bone defects and has a promising application in the treatment of bone defects.