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

Adipose-derived stem cells(ADSCs),a subset of mesenchymal stem cells,have the capacity for multilineage differentiation and self-renewal.Compared to bone marrow stem cells,adipose tissue is a more abundant and easily available source of adult stem cells,and therefore,is an ideal stem cell source in tissue engineering.Many studies have confirmed the osteogenic differentiation potential of ADSCs,and which have been considered as favorable seed cells for bone formation and regeneration.Circular RNAs(circRNAs),a kind of non-coding RNA,are highly prevalent in the eukaryotic transcriptome.CircRNAs consist of a covalent closed loop structure without a 5' cap or 3' polyadenylic acid tail,and were originally thought to be the by-product of aberrant splicing.Compared to long non-coding RNAs and micro RNAs(miRNAs),circRNAs with conserved sequences can resist RNase digestion and are relatively stable;their ability to exhibit developmental stage or tissue specific expression makes them ideal biomarkers for disease diagnosis and treatment targeting.Multiple studies have explored the important roles of circRNAs in cellular activity,embryonic development,neural development,and the development of a variety of human diseases.Functionally,circRNAs regulate gene expression through the mediation of alternative splicing or as miRNA sponges,regulating parental gene transcription and RNA processing reactions,and serving as a scaffold assembled by protein complexes or templates for translation.Previous studies have shown that circRNAs are involved in the osteogenic differentiation process and play a role in various osteogenic related signaling pathways.However,the expression pattern and functional role of circRNA in osteogenic differentiation of human ADSCs are unclear.This research is divided into the following four parts.Objective: Circular RNAs(circRNAs)are essential for stem cell differentiation.This study aimed to investigate their exact mechanism of action in human adipose-derived stem cell(hADSC)osteogenesis.Methods: We obtain adipose tissue from 3 healthy young women undergoing liposuction and extract hADSCs from adipose stem cells.Then we induce osteogenic differentiation of hADSCs,collect uninduced and induced adipose stem cell samples,extract RNA using Trizol method,detect RNA integrity by agarose electrophoresis,analyze data,construct a circular RNA expression profile,and screen for differential circRNAs online bioinformatics software.CircInteractome,miRwalk and TargetScan were used to predict miRNAs and target genes.QPCR was used to detect the expression level of circ-FTO in undifferentiated and differentiated cell samples.Interfering with circ-FTO expression in adipose stem cells,the osteogenesis index of each group was detected 14 days after induction with induction medium,and ALP staining,alizarin red staining were performed.Heterotopic osteogenesis experiments in nude mice confirm the effect of circ-FTO on osteogenic differentiation of adipose stem cells.Fluorescence in situ hybridization was used to subcellularly locate circ-FTO;miRanda database was used to find miRNAs that circ-FTO might bind to,and analyzed and screened by Targetsan database.Detection of dual luciferase reporter gene circ-FTO and predicted miRNAs,targeted binding sites and binding effects of mi RNAs and target genes were used to explore the mechanism.Results: The microarray analysis showed that compared with the non-induced group,171 circRNAs were up-regulated and 119 circRNAs were down-regulated in the induced group.The expression trend of circ-FTO is consistent with the chip result.Circ-FTO expression was significantly increased during osteogenic differentiation.After silencing circ-FTO,the osteogenic differentiation ability of adipose-derived stem cells in osteogenic induction medium decreased,and ALP staining,alizarin red staining,and mRNA of osteogenic related factors and protein expression decreased.Circ-FTO is formed by circularizing the 5th,6th,and 7th exons of the FTO gene,with a length of 344 nt and a cut point of AG.It is located in the cytoplasm of adipose stem cells and can withstand exonuclease digestion.Conclusion: Circ-FTO promotes osteogenic differentiation of adipose stem cells;it can affect the expression of LMO3 by adsorbing mi R-520 f.In addition,miR-520 f binds to LMO3 and participates in the osteogenic differentiation of adipose stem cells.