The Effect Of Circular RNA CDR1as On Osteogenic Differentiation Of Periodontal Ligament Stem Cells

Background:The biological basis of periodontal tissue regeneration is the orderly regulation of the proliferation and differentiation of periodontal ligament stem cells(PDLSCs).The osteogenic differentiation of PDLSCs is crucial to regenerate the periodontal tissue and reconstruct the mastication function.Researchers hope to artificially guide this process and promote periodontal tissue regeneration through studying the regulation mechanism.Circular RNA(circular RNA)is a type of non-coding RNA molecule with circular structure formed by special cutting and splicing of gene transcripts.Current studies have shown that circRNA play the important regulatory roles in a variety of diseases,including cancer,cardiovascular disease,nervous system diseases,diabetes,skin wounding,osteoarthritis and so on.In addition,circRNA are widely involved in tissue regeneration and differentiation of stem cells,such as the primary stage of mouse liver regeneration,the differentiation of human induced pluripotent stem cells into cardiomyocytes,the differentiation of epidermal stem cells and the osteogenic differentiation of MC3T3-E1158 induced by bone morphogenic protein 2.However,the role of circRNA in osteogenic differentiation of PDLSCs remains unclear.Purpose:The aim of this experiment was to comprehensively identify the differentially expressed circRNA and study their effects on PDLSC osteogenic differentiation,which might provide new genetic strategies for promoting PDLSC osteogenic differentiation and periodontal regeneration.Materials and Methods:1.Human PDLSCs(hPDLSCs)were isolated and cultured using the explant culture method.Then their biological characteristics were identified.2.The hPDLSCs were separately cultured with normal media and osteogenic induction for seven days.Then they were sent to the sequencing company for high throughput sequencing.The bioinformatic methods were used for identification,differentially expression analysis,quantitative real-time polymerase chain reaction(qRT-PCR)validation and function analysis of circRNA.3.Select differentially expressed circRNA and detect the osteogenesis ability of hPDLSCs by knocking down the expression of circRNA.Results:1.We successfully isolate and culture the hPDLSCs with mesenchymal stem cell phenotype and multilineage differentiation ability.2.A total of 1456 circRNA were found to be differentially expressed through high throughput sequencing.CircRNA were predicted to participate in osteogenic differentiation of hPDLSCs through parent genes or microRNAs.The qRT-PCR results of eight selected circRNA were consistent with the normalized expression of sequencing.Besides,the expression level of circRNA CDR1as was high and the most different between non-induced and induced group by qRT-PCR.3.The circRNA CDRlas was involved in the osteogenic differentiation of hPDLSCs,and the osteogenic differentiation of hPDLSCs was significantly decreased after knocking down the circRNA CDR1as.Conclusion:CircRNA are involved in the osteogenic differentiation of hPDLSCs,and circRNA CDR1as promotes hPDLSCs osteogenic differentiation.