Construction And Validation Of CircRNAs Related CeRNA In Osteogenic Differentiation Of Human Bone Marrow Mesenchymal Stem Cells

Objective: Periodontitis is a chronic inflammatory disease that occurs in the periodontal tissue and is one of the most common oral diseases that can present with alveolar bone resorption and even tooth loss.Currently,it is difficult to completely restore bone defects in periodontal tissues with conventional clinical treatments such as scaling,scraping and root planning.Bone Marrow Mesenchymal Stem Cells(BMSCs)are adult stem cells in the bone marrow with the ability to multi-directional differentiate,self-renew,migrate and colonize.As seed cells,BMSCs play a key role in the repair of bone lesions caused by periodontitis.Therefore,it is important to research the regulation of transcription factors and signaling pathways on osteogenic differentiation of BMSCs.Circular RNAs(circ RNAs)are non-coding RNAs in closed loops that are stably present in the cytoplasm of eukaryotic cells.In recent years,it has been found that some circ RNAs can act as sponges to influence MSC osteogenic differentiation,but most of them have not been identified and the specific mechanisms are not yet known.Therefore,in this study,we constructed the osteogenic differentiation-related circ RNA-mi RNA-m RNA network of human Bone Marrow Mesenchymal Stem Cells(h BMSCs)through bioinformatics,and validated the osteogenic differentiation-related circ RNA-mi RNA network of h BMSCs screened by bioinformatics analysis in vitro.The in vitro experiments validated the osteogenic differentiation-associated circ RNAs and their possible target mi RNA-m RNA axes associated with h BMSCs osteogenic differentiation,and provided a scientific basis for the future development of biomarkers that could enhance osteogenic differentiation of stem cells.Methods:1.Bioinformatics AnalysisBy mining the Gene Expression Omnibus(GEO)database for GSE135883,GSE135586,and GSE18043 datasets respectively for differential expression analysis,the resulting data were searched for pairwise relationships by circbank and mi RTar Base databases,and further,using the Gene Ontology(GO)enrichment analysis and literature base were used to construct a circ RNA-mi RNA-m RNA network associated with osteogenic differentiation of h BMSCs.2.Vitro Cellular ExperimentsAfter 7 days of osteogenic induction of h BMSCs,q RT-PCR was used to verify the expression of circ RNAs and mi RNAs screened by bioinformatics analysis.Based on the mechanism of competing endogenous RNA(ce RNA)and experimental results,hsa＿circ＿0001600with hsa-mi R-542-3p and hsa＿circ＿0005991 with hsa-mi R-424-5p were identified as the further study molecules.The circ RNAs were identified using Ribonuclease R(RNase R)enzyme digestion study,and h BMSCs after 3 days of osteogenic induction were stained with Alkaline Phosphatase(ALP)to detect the effect of knocking down hsa＿circ＿0001600 on the osteogenic differentiation ability of h BMSCs,and the effect of hsa＿circ＿0001600 on the osteogenic differentiation ability of h BMSCs and the targeting relationship between hsa-mi R-542-3p was analyzed by q RT-PCR and Western Blot(WB)at the gene and protein level.Results:1.The results of bioinformatics analysis showed that a total of 80 circ RNAs,98 mi RNAs and 814 m RNAs differentially expressed molecules were screened in three datasets;Using circ Bank and mir Tarbase databases,61 circ RNAs,56 mi RNAs and 215 m RNAs were identified based on ce RNA mechanism;Then,the 215 m RNAs were subjected to GO enrichment analysis,and 15 m RNAs associated with stem cell osteogenic differentiation were screened.The ce RNA network related to the osteogenic differentiation ability of h BMSCs was constructed with 22 circ RNAs,17 mi RNAs and 15 m RNA.2.In vitro assays showed that q RT-PCR validated 11 circ RNAs and10 mi RNAs screened by h BMSCs osteogenic induction after 7 days of bioinformatics analysis,and identified hsa＿circ＿0001600 with hsa-mi R-542-3p and hsa＿circ＿0005991 with hsa-mi R-424-5p as the study molecules.q RT-PCR was performed to detect h BMSCs osteogenesis induction on days 0,3,and 7.Hsa＿circ＿0001600 and hsa＿circ＿0005991expression gradually increased,and hsa-mi R-542-3p and hsa-mi R-424-5p expression gradually decreased;RNase R digestion assay was performed to identify hsa＿circ＿0001600 after loop formation,small interfering RNA transfection technique knocked down hsa＿circ＿0001600.q RT-PCR was detected at 3 days of osteogenesis induction.hsa-mi R-542-3p increased with the decrease of hsa＿circ＿0001600 low expression,COL1A1,Runx2 expression was decreased by hsa＿circ＿0001600 low expression The WB results showed that the expression of COL1A1,Runx2 and OCN was reduced by knocking down the hsa＿circ＿0001600.Conclusion:1.circ RNAs can influence the osteogenic differentiation of h BMSCs through a regulatory network of circ RNA-mi RNA-m RNA mechanism.2.hsa＿circ＿0001600 can promote the osteogenic differentiation of h BMSCs,and its possible mechanism is the targeted regulation of hsa-mir-542-3p.