| Background and objectivesPeriodontal ligament stem cells(PDLSCs)are a kind of adult mesenchymal stem cells derived from the periodontal ligament(PDL)with specific abilities of self-renewal and multilineage differentiation potential.Considering of its potential to differentiate into diverse functional cells under specific conditions,it is the undisputed seed cell to achieve cell therapy for periodontal tissue engineering.It has been received a widespread attention,that the usage of periodontal ligament cells to restore the soft and hard tissue defects of periodontal caused by inflammation,induced periodontal ligament cells into osteoblasts,cementoblasts or fibroblasts recover the alveolar bone of the teeth to a certain extent regeneration consequently.MicroRNA is a small endogenous nonprotein-encoding single-stranded RNA that suppresses the expression of target genes mainly by cleavage or inhibition of translation in two ways.Studies have shown that miRNAs play a key regulatory role in stem cell maintenance and multidirectional differentiation.Since studies have found more microRNAs has differentially expression in the human periodontal ligament cells osteogenic differentiation process,which suggested that miRNAs play an important role in osteogenesis differentiation.In this study,we constructed the miRNAs expression profile of the periodontal ligament stem cells with or without osteo-induction,and screened the differentially expressed miR-543.To further explore the biological role of miR-543 in osteogenic differentiation of human PDLSCs and the molecular mechanism of targeted regulation TOB2.Our results may provide a strong evidence for the molecular function of miRNAs in the process of osteogenic differentiation of mesenchymal stem cells derived from periodontal ligaments,and further optimize the miRNAs-mediated stem cell therapy.Methods1.Isolation and identification of human periodontal ligament stem cellsIn this study,premolars or third molars were collected from different individuals,and the cells were cultured by modified tissue explant collagenase method.Then the cultured cells were purified by limited dilution technique.CCK8 method was performed to examine the proliferation ability of hPDLSCs,and calculated value shows the tendency of growth.We chose the third passage of hPDLSCs to detect the surface molecular markers in flow cytometry,and the expression of the vimentin and keratin was detected by immunocytochemistry.The PDLSCs were inoculated into 10cm2 dish by the dilution method,the monoclonal formation of the cells was observed,and the cell colony formation rate was calculated.The cultured human PDLSCs were induced by osteogenesis and adipogenesis for 14 days.They were stained with alizarin red S,alkaline phosphatase and oil red O staining respectively and observed under inverted microscope.2.qRT-PCR verification and microarray analysis of gene expression during osteogenic differentiation of hPDLSCs.Trizol was added to extract the total RNA from control and osteogenic differentiation induction groups.We use NanoDropND-1000 spectrophotometer to detect the concentration and purity.Then we use miRNAURYTM LNA Array(v.18.0)chip screen miRNAs expression profile of normal culture group and osteogenic induction group were detected.The detection was repeated three times.SYBR Green method was used to detect the results of the chip and osteogenesis-related genes,and the relative expression intensity of the gene was represented by 2-△ΔCt value.3.Osteogenic function of miR-543.We use the lentivirus of miR-543 with different infection complex transfection of human PDLSCs to screen the most appropriate multiplicity of infection.qRT-PCR was used to determine the expression of miR-543 after transfected by overexpression or inhibition lentivirus.The total RNA and protein were extracted after 14 days of osteoinduction.qRT-PCR was used to detect the expression of osteogenesis-related genes.Furthermore,alkaline phosphatase and alizarin red staining reagent was used to test the ability of osteogenesis.4.Prediction and verification of target gene of miR-543.The target gene of miR-543 was predicted by Targetscan,miRMap and MiRanda databases.The target genes including TOB2,NOTCH2,ACVR2A and BMP2 were selected to confirm the change of gene and protein level after tansfected with or without lentivirus of miR-543.Moreover,miR-543 and TOB2 vector plasmids were constructed and the binding sites between them were detected by double luciferase reporter assay.5.Statistical analysis.The experimental data were analyzed by SPSS 19.0,with x±s,with two independent samples t test for comparison between the two groups,P<0.05 for the difference was considered statistically significant.Results1.Isolation and identification of human periodontal ligament stem cells.In our experiment,the human PDLSCs were isolated and cultured successfully,and a large number of mineralized nodules and orange-red lipid droplets were found in the osteogenic and adipogenic directions.Flow cytometry showed that cells were positive for expression of mesenchymal stem cell surface markers and negative expression of hematopoietic stem cell surface markers.The expression of keratin was negative and the expression of vimentin was positive,which suggested that the cells were of mesenchymal origin.2.Differentiation of miRNAs and osteogenesis-related mRNA during the osteo-induction.Microarray results showed a total of 116 miRNAs were differentially expressed,of which 30 were up-regulated and 86 down-regulated.qRT-PCR confirmed that miR-543 was selected as the key gene,and osteogenesis-related mRNA was significantly increased in induced expression.3.Cytological function of miR-543.The miR-543 overexpression of lentivirus was significantly enhanced compared with the conventional culture group.The expression of miR-543 in the lentiviral group was almost invisible.The results showed that miR-543 had obvious regulation effect on PDLSCs differentiation.4.Prediction of miR-543 target gene and double luciferase reporter.According to the intersection results of three databases of the target gene,we select TOB2,NOTCH2,ACVR2A,BMP2 as the predicted target genes for the next step of verification.The miR-543 and TOB2 binding sites were predicted to be GAAUGUU.The dual luciferase reporter confirmed that miR-543 could bind to TOB2 3 ’UTR in this sequence and play an inhibitory effect,thus promoting the process of osteoblast differentiation of human PDLSCs.Conclusions1.Our study successfully isolated and cultured human periodontal ligament stem cells.For cytological function,we verify its clonial formation and osteogenic,adipogenic differentiation ability in vitro.2.We confirmed that there is differentially expressed miRNAs in human PDLSCs before and after osteogenic differentiation,and miR-543 was identified as the key gene.3.MiR-543 plays an important role in the differentiation of human PDLSCs in osteogenesis.TOB2 was predicted as the target gene of miR-543 by bioinformatics.4.MiR-543 and TOB2 had a binding site on the 3 ’UTR.Double luciferase reported that miR-543 played a role in inhibiting TOB2 expression and promoted osteogenic differentiation. |
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