| Background and objectiveHuman periodontal ligament cells(hPDLSCs)is derived from the periodontal ligament,ectomesenchymal source of adult stem cells,belonging to the differentiated cells for tissue specific functions in the correlation of multi-directional differentiation capacity and can proliferate and form in life body organization so as to maintain its own quantity,can be cloned in vitro growth of self-renewal ability.When periodontal injury leads to the defect of alveolar bone,hPDLSCs with osteogenic potential migrated to the defect area and differentiated into osteoblasts,thus repairing the defect of alveolar bone.hPDLSCs osteogenesis potential in periodontal tissue of self-renewal and periodontal disease group played an important role in the process of repair and regeneration,but it is still not able to detailed clarify hPDLSCs osteogenetic differentiation mechanism,remains to be further explored.MicroRNA(miRNA)is a kind of micro non-coding RNA,widely exists in a variety of eukaryotic organisms,nature can restrain by degradation of target genes,target genes translation or the role of the two ways of combining the function of inhibiting mRNA expression.Current studies have shown that miRNA has important significance for the undifferentiated and self-renewal ability of stem cells.At the same time,it has been found that miRNA has the function of regulating the osteogenic differentiation of hPDLSCs.In the process of osteogenesis differentiation,specific genes are activated and closed in a specific order,controlling different stages of osteogenesis differentiation.The early stage of osteogenic differentiation will determine the direction of future cell development.Therefore,to research the miRNA hPDLSCs osteogenesis early differentiation control mechanism,for periodontal defect repair and tissue regeneration,alveolar bone defects due to periodontal disease in clinical treatment and prognosis provide new targets.Based on this,this study through the construction of miR-218-5p expression and overexpression of slow virus,study miR-218-5p in human periodontal ligament cells osteogenetic differentiation and exert the function of the early prediction and verify that miR-218-5p through targeted regulation of collagen,type ?,alpha 1(COL1A1)play the molecular mechanism of inhibitory effect of osteogenesis.The results provide strong evidence for the regulation mechanism of miRNA function in the early stage of human periodontal stem cell differentiation and provide a new way for stem cell therapy.Material and methods1.miR-218-5p expression in hPDLSC osteogenic differentiationIn this experiment,the premolars or third molars were harvested,and the cells were isolated and purified by modified enzymatic tissue block method.The cultured cells were isolated and purified by limiting dilution method.CCK8 method to detect cell proliferation,calculate data and draw cell growth curve.The third generation of human PDLSCs was taken for detection of surface molecular markers by flow cytometry.Human PDLSCs were inoculated into petri dishes by fold dilution method to observe the formation of monoclonal cells and calculate the rate of cell colony formation.The cultured human PDLSCs were stained for 7 days with alkaline phosphatase.After osteogenic and adipogenic induction for 14 days,the cells were stained with Alizarin Red S and Oil Red O,respectively,and the differentiation ability was observed under an inverted microscope.The cultured hPDLSCs were induced to osteogenesis and total RNA was extracted.The expression of miR-218-5p on the 7th day after osteogenic induction was detected by qRT-PCR.2.The function of miR-218-5p during osteogenic differentiation of hPDLSCs.2To construct miR-218-5p overexpression and inhibit the lentivirus expression,hPDLSCs were transfected by setting different multiplicity of infection,and the best multiplicity of infection was screened.According to the experimental design,hPDLSCs were transfected with the optimal multiplicity of infection,and the miR-218-5p lentivirus was stably overexpressed or inhibited in hPDLSCs by qRT-PCR.The total RNA and protein of hPDLSCs were collected after culture for 3,7 and 14 days.The expression of osteogenesis-related genes was detected by qRT-PCR and Western Blot.At the same time,the cells in each group were examined by alizarin red staining for mineralization Nodule formation.3,miR-218-5p inhibits early osteogenic differentiation of hPDLSCs through targeted targeting of COL1A1The target genes of miR-218-5p were predicted using 9 databases,and the mRNA of the target location of the predicted results was analyzed,and COL1A1 was selected to verify the changes of gene and protein level.miR-218-5p,COL1A1 mutant and wild-type vector plasmid were constructed,and the binding sites were detected by biluciferase report.4.Statistical analysis.The data obtained in this study were analyzed by SPSS 19.0 statistical software to x ± s,two independent samples t-test for comparison between the two groups,P<0.05 for the difference was statistically significant.Results1.miR-218-5p expression in hPDLSC osteogenic differentiationFlow cytometry that hPDLSCs were negative for hematopoietic stem cell surface marker and positive for mesenchymal stem cell surface markers,indicating that the cells were mesenchymal origin.The cultured hPDLSCs were induced to adipogenesis and osteogenesis,showing that a large number of lipid droplets formed in the cells and lots of mineralized nodules were observed in the cells.2.The function of miR-218-5p during osteogenic differentiation of hPDLSCs.2Compared with control cells,almost no mineralized nodules were found in the cells transfected with miR-218-5p lentivirus,and the expression of osteogenic related mRNA and protein were significantly decreased.However,miR-218-5p inhibited lentivirus-expressing cells significantly increased osteogenic capacity,osteoblast-related mRNA and protein levels were significantly increased.3.miR-218-5p inhibits hPDLSC osteogenic differentiation through targeting of COL1A1Take the intersection of a database to predict target genes results,through the literature,the final selection COL1A1 as target genes of miR-218-5p for luciferase report verification,the results showed that miR-218-5p with COL1A1 prediction in 3'UTR targeted point,the combination of miR-218-5p may by regulating target genes COL1A1,involved in the differentiation of hPDLSCs osteogenesis process.The qRT-PCR detection and blank control group cell amount of COL1A1 expression peaked in seven days,after 14 days and 21 days decreased,while the expression of miR-218-5p amount in osteogenesis minimum 7 days after induction,in 14 days and 21 days up to a certain extent,osteogenesis related mRNA and protein expression were significantly decreased;Transfection with miR-218-5p inhibits the proliferation of osteoclast,and the expression of osteo-related mRNA and protein levels increased significantly.Conclusions1.The experiment is successful isolation,identification and cultured people the periodontal ligament cells in vitro,and verify the periodontal ligament stem cells in vitro have clone formation ability,through differentiation has confirmed its ability on differentiation of osteogenesis and adipogenesis.2.The experiment confirmed that miR-218-5p in the osteogenetic differentiation of hPDLSCs have important control effect,the inhibiting function of the differentiation of periodontal ligament stem cells osteogenesis.3.Bioinformatic analysis predicted and verified COL1A1 as the target gene of miR-218-5p,which confirmed that miR-218-5p disturbed hPDLSCs osteogenesis combining with COL1A1 targeted early differentiation,inhibit the expression of COL1A1,develop the function of inhibition of osteogenesis,and predicted that miR-218-5p and COL1A1 exist in 3'UTR end binding sites in a row. |
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