| BACKGROUNDThe periodontal ligament stem cells with multi-directional differentiation potential and self-renewal ability,which can be induced to differentiate into bone tissue,cartilage tissue and adipose tissue under specific inducing conditions.And it is ideal for periodontal tissue regeneration which can be used for the treatment of periodontitis and other oral diseases.In recent years,two different non-coding RNA,lncRNA-PCAT1 and miR-106a-5p,which in the regulation of cell proliferation,migration,and stem cell differentiation have been done a lot of research.However,in the process of periodontal ligament stem cells into osteogenic differentiation,whether lncRNA-PCAT1 and miR-106a-5p can produce an interaction,and then regulate the osteogenic differentiation of periodontal ligament stem cells,and what is the molecular mechanism of its regulation,there is no correlation reported in the literature.OBJECTIVEExplore the molecular mechanism of osteogenic differentiation of periodontal ligament stem cells through endogenous competition between PCATl and miR-106a-5p,and provide a scientific basis for the repair of periodontal coloboma.METHODS1.The effect of IncRNA-PCAT1 on osteogenic differentiation of periodontal ligament stem cells.(1)The PDLSCs was induced for different time by osteoinductive medium,and then the expression of IncRNA-PCATl and osteoclastic differentiation-related genes RUNX2,OSX and ALP were detected by qRT-PCR.(2)The IncRNA-PCATl with overexpression lentiviral vector and interference plasmid were transfected into PDLSCs,respectively,and then induced for different time by osteoinductive medium.Whereafter,the expression level of osteoclastic differentiation-related genes RUNX2,OSX and ALP in PDLSCs which be transfected by different transfections,were detected by qRT-PCR and Western Blot.Next,the ALP activity of PDLSCs was detected by ALP kit,and the ability of osteogenic differentiation of PDLSCs was measured by ALP staining and ARS staining.2.The molecular mechanism of endogenous competition between lncRNA-PCATl with miR-106a-5p on osteogenic differentiation of periodontal ligament stem cells.(1)The bioinformatics and online database analysis were used to predict whether IncRNA-PCATl interacts with miR-106a-5p.(2)The binding sites between IncRNA-PCATl and miR-106a-5p were verified by luciferase reporter assay and AG02-RIP assay.(3)The IncRNA-PCATl with overexpression lenti viral vector and interference plasmid were transfected into PDLSCs,respectively,and then induced for different time by osteoinductive medium.And the expression level of miR-106a-5p in PDLSCs which be transfected by different transfections,were measured by qRT-PCR.(4)The miR-106a-5p mimics and inhibitor plasmid were transfected into PDLSCs,respectively,and then induced for different time by osteoinductive medium.And the expression of osteoclastic differentiation-related genes RUNX2,OSX and ALP in PDLSCs which be transfected by different transfections,were measured by qRT-PCR.(5)The IncRNA-PCATl and miR-106a-5p were co-transfected into PDLSCs,and then induced for different time by osteoinductive medium.Whereafter,the expression level of osteoclastic differentiation-related genes RUNX2,OSX and ALP in PDLSCs which be transfected by different transfections,were detected by qRT-PCR and Western Blot.Next,the ALP activity of PDLSCs was detected by ALP kit,and the ability of osteogenic differentiation of PDLSCs was measured by ALP staining and ARS staining.3.The molecular mechanism of osteogenic differentiation of periodontal ligament stem cells through IncRNA-PCAT1/miR-106a-5p/BMP-2/SMAD-4 signal pathway.(1)The bioinformatics and online database analysis were used to predict which target genes can be regulated by miR-106a-5p.(2)The targeted regulation between miR-106a-5p and target gene SMAD4/BMP-2 were verified by luciferase reporter assay.(3)The miR-106a-5p mimics and inhibitor plasmid were transfected into PDLSCs,respectively,and then induced for different time by osteoinductive medium.And the expression of SMAD4,BMP-2 in PDLSCs which be transfected by different transfections.(4)The IncRNA-PCAT1 and miR-106a-5p were co-transfected into PDLSCs,and then induced for different time by osteoinductive medium.And the expression of SMAD4,BMP-2 in PDLSCs which be transfected by different transfections,were detected by qRT-PCR and Western Blot.(5)The SMAD4 and BMP-2 with small interfering RNA were transfected into PDLSCs,respectively,and then induced for different time by osteoinductive medium.And the expression level of total-SMAD5,p-SMAD5 and RUNX2 in PDLSCs which be transfected by different transfections,were detected by qRT-PCR and Western Blot.Next,the ALP activity of PDLSCs was detected by ALP kit,and the ability of osteogenic differentiation of PDLSCs was measured by ALP staining and ARS staining.RESULTS1.The effect of IncRNA-PCATl on osteogenic differentiation of periodontal ligament stem cells.(1)The expression level of lncRNA-PCATI gradually increased during osteogenic differentiation of PDLSCs,and the correlation between lncRNA-PCATl and osteoclastic differentiation-related genes RUNX2,OSX and ALP in osteogenic differentiation were both positive correlation.(2)The results showed that the expression level of osteoclastic differentiation-related genes RUNX2,OSX and ALP in PDLSCs which transfected with LV-lncRNA-PCAT1 were significantly up-reguIated(P<0.05).However,after transfecting with sh-lncRNA-PCAT1,the expression level of osteoclastic differentiation-related genes RUNX2,OSX and ALP in PDLSCs were significantly down-regulated(P<0.05),respectively.After transfection of LV-lncRNA-PCATl,the ALP activity of PDLSCs was significantly enhanced.By ALP staining and ARS staining,it was found that the formation of orange-red crystals and black particles(osteoblast markers)in PDLSCs which transfected with LV-1ncRNA-PCATl,significantly increased,while PDLSCs was transfected with sh-lncRNA-PCATl,the osteoblast markers significantly reduced.2.The molecular mechanism of endogenous competition between lncRNA-PCAT1 with miR-106a-5p on osteogenic differentiation of periodontal ligament stem cells.(1)The analysis result of Bioinformatics and online database predicted that IncRNA-PCATl could bind to the binding site of miR-106a-5p,leading to direct interaction.(2)The result of Luciferase reporter assay and AG02-RIP assay demonstrated that IncRNA-PCAT1 could bind to the binding site of miR-106a-5p.(3)The results showed that the expression level of miR-106a-5p in PDLSCs which transfected with LV-IncRNA-PCAT1,was significantly down-regulated,But,after transfecting with sh-IncRNA-PCAT1-1 and sh-lncRNA-PCAT1-2,the expression level of miR-106a-5p in PDLSCs was significantly up-regulated.(4)After transfecting with miR-106a-5p mimics,the expression level of osteoclastic differentiation-related genes RUNX2,OSX and ALP in PDLSCs were significantly reduced,respectively.Whereas,the expression level of osteoclastic differentiation-related genes RUNX2,OSX and ALP in PDLSCs which transfected with miR-106a-5p inhibitor,were significantly up-regulated.(5)The result showed that the expression level of osteoclastic differentiation-related genes included RUNX2,OSX and ALP in PDLSCs which co-transfected with NC and miR-106a-5p,were significantly down-regulated,respectively.And co-transfected with miR-NC and IncRNA-PCATl,the expression level of osteoclastic differentiation-related genes RUNX2,OSX and ALP in PDLSCs were significantly increased,respectively.Moreover,after co-transfected with miR-106a-5p and IncRNA-PCATl,the expression level of osteoclastic differentiation-related genes RUNX2,OSX and ALP in PDLSCs were increased,respectively,but significantly lower than the co-transfected with IncRNA-PCAT1 and miR-NC.In addition,the results of ALP activity,ALP staining,ARS staining proved the previous results.3.The molecular mechanism of osteogenic differentiation of periodontal ligament stem cells through lncRNA-PCATl/miR-106a-5p/BMP-2/SMAD-4 signal pathway.(1)The analysis result of Bioinformatics and online database predicted that miR-106a-5p can target regulate the expression of BMP-2 and SMAD4.(2)The result of luciferase reporter assay proved that miR-106a-5p can target regulate the downstream genes included SMAD4 and BMP-2.(3)After transfecting with miR-106a-5p mimics,the expression level of downstream genes SMAD4/BMP-2 in PDLSCs were significantly reduced(P<0.05),respectively.Whereas,the expression level of downstream genes SMAD4/BMP-2 in PDLSCs which transfected with miR-106a-5p inhibitor,were significantly up-regulated(P<0.05).By correlation analysis,it is showed that there was a positive correlation between lncRNA-PCATl and target genes SMAD4/BMP-2 in the process of osteogenic differentiation of PDLSCs,while miRNA-106 and SMAD4/BMP-2 were negative related.(4)The result showed that the expression level of downstream genes SMAD4/BMP-2 in PDLSCs which co-transfected with NC and miR-106a-5p,were significantly reduced(P<0.01).And co-transfected with miR-NC and lncRNA-PCATl,the expression level of downstream genes SMAD4/BMP-2 in PDLSCs were significantly increased(P<0.01).Moreover,after co-transfected with miR-106a-5p and IncRNA-PCATl,the expression level of downstream genes SMAD4/BMP-2 in PDLSCs were increased which than that of the negative co-transfection control group,but significantly lower than the co-transfected with IncRNA-PCAT1 and miR-NC.(5)The results showed that the expression of RUNX2,a key osteogenic differentiation protein,was significantly down-regulated after the small interfecting RNA successfully suppresserd the expression of SMAD4 and BMP-2 in PDLSCs.Additionally,the phosphorylation of SMAD5 also was significantly inhibited.Simultaneously,the results of ALP staining and ARS staining showed that the ability of osteogenic differentiation of PDLSCs was significantly inhibited,after interfering with the expression of SMAD4 and BMP-2.CONCLUSIONWe can successfully verify the hypothesis proposed in this issue by a series of experiments.The IncRNA-PCATl can competitively reverse the inhibitory effect of miR-106a-5p on BMP-2 and SMAD4,and promote the expression of BMP-2 and SMAD4 during osteogenic differentiation of PDLSCs,and further promote the expression of RUNX2,OSX and ALP,and ultimately promote osteogenic differentiation of PDLSCs. |
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