The Effect And Mechanism Of Long Noncoding RNA NR＿045147 On The Function Of HPDLSCs

Objective Periodontitis is the most common inflammatory oral disease in humans.In severe cases,loose and lost teeth may occur and affected soft tissue profile.At present,traditional periodontitis therapies such as periodontal basic treatment and periodontal surgery cannot completely regenerate periodontal tissue.Human Periodontal Ligament Stem Cell(hPDLSCs)is a kind of odontogenic mesenchymal stem cells,which has multidirectional differentiation potential to regenerate defected periodontal tissue.Long non-coding RNA(LncRNA),as a key regulatory gene,plays an important role in the transcription and translation of coding RNA.Early gene chip analysis of our group showed that the expression of LncRNA NR＿045147 was significantly decreased after osteogenesis induction.However,the role of LncRNA NR＿045147 in mesenchymal stem cells and periodontal tissue regeneration is still unclear.In this study,the biological effect and molecular mechanism of LncRNA NR＿045147 on the functions of hPDLSCs could promote gene therapy in the future.Methods In this study,human PDLSCs were isolated and cultured in vitro by enzyme digestion.Subsequently,Lentiviruses were purchased,transfected,and screened.The effect of osteogenic differentiation was detected by Alkaline phosphatase activity assay,alizarine red staining,and western blot assay,the effect of migration chemotaxis was detected by scratches and Transwell assay,and the effect of mitochondrial metabolism was detected by Seahorse XF96 assay.Finally,the potential mechanism of LncRNA NR＿045147 in hPDLSCs with inflammatory factors was discussed by related pathway inhibitor and RNA pull-down experiment.Results1.After stably transfecting Consh and NR＿045147sh lentivirus into hPDLSCs,the knockdown efficiency reached 70%.Compared with the Consh group,the osteogenic differentiation ability,migration chemotaxis ability and mitochondrial metabolism ability of NR＿045147sh group were enhanced.After stably transfecting Vector and NR＿045147 lentivirus into hPDLSCs,the osteogenic differentiation ability,migration chemotaxis ability and mitochondrial metabolism ability of NR＿045147 cells were decreased in NR＿045147 group compared with the Vector group.2.The expression of NR＿045147 gene in hPDLSCs increased with IL-6and IL-8 at 0,2,4 and 8h after simulated in vitro inflammatory environment.RNA-pulldown probe was used to detect protein binding.Compared with the Consh group,the binding of LncRNA NR＿045147 to ITGB3 BP protein was reduced in the cells of NR＿045147sh experimental group.In contrast,the binding of LncRNA NR＿045147 to ITGB3 BP protein was increased in the cells of LncRNA NR＿045147 experimental group compared with the Vector group.3.The expression of ITGB3 BP was significantly increased after stable transfection of Vector and m RNA NM＿014288 lentivirus into hPDLSCs.Compared with the Vector group,m RNA NM＿014288 group had enhanced osteogenic differentiation ability and migration chemotaxis ability.Conclusion1.LncRNA NR＿045147 inhibits osteogenic differentiation,migration chemotactic and mitochondrial metabolism of periodontal ligmant stem cells.2.TNF-α can promote the expression of LncRNA NR＿045147 in periodontal ligmant stem cells.3.LncRNA NR＿045147 could regulate the expression of ITGB3 BP.4.ITGB3 BP can promote osteogenic differentiation,migration and chemotaxis of periodontal ligmant stem cells.