Effect And Mechanism Of LncRNA LOC101928855 On Osteogenic Differentiation Of Dental Pulp Stem Cells In Inflammatory Environment

Objective: dental pulp stem cells existing in human normal dental pulp tissue have been used as seed cells for various tissue regeneration in tissue engineering.Some studies have shown that stem cells also exist in dental pulp tissue in inflammatory environment,but the osteogenic differentiation ability of dental pulp stem cells in such inflammatory environment is weakened.If it can be improved,a large number of dental pulp tissues that need to be removed due to inflammation can be turned into treasure for the benefit of mankind.Long chain noncoding RNA(lnc RNA)is involved in many physiological and pathological activities such as embryonic development,inflammation and tumor.It can also participate in the regulation of stem cell differentiation.In this study,lncrna LOC101928855(raptor antisense lnc RNA)differentially expressed in odontogenic differentiation of dental pulp stem cells was screened through GEO database.Meanwhile,raptor protein,an important component of m TORC1 complex,a key target of autophagy regulation,has been proved to affect the osteogenic differentiation ability of stem cells.Therefore,this study will further explore the mechanism of LOC101928855 affecting the osteogenic differentiation ability of inflammatory human dental pulp stem cells(h DPSCs)through Raptor,and provide new ideas and basic theoretical basis for improving the osteogenic differentiation ability of inflammatory dental pulp stem cells and expanding the source of clinical dental pulp stem cells.Methods: 1.Osteogenic ability of human Dental pulp stem cells(h DPSCs)and its ability of expression of LOC101928855 under the inflammatory environment.h DPSCs were obtained from human dental pulp tissue and stimulated with appropriate concentration of LPS to simulate the inflammatory environment.The expressions of LOC101928855,Raptor and osteogenic related genes and proteins in four groups of h DPSCs(control group,osteogenic differentiation medium(OS)group,LPS treatment(LPS)group and LPS treatment osteogenic differentiation medium(LPS+ OS)group)were compared.The localization of LOC101928855 cells was determined by fluorescence in situ hybridization.2.Study the phenotype and mechanism of LOC101928855 regulating the osteogenic ability of inflammatory h DPSCs at the cell level.The overexpression and knockdown virus of LOC101928855 were constructed to interfere with the expression level of LOC10192885 in h DPSCs under normal and inflammatory conditions respectively,and the inflammatory indexes were detected by ELISA,The autophagy index LC3 was detected by immunofluorescence,The level of autophagosome was observed under electron microscope.At 7 and 14 days,the expression of genes and proteins related to osteogenesis and autophagy were detected by Western blot and RT-q PCR,alizarin red staining and ALP.3.Combined with NCBI and UCSC data analysis,it is found that Raptor has three variants(functional V1 and V3,non functional V2).It is speculated that LOC101928855 may regulate stem cell osteogenic differentiation by participating in Raptor variable shear.The RNA binding protein of LOC101928855 is predicted through the RNA binding protein prediction website,and the expression of RNA binding protein in inflammatory h DPSCs is detected by RT-q PCR,The binding proteins consistent with the expression trend of target lnc RNA were screened;RNA-pull down,RIP and Western blot experiments showed that RNA binding protein could produce specific binding with LOC101928855;Further,the impact of LOC10192885 regulating RNA-binding protein on Raptor variant expression was verified by RT-q PCR assay,gene overexpression and interference techniques.4.In vivo experiments show that LOC101928855 can improve the osteogenic ability of h DPSCs in inflammatory environment.Hydroxyapatite(HA)scaffolds combined with different treatments of h DPSCs cells [HA,HA + h DPSCs,HA + h DPSCs(LPS + loc101928855 sh RNA),HA+ h DPSCs(LPS)] were implanted subcutaneously into the back of four groups of nude mice.After 8 weeks,the materials were taken for HE staining and alkaline phosphatase activity analysis to observe the new bone formation.Results: 1.LPS in vitro 1 μg/m L stimulated h DPSCs to simulate inflammatory environment.h DPSCs had high expression of LOC101928855,decreased genes and proteins of Raptor and osteogenic related protein factors,and decreased osteogenic ability of cells.In immunofluorescence,LOC101928855 was mainly located in the nucleus.2.The overexpressing of LOC101928855 in h DPSCs resulted in decreased h DPSCs’ activity,the lower expression of Raptor and osteogenic related factors,the higher level of autophagy factors’ genes and proteins’ expression,while that did not significantly change h DPSCs’ releasing of inflammatory factors,but decreased its’ osteogenic differentiation potential;knockdown of LOC101928855 in inflammatory environment significantly increased the activity of h DPSCs,decreased its’ release of inflammatory factors,significantly increased the gene and protein expression of Raptor and osteogenic related factors,decreased the expression of autophagy genes and proteins,significantly reduced the phenomenon of autophagy under electron microscope,and increased the osteogenic differentiation potential of h DPSCs.3.RNA-pull down,RIP and Western blot experiments demonstrated that LOC101928855 could specifically bind to SFRS1 protein,enhancing the stability of SFRS1 protein in h DPSCs.SFRS1 was significantly increased in LOC101928855 overexpressed h DPSCs,while functional Raptor variants(V1 and V3)were significantly decreased.When LOC101928855 of h DPSCs were knocked down,SFRS1 was significantly decreased,while Raptor(V1and V3)was significantly increased.Overexpression of SFRS1 inhibited the expression of functional Raptor variants(V1 and V3),while knockdown of SFRS1 promoted the expression of functional Raptor variants(V1 and V3).Complementary experiments of LOC101928855 and SFRS1 confirmed that LOC101928855 participated in the shearing of Raptor variants by regulating SFRS1.4.Experiments in vivo showed that compared with non-knockdown group(HA+h DPSCs+LPS group),alkaline phosphatase activity and new bone mass were significantly increased in LOC101928855-knockdown h DPSCs under inflammatory environment(LPS+LOC101928855 sh RNA group),indicating that osteogenic differentiation ability was significantly improved in inflammatory knockdown groupConclusions: 1.As for h DPSCs under inflammatory environment,the osteogenic ability was decreased,LOC101928855 was overexpressed,and osteogenic factors and Raptor and its proteins were down-expressed.LOC101928855 is primarily located in the nucleus.2.With interference of lentivirus,the overexpression of LOC101928855 in normal h DPSCs decreased cell activity,the level of Raptor and osteogenic related proteins,and increased autophagy activity.Knockdown of LOC101928855 in h DPSCs under inflammatory environment increased the expression of Raptor and osteogenic related proteins,reduced autophagy activity,and reduced the release of inflammatory factors.3.LOC101928855,as an antisense lnc RNA of Raptor,specifically binds to SFRS1 protein to participate in variable shearing of Raptor pre-m RNA,reduced the level of functional Raptor(V1 and V3),increased autophagy activity,and reduced the osteogenic differentiation potential of pulp stem cells.4.Under inflammatory environment,the bone formation ability of the LOC101928855-knockdown h DPSCs combined with hydroxyapatite scaffold was significantly higher than that of the non-knockdown hDPSCs.