LncRNA H19 Promotes The Committed Differentiation Of Stem Cells From Apical Papilla Via MiR-141/SPAG9 Pathway

Objective:Long noncoding RNAs(lnc RNAs)exert significant roles at transcriptional and post-transcriptional levels.As one of the most conserved noncoding transcripts in mammalian development,lnc RNA-H19 was confirmed to be involved in the differentiation of myofibroblast,keratinocyte and the regeneration of the intestine epithelium.Stem cells from apical papilla(SCAPs)are a major kind of neural crest derived mesenchymal stem cells(MSCs),which are essential for the developing alveolar bone,tooth root and dental pulp-dentin complex.The aim of this thesis is to elucidate the effects of lnc RNAH19 in the proliferative behaviors and osteo/odontogenesis of SCAPs,as well as its specific mechanism.Methods:SCAPs were isolated,cultured and purified according to the reference(Sonoyama,PLOS ONE 2006),FCM surface molecules detection was conducted to characterize the SCAPs.Lentivirus infection and mi RNA mimics/inhibitors transfection were performed to over-express/down-regulate lnc RNA-H19 and mi R-141,respectively.CCK-8 detection,Ed U assay and flow cytometry analysis were used to assess the proliferation ability of SCAPs.Western blot,q RT-PCR,immunofluorescence staining,alizarin red S/ALP staining,Ca²⁺and ALP activity quantification assays were conducted to evaluate the osteo/odontogenic differentiation of SCAPs.Micro-CT analysis,H&E staining,Masson's trichrome and immunohistochemical staining experiments were performed to evaluate the effects of lnc RNA-H19 on the osteogenic differentiation of SCAPs in vivo.Moreover,dual-luciferase reporter assay and rescue assays were performed to verify the regulatory mechanism of lnc RNA-H19,that is:regulate the osteo/odontogenic differentiation of SCAPs by mi R-141/SPAG9 axis.Results:FCM analysis results indicated that the SCAPs showed positive expressions of mesenchymal stem cell markers(CD29,CD90 and CD105),but hematopoietic makers(CD34 and CD45)were negatively expressed.CCK-8,Ed U and Flow cytometry results suggested that lnc RNA-H19 could not affect SCAPs proliferation.Western blot,q RT-PCR,ALP,Alizarin red staining and quantification demonstrated that H19 promoted the osteo/odontogenic differentiation of SCAPs in vitro.While opposite results were observed when lnc RNA-H19 down-regulated.Micro-CT,H&E and Masson staining showed lnc RNA-H19 could promote osteogenesis in vivo.On the other hand,mi R-141 inhibited the osteo/odontogenic differentiation of SCAPs.Luciferase report assay validated the binding condition between mi R-141 and H19.Moreover,mi R-141 down-regulated SPAG9 expression in SCAPs.Furthermore,rescue assay indicated that lnc RNA-H19 could sponge mi R-141,led to the up-regulation of SPAG9 expression,activating P38 and JNK pathways and promoting the committed differentiation of SCAPs.Conclusion:The osteo/odontogenesis of SCAPs was upregulated by overexpression of H19 via mi R-141/SPAG9 pathway...