Quaking Promotes The Odontoblastic Differentiation Of Human Dental Pulp Stem Cells

1.QKI promotes the odontoblastic differentiation of hDPSCs Objectives:Odontoblastic differentiation of human dental pulp stem cells(hDPSCs)is essential for the formation of reparative dentin after dental caries or injury.Quaking(QKI)is an RNA-binding protein which functions in cell metabolism by bindng to tareget genes.Multiple roles have been reported in physiological or pathological processes,but its role in the odontoblastic differentiation of hDPSCs remains unknown.Our study aims to investigate the expression pattern and function of QKI during the odontoblastic differentiation of hDPSCs.Methods:hDPSCs were isolated and identified from human healthy molars.hDPSCs were induced by odontoblastic induction medium in vitro.QKI expression levels were assessed by Immunohistochemistry in vivo and by qRT-RCR,Western blot,Immunofluorescence staining during the odontoblastic differentiation of hDPSCs in vitro.Overexpression and knockdown of QKI were performed to investigate its regulatory function.qRT-PCR,Western blot were performed to detect the expression of the odontoblastic differentiation related genes.ALPase acitivity and mineralized nodules were also detected.Results:We found QKI was up-regulated during the odontoblast differentiation in mice in vivo and odontoblastic differentiation of hDPSCs in vitro.Overexpression or knockdown of QKI in hDPSCs led to the increase or decrease of odontoblast marker genes' expressions,indicating its positive role in odontoblastic differentiation.Conclusions:RNA-binding protein QKI was up-regulated during odontoblastic differentiation in vivo and in vitro.QKI was able to promote the odontoblastic differentiation of hDPSCs.2.QKI functions as a ceRNA of KLF4 to promote the odontoblastic differentiation of hDPSCsObjectives:Odontoblast differentiation is an identical and complex process,which is regulated by multiples of molecular mechanisms,including growth factors,transcriptional factors,microRNAs.Different signal pathways relate with each other to form a network and regulate odontoblast differentiation in different expression levels.CeRNA network illuminates an emerging area of investigation in another post-transcriptional regulatory way for developmental research or disease treatment.In this study,we want to validate the network of ceRNA by QKI and KLF4 and its function in the odontoblastic differentiation of hDPSCs.Methods:qRT-PCR and Western blot were used to detect the expression patterns of QKI and KLF4 during the odontoblastic differentiation of hDPSCs.SiRNAs of QKI and KLF4 were transfected into hDPSCs respectively to confirm their mutual expression relationships.Analysis of the microRNA binding sites within the 3'-UTR of KLF4 and QKI revealed that QKI shared common microRNAs with KLF4.MicroRNAs were overexpressed in hDPSCs.Western blot and dual luciferase activity assays were used to confirm whether these microRNAs could function.Dual luciferase astivtity assays were performed to detect whether their relationships were microRNA independent.Results:QKI and KLF4 had mutual regulatory relationships during the odontoblastic differentiation of hDPSCs.Mir-124-3p,mir-128-3p,mir-145-5p and mir-429 obviously reduced the protein levels of both QKI and KLF4 compared with the scrambled group.And they significantly repressed the luciferase activities of KLF4 3'-UTR and QKI 3'-UTR,whereas the mutant constructs had no effect.And we found that knockdown of QKI significantly reduced KLF4 3'-UTR luciferase activities.However,the reduction was abolished when the MREs was mutated.Conclusions:QKI mRNA is a ceRNA of KLF4 mRNA and competes for their common microRNAs including mir-124-3p,mir-128-3p,mir-145-5p,and mir-429,by which QKI can upregulate KLF4 during the odontoblastic differentiation of hDPSCs.3.QKI binds to DSPP mRNA to promote the odontoblastic differentiation of hDPSCsObjectives:As an RNA-binding protein,QKI can bind to its target genes(pre-mRNA or mature mRNA)through the QKI response elements,functioning as an activator or a suppressor in multiple manners,for example,alternative splicing of pre-mRNA,mRNA stabilization,etc.Cell differentiation and human diseases are both involved in this mechanism.In this study,we asked whether QKI,as an RNA-binding protein,were able to promote the odontoblastic differentiation of hDPSCs through binding to any odontoblast-related mRNA,besides its role as a ceRNA of KLF4.Methods:We used the bioinformatic approach to perform the prediction of Quaking response elements.We employed RIP assay and RNAs eluted from the immunoprecipitation were detected by qRT-PCR.hDPSCs were transfected with QKI siRNA(siQKI),or scramble siRNA(scramble)and were cultured with ActD for 12 hr.RNAs were collected at different time and qRT-PCR was performed for the detection of the half-life time of target RNA.Results:QKI response element was predicted on DSPP mRNA coding sequence by bioinformatic analysis.RIP assays showed that QKI protein was able to recruit more DSPP mRNA than the IgG group.Half-life time assay showed that less DSPP mRNA remained in the siQKl group than in the control group.Conclusions:QKI protein translated from QKI mRNA can promote the odontoblastic differentiation of hDPSCs by binding to and stabilizing DSPP mRNA.Increased accumulation of DSPP mRNAs results in enhanced DSP proteins.