Sp1 Functions As A CeRNA Of Klf4 To Regulate Odontoblast Differentiation

1. Spl mRNA functions as a Competitive Endogenous RNA of Klf4 to promote Odontoblast DifferentiationObjectives:Competitive endogenous RNAs (ceRNAs) are a group of transcripts post-transcriptionally regulating each other by competing for binding to the common microRNAs (miRNAs). The differentiation of odontoblasts is a complex process regulated by a battery of transcription factors and signaling molecules. Our previous studies have found that Klf4 is a critical transcription factor that promotes the odontoblast differentiation via the up-regulation of Dmpl. In this study, we try to explore the ceRNAs networks for Klf4 during the odontoblast differentiation.Methods:To predict and characterize the Klf4 ceRNA network in the mouse genome, a multifaceted scheme involving bioinformatics analysis and experimental validation were devised. Then, qRT-PCR was used to test the expression pattern of Klf4, along with six candidate ceRNAs during odontoblast differentiation of mDPC6T cells. ISH were performed to detect Spl and Klf4 mRNA expression pattern in PN2 mouse lower incisors. We performed siRNA-mediated gene silencing of Spl or Klf4 in mDPC6T cells to test whether there was a correlation between Spl and Klf4. Western blot ans qRT-PCR were used to test the expression of Spl and Klf4 in protein and mRNA level. Dual luciferase assays were carried out in mDPC6T cells to further detect interaction between Spl and Klf4. Silencing Dicer was used to test whether the interaction of Spl and Klf4 are microRNAs dependent. Overexpression of miR-7a, miR-29b, miR-128, miR-135a, miR-137, miR-200c, miR-363 and miR-375 in mDPC6T cells, western blot was used to test the expression of Spl or Klf4. Dual luciferase assays were performed to confirm the interaction between miR-la, miR-7a, miR-29b, miR-135a with Spl and Klf4. Overexpression of Spl 3’UTR to test the function of Spl mRNA during odontoblast differentiation.Results:We acquired 30 candidate Klf4 ceRNAs. The mRNA expression levels of Spl, Qk, Tnrc6b and Klf4 showed a significant up-regulation, while Nfib, Noval and Bcllla were obviously decreased after induction. Spl and Klf4 exhibit identical expression pattern in the odontoblast layer of PN2 mouse lower incisor. Knockdown of Spl significantly reduced Klf4 mRNA and protein expression. Meanwhile, silencing Klf4 results in an obvious decrease of Spl mRNA and protein expression. Silencing Spl significantly suppressed the Luc-Klf4-3’UTR activity. Meanwhile, silencing Klf4 significantly suppressed the Luc-Sp1-3’UTR activity. And we found that knockdown of Spl significantly reduced KLF4 expression levels. However, the reduction was abolished when Dicer was knockdown. KLF4 expression had no significant decrease after co-transfection of siSpl+siDicer. miR-la, miR-7a, miR-29b and miR-135a obviously reduced the protein levels of both SP1 and KLF4 compared with scrambled group. miR-la, miR-7a, miR-29b and miR-135a significantly repressed Luc-Klf4-3’UTR and Luc-Spl-3’UTR activity, whereas the mutant construct had no effect in mDPC6T cells.Conclusion:Spl functions as a ceRNA of Klf4 through competing for miR-la, miR-7a, miR-29b and miR-135a to promote odontoblast differentiation.2. SP1 promotes odontoblastic differentiation of mouse dental papilla cells through activating Dmpl promoterObjectives:Odontoblasts are a kind of terminally differentiated and polarized cells, which are derived from cranial neural crest. Dental papilla cells (DPCs), precursors of odontoblasts, have the potential to differentiate into odontoblasts. Diverse signaling molecules and transcription factors play key roles in this differentiation process. The transcription factor specificity protein 1 (SP1) is a member of the specificity protein/Kruppel-like factor (SP/KLF) family. SP1 has been found to be involved in the differentiation of osteoblasts, neuronal cells and lens cells. The aim of this study was to investigate the role of SP1 in odontoblastic differentiation of dental papilla cells.Methods:Immunohistochemical assay was used to detect the expression of SP1 in vivo. The dental papilla cell line (mDPC6T) previously established by our group was used to do the in-vitro test. Western blot and immunofluorescence were used to verify the expression of SP1 during the odontoblast differentiation of mDPC6T cells. pcDNA3.1-SP1 plasmid encoding SP1 coding sequence was constructed to overexpress SP1 while SP1 siRNA was used to knock down SP1. Then the biologic effects of SP1 on mDPC6T cells were determined by the evaluation of ALPase activity, the detection of ALP, DSPP, and DMP-1 expression and the formation of mineralized nodules. Dual luciferase activity assays were used to test whether SPl can transactivate DMP1 promoter. Forced expression of Dmpl in the SP1 knock-down mDPC6T cell line was used to test whether DMPI could rescue odontoblast differentiation ability.Results:Immunohistochemistry verified that SP1 was specifically expressed in polarizing and secretory odontoblasts in vivo. qRT-PCR and immunofluorescence revealed that the expression of SP1 was significantly upregulated during odontoblastic differentiation of mDPC6T cells in vitro. Overexpression of SP1 significantly increased odontoblast-related genes including DSPP, DMP1 and ALP and promoted the formation of mineralized nodules. Meanwhile, knock-down of SP1 decreased the expression of these odontoblast-related genes and suppressed the formation of mineralized nodules. Dual luciferase activity assays confirmed that SP1 was able to specifically bind to the Dmpl promoter and transactivate its expression. Forced expression of Dmpl in the SP1 knock-down mDPC6T cell line significantly recovered its odontoblastic differentiation ability.Conclusion:SP1 promotes odontoblastic differentiation of mouse dental papilla cells through activating Dmpl promoter.