Sp1 is a competitive endogenous RNA of Klf4 during odontoblast differentiation

Our previous studies have demonstrated that KLF4 is a critical transcription factor that promotes the odontoblastic differentiation of dental papilla cells. Klf4 mRNA was found to be regulated by multiple microRNAs (miRNAs). Competitive endogenous RNAs (ceRNAs) are a group of transcripts post-transcriptionally regulating each other by competing for their common miRNAs. However, the regulation of Klf4 by ceRNAs in odontoblastic differentiation remains unknown. In this study, we predicted a group of potential Klf4 ceRNAs with bioinformatics approach, and examined the expression of Klf4 and five interested potential ceRNAs including Sp1 using real-time PCR during odontoblastic differentiation of mDPC6T. The expression levels of both Sp1 and Klf4 were significantly upregulated during this process. In situ hybridization verified that Sp1 was co-expressed with Klf4 in the differentiating and the mature odontoblasts in vivo. Knockdown of Sp1 using siRNA resulted in a significant reduction of Klf4 and vice visa. This interaction was further confirmed to be miRNA dependent. Common miRNAs of Klf4 and Sp1 were predicted, among which miR-7a, miR-29b and miR-135a were able to downregulate both Klf4 and Sp1 expression after their separate overexpression in the mDPC6T cells. Dual luciferase assays showed that these miRNAs separately regulated the 3'UTRs of both Klf4 and Sp1, and the down-regulation of Klf4 3 'UTR by Sp1 siRNA was abolished when these three miRNAs' binding sites were mutated in the Klf4 3 'UTR. Therefore, our results indicate that Sp1 functions as a ceRNA of Klf4 during odontoblastic differentiation through competing for miR-7a, miR-29b and miR-135a.