miRNAs are important regulators of signaling pathways during morphogenesis and organogenesis, including tooth development 
. The importance of Dicer and miRNAs during tooth development has been demonstrated, but there has been relatively little progress in the identification and characterization of the roles of specific miRNAs in tooth development.
The miRNA microarray analysis from early and late bell stage of human tooth germs yielded numerous differentially expressed miRNA transcripts. Among the differentially expressed miRNAs, miR-34a was chosen for validation by qPCR and was indeed differentially expressed in the early/late bell stages. Interestingly, we found that many target genes were transcription factors that participated in organ development and were associated with most of the tooth development signaling pathways.
miRNAs function as fine regulators during biological events by posttranscriptional mechanisms 
. In this study, when cells were transfected with specific miRNA oligonucleotides, both the mRNA levels and the protein levels of predicted target genes were shifted. Interestingly, the fold changes of some target genes were not concordant at mRNA (
) and protein levels ( NOTCH1). This may be because miRNAs either promote degradation of target mRNA or inhibit translation posttranscriptionally.
Target prediction tools revealed that LEF1
(WNT pathway), FGF2
(FGF pathway), BMP7
(TGF-beta pathway), NOTCH 1
(NOTCH pathway) and GLI2
(SHH pathway) were predicted target genes of miR-34a, and all of them are members of crucial signaling pathways involved in tooth development. Our findings revealed that at both the mRNA level and protein level both BMP7 and NOTCH1 in cells were affected after oligonucleotide transfection. This result supported the prediction that these genes can be recognized at their 3′UTRs and targeted by miR-34a. It has been well documented that activation of NOTCH signaling can inhibit cell differentiation, while suppression of the pathway leads to cell differentiation 
. We propose () that by targeting NOTCH1
, miR-34a suppresses the NOTCH signals in developing human dental mesenchyme, resulting in cell differentiation and up-regulation of DSPP in human dental papilla cells. Meanwhile, miR-34a can target BMP7
, which is a member of the TGF-beta signaling pathway. Involvement of BMP7 in the developmental events was suggested as early as 1995 that BMP7 deficient mice
resulted in skeletal defects restricted to the rib cage, the skull and the hindlimbs 
Other investigations indicated that BMP7
was expressed in dental papilla cells which will further differentiate into odontoblasts 
. Tooth phenotypes were exhibited in Bmp7
-conditional knockout mice generated by Zouvelou. Conditionally knockout of Bmp7
in dental mesenchyme resulted in missing maxillary incisors and deformed/hypoplastic mandibular incisors 
. In the present study, by suppressing the expression of BMP7, miR-34a down-regulates the expression of ALP. Previous study pointed out that ALP
in odontoblasts decreased at late odontogenic developmental stage.
NOTCH signaling and TGF-beta signaling pathway are well-known to have crosstalk with each other in organogenesis through a spatial and temporal pattern 
. In our model, the overall effect is that miR-34a regulates the cytodifferentiation of human dental papilla cells by down-regulating ALP and up-regulating DSPP.
microRNA-34a regulates cytodifferentiation of human dental papilla cells via NOTCH and TGF-beta signaling pathways.
Since there were no changes in either mRNA level and protein level of miR-34a predicted target genes LEF1, FGF2 and GLI2, our data indicate that miR-34a does not indiscriminately affect all the pathways involved in tooth development. This further supports our hypothesis that miR-34a specifically targets NOTCH and TGF-beta signaling pathways and regulates dental cell differentiation in developing tooth germ.
In conclusion, our analyses utilizing microarray technology, qPCR, western blotting, immunofluorescence, and target prediction tools have indicated that miRNA-34a may play an important role in dental papilla cells differentiation during human tooth development by targeting several signaling pathway.