Gene Regulation By MicroRNA, BHLH Factors And PcGs During P19 Cell Differentiation

Neuogenesis is the most complicated and rigorous event of the vertebrate development processes.Vertebrate central nervous system contains many different types of nerve cells,neurons and glia cells.It is a very long,complicated,precise process from nerve stem cells,nerve precursor cells,to mature nerve cells.Furthermore,there are a great quantity of cell subgroups and synaptic linkages,that means it is not enough for the regulation of neural development only with the definite protein coding genes.The discovery of miRNA(microRNA) established a new area for the regulation of neural development.With the profound research on miRNAs,people found that regulation contribution exists between the important transcription factor(TF) and miRNAs during many important life processes.Epigenetic events,including DNA methylation and post-transcriptional modifications of the histones control the transcriptional program of each cell by regulating chromatin structure.Objective:To investigate the role of miRNA,TF,PcGs during the neural differentiation, investigate the relation and regulation between miRNA and TF,miRNA and PcGs,TF and PcGs.To try to explain the mechanism of neural differentiation from different level. Methods:We used P19 cells as a in vitro model for neural differentiation,firstly we detected the expression of important TF,PcGs during neural differentiation.Then,we predicted the target genes and transcription factor bind site of neural specific miRNA.We ascertained the target gene of miRNA by using semi-quantitive RT-PCR,western blot and dual luciferase activity assay.Finally,we looked for the relation between TF and miRNA,TF and PcGs by using RNAi,semi-quantitive RT-PCR,western blot analysis.Results:1.After RA treated,the expression of PcGs protein and H3K27me3 protein decreased with the differentiation of P19 cells;2.We predicted that Ezh2(Enhancer of Zeste homolog2) may be the target gene of mmu-miR-124 by software TargetScan;so we detected the mRNA and protein level of Ezh2 in P19 cells after transfection of miR-124 and LNA-miR-124.Finally we found that in the presence of miR-124,Ezh2 mRNA level was not changed,however, the Ezh2 protein level decreased.In contrast,the level of Ezh2 protein increased when miR-124 was knocked down;however,the level of Ezh2 mRNA was unchanged.Next,we constructed a PGL3-promoter plasmid with the 3'UTR region which complemented with the "seed" of miR-124 and the mutation plasmid, co-transfected P19 cells with miR-124 RNA duplex.Thus using dual luciferase reporter assays,we proved that Ezh2 is the target gene of mmu-miR-124,and the target region is in 3'UTR.3.After RA treated,the expression of bHLH(basic Helix-Loop-Helix) factors had a regular change.Among them,Mash-1(Mammalian achaete-scute homologue-1) increased with RA treatment,it reached the peak in 2 days after differentiation,but it decreased after that,Hes-1(Hairy and E(spl) homolog-1) was highly expressed in the undifferentiated P19 cells,and decreased with RA treatment,finally it can scarcely be detected in differentiated P19 cells.4.We predicted the promoter of mmu-miR-124 with the documents published,and predicted that there might be a bind site of Mash-1 upstream of mmu-miR-124.5.We transfected differentiated P19 cells with Mash-1 siRNA,and detected the level of miR-124 with TaqMan MicroRNA Assay,and finally found miR-124 decreased with the degression of Mash-1.Thus,Mash-1 perhaps regulates the expression of mmu-miR-124.6.We detected the mRNA and protein level of Hes-1 in P19 cells after transfection of LNA-miR-124.Finally we found that the level of Hes-1 protein increased when miR-124 was knocked down;however,the level of Hes-1 mRNA was unchanged. However,we could not predicted that relation between Hes-1 and mmu-miR-124 by the software.We blasted the sequence of mmu-miR-124 and 3'UTR of Hes-1,and finally found that they had 50%complementation between them.So the target site might be in the 3'UTR region,but we couldn't eliminate the other situation.Hes-1 might be a target gene of mmu-miR-124.7.We transfected differentiated P19 cells with Ezh2 siRNA,and detected the level of Mash-1,and finally found Mash-1 increased with the degression of Ezh2 and H3K27me3.Thus,Mash-1 perhaps was the target gene of Ezh2.Conclusions:1.mmu-miR-124,Hes-1,Mash-1 are in dynamic balance during the differentiation of P19 cells.Before P19 cells differentiate,Hes-1 perhaps binds with N-box of Mash-1 promoter,or form inactive heterodimers with Mash-1 and inhibits the activity of Mash-1,so the expression fo miR-124 is also inhibited,thus neuronal differentiation is inhibited.After RA treated,P19 cells begin to differentiate,Mash-1 transient expresses,promotes and maintains the expression of miR-124,miR-124 inhibits the expression of Hes-1,thus relieve the inhibition of downstream genes of Mash-1,so miR-124 begins to express.2.mmu-miR-124,PcGs,Mash-1 are in dynamic balance during the differentiation of P19 cells.Before P19 cells differentiates,Ezh2,the catalytic activity element of PRC2,catalysises the K27 of histone H3 as methylation transferring enzyme,and inhibits the transcription of Mash-1,so the expression fo miR-124 is also inhibited, thus neuronal differentiation is inhibited.After RA treated,P19 cells begin to differentiate,Mash-1 transient expresses,promotes and maintains the expression of miR-124,miR-124 targets Ezh2 by post-transcriptional silencing,thus relieve the inhibition of downstream genes Mash-1,so miR-124 begins to express.In a word,we studied the interaction of genes,proteins,and micromoleculars during the neuronal differentiation,and preferred a new network regulation mechanism of miRNA,PcGs,TF.This offered a rationale for deep research for neural development, offered a method for research on the mechanism of great birth defects,provided new treatment targets for curing nervous system diseases.