Study On The Regulatory Relationship Between MiR-200a And ATF5,AC3 And TET3

microRNA(miRNA)is a non-coding RNA,contains 18-24 nucleotides,the transcription by binding the target mRNA 3'UTRs regulate target gene expression.The miR-200 family is one of the most closely-watched miRNA family,it occurred during the migration of the tumor cells and epithelial mesenchymal transition(EMT)plays an important role,but also in the olfactory neural development.ATF5 is Activating transcription factor 5,which is a part of transcription factor ATF/CREB.it plays an important role in the differentiation of the nervous system,but also the cell apoptosis.Using bioinformatics analysis,we can see that there is a miR-200 a binding site in the 3' untranslated region of transcription factor ATF5,but it is unknown whether it is a direct target of miR-200 a.AC3(adenylate cyclase 3)gene plays an important role in the process of mouse neural development and ATF5 plays a role as an upstream transcription factor.However,no specific molecular mechanism of action has been reported yet.TET is an abbreviation of ten-eleven translocation and is a dioxygenase in vivo.It has an ability to oxidize 5-methyl cytosine(5-MC)into the serotonin-cytosine(5-HMC),which plays a key role in DNA methylation.So whether it has an effect of methylation on the promoter region of miR-200 family remains to be studied.In this dissertation,Hela cells,3T3 cells,CT26 cells and 293 T cells were used as experimental materials.And we studied from the following three sections:1.Construction of wild-type fluorescent enzyme-containing ATF5 gene 3'UTR full length fluorescent enzyme report plasmid and ATF5 Gene 3'UTR seed sequence(in combination with miR-200a)mutation in the gene plasmid.The mi R-200 a mimic was transfected into HeLa cells with one of the plasmids.After a certain period of time,a reporter gene detection system was used.The result was that miR-200 a mimic significantly decreased the expression of wild type plasmid,but had little effect on the mutation plasmid,illustrates that miR-200 a binds to the 3'UTR of the ATF5 gene.The mRNA and protein expression of ATF5 gene after miR-200 a mimic/inhibitor transfected 3T3-L1 cells and CT26 cells were then detected,and the results showed that after transfection of miR-200 a mimic,the expression of ATF5 gene was silenced,and the expression of ATF5 was increased after miR-200 a inhibitor was transfected,further indicating that miR-200 a regulates the expression of ATF5.2.The ATF5 Cdna was cloned into the eukaryotic expression vector and named ATF5.The empty vector was used as the control and named NC.The eukaryotic expression vector was resistant to the neo drug resistance gene.After transfection of 3T3 cells with the appropriate concentration of G418 for drug screening,and then detect the mRNA and protein expression of ATF5 and AC3 gene.CHIP and the luciferase reporter system were used to detected the interaction of the AC3 and ATF5.It can be seen that after transfection compared with the control group,ATF5 and AC3 gene in m RNA and protein levels are rising trend.The ATF5 is combined with the position of the AC3 's promoter area about 400-1200 bp.3.3T3 cells were used as experimental materials,transfection with synthetic Tet3-siRNA,the expression of TET3 was silenced.Then we detected the methylation of miR-200 a promoter region and non methylation level,The results showed that the methylation level of mir-200 a promoter region increased after silent TET3,and the non methylation was the opposite.Conclusion:1.The ATF5 gene is one of the targets of miR-200 a.2.Up-regulation of ATF5 gene expression promoted the expression of AC3 gene.3.Knockdown of the TET3 gene caused elevated methylation levels in the 200 a promoter region and the non methylation was the opposite.