Applying Dual-Luciferase Reporter Gene System To Detecting The MiRNA-mRNA Target Interaction Between Ssc-miR-369 And TNF-α Gene

Through the sequences alignmented between human TNF-a gene and sus scrofa TNF-a gene,and through the miR-369 targets seeking and matching useing the seed sequences,2nd-8th bases of miR-369,we predicted that ssc-miR-369 also regulated the TNF-αgene.Finally,we used the Dual-Luciferase(?) Reporter Gene System to identify the result if ssc-miR-369 regulate the expression of sus scrofa TNF-a gene. The results revealed that:(1) TNF-a gene had high similarity among Homo sapiens,Sus scrofa,Bos taurus,mus musculus,Kattus norvegicus and Orytolagus cuniculus.Among theses,Sus scrofa had the highest similarity with Bos taurus,higher with Homo sapiens.(2) Sus scrofa and Homo spaiens TNF-a gene both had an AREs,which were the same length nucleotide sequences(34 nt) in their 3'UTR area,and a high similarity happened between the two AREs. By the base pairing of seed sequences of miRNA-369 2nd-8th base,we found that Homo sapiens and Sus scrofa AREs area both had two target sites with high similarity.(3) The wild reconbination plasmid and mutation reconbination plasmid were gained,it also showed that the wild AREs sequences and the target sites mutated AREs sequences were correctly and successfully ligated to the plasmid vector psiCHECKTM-2,which had the dual-luciferase reporter gene.(4) When transfected the porcine vascular endothelial cell line(PIEC),we found that,only the cell proliferate to 90% confluence,and the final concentration are 50 nM and 150 ng of mimics(miRNA simulacrum) and plasmid vector sepreately, the optimalizing result of transfection would be reached.(5) When compare the luciferase ratioes (Renilla/Firefly) between mutation reconbination plasmid transfected only and wild reconbination plasmid transfected only,the result showed extremely significantly difference,p<0.01,indicating that AREs influences the stable of mRNA. Compare with WT,MT lack three consecutive AUUUA pentamers,it indicated the AUUUA pentamers had a close relation with the stable of mRNA, and we speculated the stable of mRNA maybe have a positive correlation with the continuity of AUUUA pentamers.It also showed that, for the same amount AUUUA pentamers,the better continuity of which,the stronger effect to the stable of mRNA;the worse continuity of which,the weaker effect to the stable of mRNA.(6) When miRNA over-expression(Group C,mimics+WT), p<0.01,the luciferse ratio of which was extremely significantly lower than the control group (control,WT), indicating that miRNA-369 inhibited the expression of renilla luciferse and miRNA-369 down-regulates the expression of TNF-a gene.(7) When miRNA inhibition (Group D,inhibitor+WT),the luciferase ration of which compared with control group, P>0.05, showing insignificantly difference with control;however,when it compared with the group C, P<0.05,showing significantly difference. The result further comfirmed that miRNA-369 down-regulates the expression of TNF-a gene.(8) In the procession of regulating mRNA post-transcription,AREs had some similarity with miRNA. However, when they coexsiting,the regulation affect of which didn't interfere each other,or the effection of each other were little,even the target site of miRNA located in the AREs area.(9) The success of our research suggested that a new way to screen animal function gene was gained,and which could better utilize the success results of human,mouse,or other species to our animal research.Beyond that,maybe this research also provided some usefull reference data for the fundamental researches of ARE(AU-rich elements)and miRNAs(microRNA).