Research On Target Relationship Between MiR-185 And AKT1 By Dual Luciferase Report Gene Assay

Objective: Our team found that the expression levelof mi R-185 was significant down-regμlation in A549, NCI-H1975, NCI-H1650 and LTEP-a-2 cell lines in previous studies.It is a negative correlationbetween theexpression level of AKT1 protein and theexpression level of mi R-185, and it is also suggesting that mi R-185 can regulate the expression of AKT1, and AKT1 maybe a potential target gene of mi R-185,there is noenough evidence to prove it now.In this study,we research how mi R-185 regulate AKT1 3’- untranslated region(3’-untranslated region,3’-UTR) by dual luciferase reporter assay,to provide the theoretical basis for the direct relationship between mi R-185 and AKT1,further to provide the theoretical basis for the possible mechanism of mi R-185 and AKT1 in lung cancer occurrence and development.Methods:AKT1 m RNA bearing mi R-185 binding sites was predicted by bioinformatics software,design and synthesize AKT1 gene 3’-UTR sequenceand mutation of AKT1 gene 3’-UTR sequence with the chemical synthesis method.Two kinds of target gene fragments were cloned into a pmir GLO vector with dual luciferase report gene,resulting intwo kinds of recombinant vector,which were called the AKT13’-UTR dual luciferase report gene vector(pmir GLO-AKT1) and itsmutant vector(pmir GLO-mut-AKT1).The recombinant vectors were successfully constructed,then these two recombinant plasmid vector and mi R-185 mimics or mi R-185 Negative Control(NC) were transfected into 293 T cell by LipofectamineTM2000,and the dual luciferase assay system was used todetect theirs activity.Thedual luciferase reporter gene vector contains two kinds of luciferase genes,one is a firefly luciferase reporter gene,and the other is a renilla luciferase report gene,which could express in the same cell.Firefly luciferase reporter gene as the main report gene, the target gene fragments was cloned into the multiple cloning sites(MCS),which was located in the downstream of the firefly luciferase report gene.If the level of target gene expression was inhibited, the firefly luciferase transcription process would be blocked;therefore,firefly luciferase protein translationprocess was inhibited, and firefly fluorescence value would fallen,while the expression level of renilla luciferase gene servedas the internal reference was not affected,and meanwhile the firefly luciferase activity/Renilla luciferase activity decreased.Results:The results of PCR electrophoresis and gene sequencing showed that the size and sequence of target genefragments containing 3’-UTR of AKT1 and mutated AKT1 sequence which were amplified by PCRwere consistent with the Gene Bank reported, and inserted into the right direction.Therefore,the two kinds of recombinant vetor which contained the mi R-185 binding site and the mutation site were constructed successfully.The results of dualluciferase assay system showed that the luciferase activity had no significant difference in the mi R-185 group compared with the mi R-185 NC group in the experiments of transfection withmutant plasmid vector(F=2.29, P>0.05);In the experiments of transfection with recombinant plasmid containing the AKT1 3’-UTR,the luciferase activity was apparently inhibited in the mi R-185 group compared with the mi R-185 NC group,and luciferase activity was significantly different(F=80.39, P<0.05).Conclusions:The two kinds of recombinant vetorcontaining the 3’-UTR of AKT1 and mutated AKT1 sequence were constructed successfully; AKT1 may be a potential target gene for mi R-185,whichis combined in AKT1 3’-UTR,and has a direct inhibitory effect on AKT1 atthe post transcriptional level.