Research On Target Relationship Between MiR-147a And PDPK1 By Dual Luciferase Reporter Gene Assay System

Background: With the continuous improvement of economic and technological level,ionizing radiation has been completely filled with people's daily life.Micro RNA(mi RNA)has been found to be differentially expressed in different types of radiation damage.It has been found that mi R-147 a is an important differential expression gene after radiation injury,which can promote the apoptosis of normal cells leading to radiation damage sensitivity.PDPK1 mediates the phosphorylation of AKT,PI3K/PDPK1/AKT signaling pathway plays an important role in the protection of radiation damage.After irradiation,mi R-147 a has the opposite relationship with PDPK1.Objective: To study the relationship between mi R-147 a and PDPK1,we can further explore the possible basis of mi R-147 a and PDPK1 gene in the pathogenesis and development of radiation carcinogenesis.Methods: Mi R-147 a was used to predict the binding sequence of PDPK1 gene by bioinformatics software(Target Scan,mi Randa).The target gene fragment of PDPK1 3'-UTR was designed and cloned into GV272 luciferase reporter gene vector.The PDPK1 3'-UTR double luciferase reporter gene wild type vector(GV272-PDPK1 3'-UTR)was constructed by T4 ligase reaction.(GV272-PDPK1 3'-UTR-mut).In addition,the mi R-147 a PCR amplified fragment was ligated with the GV268 luciferase reporter gene vector,and the GV268-mi R-147 a recombinant vector was constructed by exchanging the exchange reaction.The recombinant vector was identified by gene sequencing to demonstrate that the recombinant vector was successfully constructed.The recombinant plasmids were transfected into 293 T cells using the transfection reagent Lipofectamine TM 2000 Reagent,then the activity was detected by double luciferase gene detection system,and the relationship between them was verified by western blot and fluorescence quantitative PCR.And then determine whether mi R-147 a and PDPK1 have a direct regulatory effect.Result: The results of PCR gene sequencing showed that the size and sequence of PDPK1 3'-UTR and PDPK1 3'-UTR sequences after PCR amplification were consistent with the reported sequences of Gene Bank.Thus,two recombinant plasmids containing mi R-147 a binding sites and their mutant recombinant plasmids were successfully constructed.The results of double luciferase reporter assay showed no significant difference in luciferase activity compared with negative control group compared with mi R-147 a group transfected with PDPK1 3'-UTR mutant vector plasmid.The activity of luciferase in mi R-147 a group was significantly inhibited by transfection of PDPK1 3'-UTR vector.There was a significant difference between the luciferase activity and the negative control group(P <0.05).In western blot,the expression of PDPK1 and mi R-147 a was detected in western blot.The expression of mi R-147 a was significantly lower than that of negative control group(P <0.05).After adding inhibitor,mi R-147 a Group expression increased,with significant difference(P <0.05).Conclusions: The PDPK1 gene is the target gene of mi R-147 a.Mi R-147 a binds to the 3'-UTR region of PDPK1 gene and has a direct inhibitory effect on PDPK1 gene at post-transcriptional level.