The Association Of Polymorphism In MiRNA-625 Binding Site Polymorphism And The Targeted Regulation To AKT2 |
Posted on:2017-05-04 | Degree:Master | Type:Thesis |
Country:China | Candidate:B Wei | Full Text:PDF |
GTID:2284330509452646 | Subject:Internal Medicine |
Abstract/Summary: | PDF Full Text Request |
ObjectiveThe aim of this experiment was to verify the targeting regulation between miRNA-625 and AKT2 by constructing the luciferase reporter plasmid of the 3’- UTR in AKT2 and evaluate the role of the SNP rs2304186 in the regulation to AKT2 from miRNA-625 by constructing the mutant luciferase reporter vector of the rs2304186.Methods:The AKT2 gene was predicted as miRNA-625 target gene by the biological software TargetSan. The 3’- UTR of the AKT2 were amplified by PCR and the wild type sequences of rs2304186 were cloned into luciferase reporter vector pMIR-Report(pMIR-AKT2-WT). The mutant sequences in the target site of the miRNA-625 and the rs2304186 were also cloned into luciferase reporter vector pMIR-Report(pMIR-AKT2-MUT1 and pMIR-AKT2-MUT2). The recombinant plasmids were transfected into the 16 HBE cell line、HEK-293 T cells line and A549 cells line together with miRNA-625 mimics or miRNA negative contrast(NC). The pRL-SV40 was ransfected into every group as a correction. The relative luciferase activity was detected by dual luciferase reporter gene system.ResultsThe recombinant plasmids were identified by PCR、double enzyme digestion and sequencing correctly. Dual-luciferase reporter assay system revealed that the luciferase activity of the pMIR-AKT2-WT together with miRNA-625 group was significantly decreased compared to the pMIR-AKT2-MUT2 together with miRNA-625 group(P<0.05). The luciferase activity of the pMIR-AKT2-MUT2 together with miRNA-625 group was significantly decreased compared to the pMIR-AKT2-MUT1 together with miRNA-625 group(P<0.05).ConclusionsThe results of dual luciferase reporter assay system suggested that the luciferase activity of the recombinant wild type luciferase reporter gene vector of rs2304186 inthe AKT2 3 ’-UTR could be inhibited by the miRNA-625 and miRNA-625 could regulate the AKT2 gene.The rs2304186 located in the targeting sequences of the miRNA-625 could reduce the efficiency in the regulation to AKT2 from miRNA-625. |
Keywords/Search Tags: | miRNA-625, AKT2 gene, luciferase reporter gene, 3’-UTR, SNP |
PDF Full Text Request |
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