Dissertation > Medicine, health > Internal Medicine > Endocrine diseases and metabolic diseases > Islet disease > Diabetes

Study on the Interaction between Transcription Factor TCF7l2and Ide

Author XuDan
Tutor RenWei
School Chongqing Medical University
Course Internal Medicine
Keywords transcription factor TCF7L2 IDE gene Chromatinimmunoprecipitationlentivirus vector TCF7L2 RNA interference insulin degrading enzyme
CLC R587.1
Type Master's thesis
Year 2012
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PART1DETECTION OF TCF7L2BINDING TO IDE GENEPROMOTERS BY CHROMATINIMMUNOPRECIPITATIONOBJECTIVE: To identify the region in IDE gene promoter where thetranscriptional factor TCF7L2can bind.METHODS: Human hepatoma HepG2cell were obtained as the studysubjects. Chromatin immunoprecipitation and PCR were performed usingthe antibody specific for TCF7L2to verify the binding of TCF7L2to IDEpromoter.RESULTS: IDE gene specific fragments were detected in the DNAfragments immunoprecipitated by antibody specific for TCF7L2.CONCLUSIONS: TCF7L2protein could bind to the promoterregions of IDE gene in HePG2cells, and may involved in regulation ofIDE gene expression.The result may lay a theoretical foundation forstudying the effect of TCF7L2RNA interference on expression of insulin degrading enzyme. PART2EFFECTS OF GENE SILENCE OF TCF7L2-TARGETINGON EXPRESSION OF INSULIN DEGRADING ENZYMEOBJECTIVE: To study the effect of TCF7L2on expression of insulindegrading enzyme, we constructed the shRNA lentiviral RNAi vectortargeting TCF7L2gene (LV-TCF7L2-shRNA), and detect the silencingeffect to TCF7L2gene in the Human hepatoma HepG2cells,then furtherstudied the effect of TCF7L2on expression of insulin degrading enzyme.METHODS:1. Specific shRNA lentiviral vetor targeting a sequenceof human TCF7L2mRNA coding region (LV-TCF7L2-shRNA) andnegative control vector (LV-NC-GFP-shRNA) were constructed.2. HePG2cells were divided into3groups: interference group,negative control group and blank control group. The groups wereadministrated with LV-TCF7L2-shRNA and LV-NC-GFP-shRNA andnothing respectively.3. The changes of mRNA of TCF7L2and IDE were analyzed usingFQ-PCR,the Protein level of TCF7L2and IDE were detected by Western blot.RESULTS:1.TCF7L2mRNA and protein expression inLV-TCF7L2-shRNA group cells were down regulated compared withLV-NC-GFP-shRNA group and blank Control group cells (P<0.05).2. The level of IDE mRNA and protein expression was significantlydecreased after the suppression of TCF7L2expression in HePG2cells(P<0.05).CONCLUSIONS: The lentivirus RNAi vector of LV-TCF7L2-shRNAwas constructed successfully. LV-TCF7L2-shRNA effectively inhibitedIDE expression. Thus, it suggested that TCF7L2may be an importanttranscription factor involving in regulation of IDE gene expression

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