Construction And Identification Of HIF-2α Gene RNAi Lentivirus Vector

Background and objectiveColorectal cancer is one of the most common malignant tumor, currently the comprehensive treatment of colon cancer including operations and adjuvant chemotherapy is still the main means,but its efficacy is currently in a state of relative stagnation,so we must find a new breakthrough by basic research in order to enhance the effectiveness of treatment.Hypoxia is a universal phenomenon that exist in Colorectal cancer.Hypoxia-inducible factor 2α(HIF-2α)is one of the most important regulatory factor in the tumor hypoxia responsive reaction.Recent studies show that HIF-2αplays an important role in tumor proliferation, metabolism,angiogenesis and metastasis,but the regulatory mechanism of HIF-2αis still unclear.This study is to construct the HIF-2αgene RNAi lentivirus vector,and use it to transfect the 293T cells,then select the optimal target.It can be a preliminary preparation for observing the expression of HIF-2αgene RNAi lentivirus vector in Colorectal cancer cells line and an in-depth study of the regulatory mechanism and role of HIF-2αin colorectal cancer cells in the hypoxic microenvironment.Method1.Construction of the HIF-2αgene RNAi lentivirus vector.Design several RNAi target sequences on the basis of the HIF-2αgene according to RNAi sequence designing principle,and synthesize double strands DNA oligo including interference sequence,then construct the HIF-2αgene RNAi lentivirus vector by digesting and ligatting.Then analyze the HIF-2αgene RNA interferential lentivirus vector by DNA sequencing.2.Construction of the HIF-2αover-expression vector.Attain the HIF-2αfrom the plasmid clone template containning the HIF-2α,then digest the HIF-2αand the target vector respectively and ligate them to construct the HIF-2αover-expression vector.Then analyze HIF-2αover-expression vector by DNA sequencing3.Select effective targets by RNAi and identificationTransfect the 293T cells with the constructed over-expression vector containing the HIF-2αand the RNAi virus vectors aiming at different interferential targets of HIF-2αtogether,then observe the transfection effect with fluorescent microscope 24 hours after transfection and collect the cells to extract protein 36 hours after transfection.Then detect the expression result of the targets protein by Western blot to judge the interferential effect of different targets.ResulDNA sequencing and target-selection show that the HIF-2αgene RNAi lentivirus vector is constructed correctly,it suppress the expression of the HIF-2αgene significantly by Western Blot probation.ConclusionsThis study successfully constructs the HIF-2αgene RNAi lentivirus vector...