The Study Of Interaction Between AhR,HIF-1α And ARNT By FRET

Objective The study of interaction between Ah R,HIF-1α and ARNT by using FRET technology in A549 cells under different concentrations of Ba P with using Co Cl2 as the agonist of HIF-1α.The aim of this study is to provide some reference for the study of the relationship between these two important signal pathways,and to provide some experimental evidence for scientists to find drugs to block Ah R and HIF-1 signaling pathways to prevent and treat cancer.Methods(1)Total RNA was extracted from A549 cells by using molecular cloning gene recombination technique.The target gene Ah R and ARNT fragments were cloned by RT-PCR.The two target genes Ah R and ARNT were recombined into fluorescent expression by plasmid construction p Ac GFP1-N1-Ah R and p Ds Red-Monomer-N1-ARNT were cloned into p Ac GFP1-N1 and p Ds Red-Monomer-N1 plasmids.(2)Fluorescence resonance energy transfer assay was used to investigate the study of interaction between Ah R,HIF-1α and ARNT.p Ac GFP1-N1-Ah R is the target gene Ah R and GFP fusion expression.p Ds Red-Monomer-N1-ARNT is a fusion expression of ARNT and p Ds Red-Monomer-N1.Detection of Ah R and ARNT interaction,which is equivalent to detection FRET occurred of p Ac GFP1-N1 and p Ds Red-Monomer-N1.Liposome transfection mediated the expression of p Ac GFP1-N1-Ah R and p Ds Red-Monomer-N1-ARNT in A549 cells.The cells were incubated with Co Cl2(300μM,24h)for different concentrations of Ba P(0μM、2μM、4μM、8μM,24h).By using the fluorescence imaging module of high content cell screening system,we determined whether Ah R and HIF-1α competed with ARNT / HIF-1β based on the fluorescence intensity of the fluorescent recombinant plasmid in A549 cells with Co Cl2 excited HIF-1α into the nucleus.Results(1)We successfully constructed two recombinant plasmids,which are pAc GFP1-N1-Ah R and p Ds Red-Monomer-N1-ARNT.The recombinant plasmids were successfully expressed in A549 cells after transfection.The transfection rates were respectively 15% and 19%.(2)We found that in the cytoplasm can be observed in the green fluorescence signal in the nucleus can be observed in the red fluorescent signal.The results showed that the two recombinant plasmids were successfully expressed in A549 cells.(3)When p Ac GFP1-N1-Ah R and p Ds Red-Monomer-N1-ARNT were transfected into A549 cells with different concentrations of Ba P(0μM,2μM,4μM,8μM,24h).FRET fluorescence signal intensity compared with the control group was significantly increased.When the concentration of Ba P was 8μM,the fluorescence intensity of FRET was about 2.5 times of that of the control group,and the difference was significant(P<0.05).(4)After treatment with 300μM CoCl2 and BaP 8μM for 24 h,the intensity of fluorescence signal intensity of FRET was decreased by 0.6 times compared with Ba P 8μM for 24 hours.Conclusion Ba P could up-regulate the FRET fluorescence signals of p Ac GFP1-N1-Ah R and p Ds Red-Monomer-N1-ARNT in a dose-dependent manner.The decrease of FRET fluorescence intensity after Co Cl2 treatment showed that HIF-1α competed with Ah R to bind to ARNT protein.Furthermore,the activation of the HIF-1α signal pathway can inhibit the Ah R signaling pathway.