The Study Of Interaction Between HIF-1 And AHR Signaling Pathways In A549 Cells

Objective: To study the interaction between HIF-1 and AhR signal pathways in A549 cells under hypoxic conditions. Methods:(1) Real-time Quantitative PCR was used to screen the optimal concentration and action time of CoCl2 in A549 cells.(2) MTT technique was used to screen the optimal concentration and action time of B(a)P in A549 cells.(3) A549 cells(1×106) were cultured in Dulbecco’s modified Eagle’s medium maintained at 37°C in a 5% CO2 atmosphere. After a further 24 h, cells were treated with cobalt chloride and B(a)P, after a certain period of time, the cells were harvested. Real-time Quantitative PCR and western blot were used to detect the mRNA and protein expression of VEGF, CAⅨ, CYP1A1 and CYP1B1; Immunoprecipitation was used to detect the binding capacity of AhR/HIF-1α and HIF-1β/ARNT. Results:(1) Detecting the expression of HIF-1α mRNA by Real-time Quantitative PCR, the optimal concentration and action time of CoCl2 were screened which were 300μM and 24h;(2) Detecting the influence of B(a)P in effect on A549 cell proliferation by MTT assay, the optimal concentration range and action time were screened which were 0~8μM and 24h;(3) Cells were treated with CoCl2(300μM), and then different concentrations of B(a)P was added to the medium for 24 h, the m RNA expression of VEGF, CAⅨ, CYP1A1 and CYP1B1 were higher than the control group, specifically, in the presence of 8μΜ B(a)P, the mRNA expression of each gene was about 5, 7, 7, 12-fold of the control group approximately, the differences were statistically significant(P<0.01); The protein expression of VEGF, CAⅨ, CYP1A1 and CYP1B1 were higher than the control group, specifically, in the presence of 8μΜ B(a)P, the protein expression of each gene was about 4, 5, 11, 8-fold of the control group approximately, the differences were statistically significant(P<0.01); The binding capacity of AhR and HIF-1β/ARNT was lower than control group, specifically, in the presence of 8μΜ B(a)P, the binding amount of AhR was about 32% of the control group, the differences were statistically significant(P<0.01), but the binding capacity of HIF-1α and HIF-1β/ARNT was higher than the control group, specifically, in the presence of 8μΜ B(a)P, the binding amount of AhR was about3-fold of the control group approximately, the differences were statistically significant(P<0.01). Conclusion: Under hypoxic conditions, B(a)P up-regulated in a dose-dependent manner the m RNA and protein expression of HIF-1 signaling pathway downstream genes(VEGF, CAⅨ), increased the the binding capacity of HIF-1α and HIF-1β/ARNT; B(a)P up-regulated in a dose-dependent manner the m RNA and protein expression of Ah R signaling pathway downstream genes(CYP1A1, CYP1B1), while the binding capacity of Ah R and HIF-1β/ARNT is inhibited by HIF-1 signaling pathway.