The Relationship Of BaP To CMA With HSP90 On The Hypoxia Environment

Objective:In this study,A549 cells were treated with different concentrations of BaP on the hypoxia environment to investigate whether BaP acts on chaperone-mediated autophagy（CMA）related with HSP90.Methods:（1）The cell hypoxia environment was constructed by CoCl₂,and the mRNA expression of HIF-1αwas detected by qPCR method to determine whether the hypoxic environment was successfully constructed;（2）The mRNA and protein expressions of HIF-1α,HSP90,HSC70 and LAMP-2A in A549 cells under hypoxia were detected by qPCR and Western blot;（3）The mRNA and protein expressions of HIF-1α,HSP90,HSC70 and LAMP-2A in A549 cells under hypoxia in the different concentration of BaP were detected by qPCR and Western blot;（4）The mRNA and protein expressions of HIF-1α,HSP90,HSC70 and LAMP-2A in A549 cells treated with HSP90 inhibitor Geldanamycin（GA）and silencing HSP90αwere detected by qPCR and Western blot;（5）Alkaline comet assay,Immunofluorescence detection ofγ-H2AX focus experiment,qPCR and Western blot were used to detect the effects of different concentrations of BaP on DNA damage and mRNA and protein expression of HSP90,HSC70 and LAMP-2A in A549 cells,and further explore the relationship between DNA damage and CMA.Results:（1）Compared with the normoxic group,the expression of HIF-1αmRNA was increased to some extent after hypoxia at different time.The expression of HIF-1αmRNA was the highest after 24h in hypoxia,and the difference was statistically significant（P<0.001）;（2）Under hypoxic conditions,the mRNA and protein expression levels of HIF-1α,HSP90,HSC70 and LAMP-2A were significantly increased compared with the normoxic group,and the difference was statistically significant（P<0.05）;（3）Under hypoxic conditions,the mRNA and protein expression levels of HIF-1α,HSP90,HSC70 and LAMP-2A were significantly decreased when 1μM BaP was applied to A549 cells for 24h,and the difference was statistically significant（P<0.05）;（4）Under hypoxic environment,Geldanamycin and silencing HSP90αin A549 cells can enhance the inhibitory effect of BaP on the mRNA and protein expression of HIF-1α,HSP90,HSC70 and LAMP-2A,and the difference was statistically significant compared with BaP group（P<0.05）;（5）Under normal-oxygen environment,when BaP was applied to A549cells for 24h,the degree of DNA single and double-strand break and the mRNA and protein expression of HSP90,HSC70 and LAMP-2A increased with the increase of BaP concentration,the difference was statistically significant（P<0.05）.Conclusion:1.Hypoxia can promote CMA in A549 cells;2.Under hypoxic environment,BaP has an inhibitory effect on CMA;3.Under hypoxic conditions,HSP90 inhibitors and silencing HSP90αin A549 cells can enhance the inhibitory effect of BaP on CMA;4.Under normoxic environment,BaP can cause DNA damage and correlate with CMA.