Regulatory Mechanism Of Heat Shock Protein 90 On Autophagy Regulator Transcription Factor EB In Human Hepatocellular Carcinoma Cells

BackgroundHepatocellular carcinoma（HCC）is a malignant tumor disease that seriously threatens human health.As an indispensable molecular chaperone of eukaryotic cells,Hsp90 is involved in the regulation of complex protein homeostasis system and is a star molecule in the development of anticancer drugs.Some studies have found that Hsp90 and other molecular chaperones are overexpressed in carcinoma tissues,and the expression of Hsp90a in hepatocellular carcinoma tissue is closely associated with the degree of tumor differentiation and neoplasm size.Hypoxia is a universal stimulus in solid tumors,which can induce autophagy and is conducive to the survival of tumor cells.Autophagy is closely related to malignant transformation,survival and proliferation of tumor cells.TFEB is an critical transcription factor in autophagy process,it plays an important role in the initial stage and development of various diseases,and is a potential therapeutic target for cardiovascular diseases,neurodegenerative diseases,and tumors.Objectives1.To investigate the effects of different concentration gradients of Hsp90 N-terminal inhibitor STA9090 and C-terminal inhibitor Novobiocin on the activity of HCC HepG2 cells;2.To investigate the effect of hypoxic stress on the expression levels of Hsp90 and TFEB proteins in HepG2;3.To investigate the effect of Hsp90 N-terminal inhibitor STA9090 and C-terminal inhibitor Novobiocin on the protein levels of TFEB and downstream molecules LC3 and P62 in HCC HepG2;4.To investigate the effect of transient transfection of full-length Flag-Hsp90 expression plasmid on TFEB protein expression level in HepG2 cells（HepG2 WT）and HepG2 cells（HepG2 KO）knocked out with Hsp90AA1,respectively.5.To investigate the effect of Hsp90 N-terminal inhibitor STA9090 and C-terminal inhibitor Novobiocin on TFEB transcription level;6.Explore whether Hsp90 binds in the proximal promoter region of TFEB and the specific binding location.Methods1.Hsp90 N-terminal inhibitor STA9090 and C-terminal inhibitor Novobiocin were respectively given with different concentration gradients to treat HCC cells HepG2,and cell viability was detected by MTT method.2.Under both normoxic and hypoxic conditions,Western Blot was used to detect the effects of certain concentration of Hsp90 on the expression levels of n-terminal inhibitor STA9090 and C-terminal Novobiocin on TFEB and its downstream autophagy-related genes LC3 and P62.3.The effects of Hsp90 inhibitor STA9090 with different concentration gradients on TFEB protein expression levels were detected by Western Blot under both normoxic and hypoxic conditions.4.After STA9090 treatment of nude mice,the expression of TFEB protein in tumor tissues was detected by immunohistochemistry;5.HepG2 cells（HepG2 WT）and HepG2 cells（HepG2 KO）were transfected with full-length Flag-Hsp90 expression plasmids,and TFEB protein expression levels were detected by Western Blot.6.Different combinations were set with STA9090（Hsp90 inhibitor）,CHX（protein translation inhibitor）and MG 132（protein degradation inhibitor）,and TFEB protein expression was detected by Western Blot.7.The effect of Hsp90 inhibitor STA9090 on the expression level of TFEB mRNA was detected by Reverse Transcription PCR under normoixa and hypoxia conditions.8.The effects of N-terminal inhibitor STA9090 and C-terminal Novobiocin on the binding ability of Hsp90 to the proximal.promoter region of TFEB were observed by chromatin immunoprecipitation（ChIP）.9.Plasmids including three different sequence of TFEB proximal promoter region were constructed.The dual-lucifase reporter assay verified Hsp90 affects the transcription activity of TFEB proximal promoter region.Results1.The MTT test results showed that compared with the control group,the cell viability decreased after Hsp90 N-terminal inhibitor STA9090 or C-terminal inhibitor Novobiocin treatment of HCC cells HepG2 for 24 hours（p<0.05）,and the cell viability gradually decreased with the increase of STA9090 concentration.The IC50 of STA9090 and Novobiocin were 0.1 mol/L and 0.5mmol/L,respectively.2.Western Blot results showed that under both normoxic and hypoxic conditions,the protein expression levels of TFEB,LC3 and P62 decreased in N-terminal inhibitor STA9090 and C-terminal inhibitor Novobiocin treated HepG2 cells,3.Western Blot results showed that under both normoxic and hypoxic conditions,the Hsp90 inhibitor STA9090 down-regulated the TFEB protein expression level and was concentration-dependent.4.Immunohistochemical results showed that after STA9090 treatment,the expression level of TFEB protein in tumor tissues decreased.5.Western Blot results showed that the full-length Flag-Hsp90 protein expression plasmid was transfected transiently in HepG2 cells（HepG2 KO）with wild HepG2 WT and HepG2 cells（HepG2 KO）with Hsp90AA1 gene knockout for 24h,respectively,and the expression level of TFEB protein increased.6.Western Blot results showed that the decrease in TFEB protein expression caused by Hsp90 inhibitor STA9090 was due to the inhibition of TFEB protein synthesis rather than the promotion of TFEB protein degradation.7.The results of Reverse Transcription PCR showed that Hsp90 inhibitor STA9090 down-regulated the expression level of TFEB mRNA under both normoxia and hypoxia.8.ChIP experiment proved that Hsp90 binds to the TFEB proximal promoter region（-179～-57）and is involved in regulating its transcription process.Hsp90 inhibitors STA9090 and Novobiocin inhibit its binding ability.9.The dual-lucifase reporter assay demonstrated that the TFEB proximal promoter region（-500～0）is essential for the transcription of TFEB.The N-terminal inhibitor of Hsp90,STA9090,and the C-terminal inhibitor,Novobiocin,inhibit the TFEB transcription by the TFEB proximal promoter region.Conclusions1.The expression levels of Hsp90 inhibitor induced TFEB and its downstream autophagy-related target genes LC3 and P62 were decreased;The expression levels of hypoxia-stress-induced TFEB and its downstream autophagy-related target genes LC3 and P62 were increased;2.Hsp90 has a regulatory effect on TFEB,regulating the protein synthesis process of TFEB rather than the degradation process,and regulating the transcription level of TFEB during the protein synthesis process of TFEB;3.Hsp90 regulates TFEB transcription levels by binding Hsp90 to the TFEB promoter region,thereby affecting TFEB transcription activity.Hsp90 binds to the DNA sequence region of the TFEB promoter region（-500～0）.