The Mechanism Of Hsp90α And Its Substrate Protein DNA-PKcs Regulating Hsp70 Transcription Under Heat Stress

BackgroundLiver cancer is one of the most common malignant tumors in China.In 2014,there were about 400,000 new cases of liver cancer in China and about 380,000 deaths caused by liver cancer.The incidence and mortality of liver cancer ranked the second among all kinds of cancers.Primary liver cancer is divided into six types.Hepatocellular carcinoma(HCC)accounts for more than 90%of the primary liver cancer.Hyperthermia is a method of treating tumor by heating,which causes the growth of tumor cells to be hindered and tumor cells to be killed without inducing serious damage to normal cells.It uses the related physical energy to precipitate in tissues and produces heat effect,so that the temperature of tumor tissue rises to the effective treatment temperature(40℃-44℃)for a period of time.On the one hand,hyperthermia can damage DNA,and DNA damage reponse is strongly activated at the DNA double-strand breaks(DSBs).On the other hand,hyperthermia can lead to the initiation of protective measures for cancer cells,especially the synthesis of heat shock proteins such as Hsp70.It has been reported that a tumor patient with a Hsp90α gene defect has a better curative effect and prognosis than tumor patients with ordinary level of Hsp90α.Among Hsp90α-deficient tumor patients,Hsp70 is the most important protective molecular to resist hyperthermia.Hsp70 can inhibit the apoptosis of tumor cells,stabilize the lysosome membrane,inhibit the death of autophagic cell and affect the therapeutic effect of the tumor.It has been revealed that HSF1 is the transcriptional factor of Hsp70,furthermore,the transcriptional regulation of Hsp70 conforms to the Pol Ⅱ release pausing.However,whether Hsp90α can regulate the release of PolⅡ on Hsp70 gene still need to be further explored.DNA dependent protein kinase catalytic subunit(DNA-PKcs)is a key component of DNA repair.It can repair damaged DNA and maintain the integrity of the genome.What is the role of DNA-PKcs in the transcriptional regulation of Hsp70 in the hepatoma cells and the effect of Hsp90α on it under heat stress?This study explored the transcriptional regulation of Hsp70 under heat stress,and will figure out the above questions.Objective1.To observe the changes of Hsp70 in wild type hepatoma cell and Hsp90αknockout hepatoma cell.2.To observe the protein and transcription of Hsp70 in two hepatocellular carcinoma cells under heat stress.3.To investigate the possible mechanism of hyperthermia on the changes of Hsp70 in two types of hepatocellular carcinoma cells.4.To investigate the possible mechanism of DNA damage caused by heat stress on PolⅡ arrest and release.Method1.Western Blot analyzed the expression of different proteins in two types cells.2.Polymerase Chain Reaction analyzed the mRNA expression of different fragments of HSPA1B.3.Chromatin Immunoprecipitation was used to analyze the binding of proteins to the HSPA 1B in two types cells under heat stress.4.The rate of apoptosis was analyzed by flow cytometry.5.Plasmid transfection established an HSF1 overexpression model.6.Immunofluorescence was used to analyze the expression and co-localization of both proteins in the cells.7.DNA damage was analyzed by Comet Assay8.Using SPSS 21.0 software for statistical analysis.Multiple groups were compared using one-way ANOVA(One Way ANOVA).The experimental group and control group compared with each other by Dunnett method.Two independent sample groups were compared using t test.The rate between the two groups was compared using the chi-square test.The test level was P<0.05.Result1.Under heat stress at 42℃,the amount of newly synthesized Hsp70 protein in wild-type human hepatoma HepG2 cells was greater than Hsp90α-/-cells,and the apoptosis rate of wild-type cells was lobuer than that of Hsp90α knockout cells.2.Under heat stress at 42℃,the transcription level of HSPA 1B mRNA in wild-type hepatoma cells was higher than that in Hsp90α knockout cells,and Hsp90α promoted the recruitment and pausing release of PolⅡ.3.Under heat stress at 42℃,HSF1 promoted the transcription of HSPA 1B.HSF1 increased the amount of PolⅡ bound to HSPA 1B.The deletion of Hsp90αinhibited the transcriptional activity of HSF1.4.Under heat stress at 42℃,DNA-PKcs promoted transcription of HSPA 1B.The deletion of Hsp90a led to a decrease in DNA-PKcs protein.5.Under heat stress at 42℃,DNA double-strand breaks of Hsp90α knockout cells was more severe than that of wild-type cells.Conclusion1.Under heat stress,the deletion of Hsp90α inhibited the synthesis of Hsp70 and promoted the apoptosis.2.The deletion of Hsp90α affected the binding of HSF1 at the promoter region of HSPA1B,which affected the recruitment and pausing release of Pol Ⅱ under heat stress.3.Under heat stress,DNA-PKcs promoted the transcription of Hsp70 by DNA damage repair response which required the participation of Hsp90α.