Study On The Mechanism Of HSF1 Regulated PD-L1 Expression In Hepatocellular Carcinoma

Objectives:1.To study the relationship between heat shock transcription factor(HSF1)and programmed death receptor-ligand 1(PD-L1)in the hepatocellular carcinoma(HCC)tissues and cells.2.To study the regulation mechanism of HSF1 on PD-L1 expression in HCC.Methods:1.The relevance between the expression of HSF1 and PD-L1 in HCC tissues of 105 clinical patients were analyzed by IHC method.2.The total RNA of HCC cells was extracted,and the mRNA expression levels of HSF1 and PD-L1 in HCC cells were detected by qRT-PCR;The total proteins of HCC cells was extracted and the protein expression levels of HSF1 and PD-L1 were detected by western blot.3.The overexpression plasmid vector of HSF1 was constructed and the HSF1 gene was overexpressed by plasmid vector transfection in Huh7 cells.After 48 hours transfection,total RNA was extracted and the mRNA expression levels of HSF1 and PD-L1 were detected by qRT – PCR.The total protein was extracted and the expression levels of HSF1 and pd-1 protein expression were detected by Western Blot and the expression level and phosphorylation level of the correlated signal molecules of Clusterin and JAK signaling pathway.4.The lentivirus vector interfering with HSF1-shRNA was constructed,the expression of HSF1 in Huh7 cells was silenced by RNA interference.After 48 hours,contrasted with the empty vector,the mRNA expression levels of HSF1 and PD-L1 were detected by qRT – PCR;Western Blot was used to detect the protein expression levels of HSF1 and PD-L1 and the expression level and phosphorylation levels of the correlated signal molecule STAT in Clusterin and JAK signaling pathway.5.Contrasted with the empty vector group of HepG2,the HepG2 cells with HSF1 overexpression were devided into four groups,the transfected and nontransfected with Clusterin downregulating plasmid groups,and with or without STAT3 signaling pathway inhibitor(C188-9)groups.the total proteins in these four groups of HepG2 cells were extracted,and the protein expression levels and the phosphorylation levels of PD-L1,Clusterin and STAT3 were detected.6.The animal model of the tumor-bearing nude mices with HSF1 Knockdown were established,the tumor was taken out of the nude mice after raising a certain period of time,and the expression levels of HSF1 and PD-L1 in HCC tissues was detected by ICH.Results:1.The HCC tissues contained 50 cases of HSF1(+),55 cases of HSF1(-),33 cases of PD-L1(+),72 cases of PD-L1(-)and 29 cases of HSF1(+)PD-L1(+).The expression of HSF1 was significantly correlated with PD-L1,and the correlation coefficient R2=0.889.2.The mRNA expression levels of HSF1 and PD-L1 were consistent with the protein expression levels of them in HCC cell lines,and the expression levels between HSF1 and PD-L1 were positively correlated.3.In the Huh7 cells with overexpressing HSF1,the mRNA and protein expression levels of PD-L1 were all elevated,in addition,the protein expression levels of Clusterin and the phosphorylation levels of STAT3(p705)were significantly increased.4.The mRNA and protein expression levels of PD-L1 were decreased in HepG2 cells with HSF1 silence.The protein expression levels of Clusterin and STAT3 were significantly decreased while the phosphorylation level of STAT3 had no obvious change.5.The expression levels of STAT3 and PD-L1,and the phosphorylation level of p-STAT3(Tyr)were reduced after transfected with Clusterin downregulating plasmid in Hep G2 cells.The expression levels of STAT and PD-L1,and the phosphorylation level of p-STAT3(Tyr)were increased after covering the HSF1 gene.When adding the inhibitor of the STAT3 pathway,the expression of STAT3 was successfully suppressed,and the phosphorylation level of p-STAT3(Ser)and p-STAT3(Tyr)were reduced,meanwhile,the expression level of PD-L1 was decreased.After transfected with HSF1 overexpression plasmid,the expression levels of the CLU,STAT3 and PD-L1 were increased,and the expression of pSTAT(Tyr)was also increased.6.The results of the tumor-bearing nude mices model with HSF1 knockdown showed that the proliferation of HCC cells were inhibited and the expression level of PD-L1 was decreased.Conclusions:1.The expression of HSF1 are highly positivly correlated to PD-L1 in HCC tissues.2.After overexpressed the HSF1 gene,the protein expression level of Clusterin and the phosphorylation levels of STAT3(p705)are significantly increased,and the expression of PD-L1 is also increased.While the protein expression levels of Clusterin,STAT3 and PD-L1 are significantly decreased with HSF1 knowdown,indicating that HSF1 could regulate the expression of PD-L1 by activating the Clusterin/STA3 signaling pathway.3.Inhibiting the Clusterin or STAT3 signaling pathway in Hep G2 cells with HSF1 overexpression,the expression of PD-L1 is decreased,which further demonstrated that HSF1 could regulate the expression of PD-L1 through the Clusterin/STAT3 signaling pathway.4.This finding can provide theoretical basis for the regulation of the expression of PD-L1 in HSF1 and promote the development of hepatocellular carcinoma.It also provides new findings for the immunotherapy of tumors.