Study On The Role Of HSPA8 In Promoting The Invasion And Metastasis Of Ovarian Cancer

BackgroundOvarian cancer is a disease that has high incidence and mortality rate in women,with intraperitoneal dissemination and distant metastasis being important causes of death,but the mechanisms of invasion and metastasis are still unclear.Our research team previously found that STEAP1 was highly expressed in ovarian cancer and was related to the invasion and metastasis of cancer cells.The co-immunoprecipitation result showed that HSPA8 can bind to STEAP1,and HSPA8 was the first gene identified in the mass spectrometry.HSPA8 had been shown to participate in the proliferation and differentiation of tumor cells.HSPA8 is highly expressed in a variety of malignant tumors,but its role in ovarian cancer is still unclear.Therefore,this study carried out a series of studies on the role of HSPA8 in ovarian cancer from four levels:clinical,organization,cellular and animal,aiming at exploring the gene targets that can prevent the invasion and metastasis of ovarian cancer.Research purpose1.To explore the relationship between HSPA8 and the survival and prognosis of ovarian cancer patients.2.To explore the expression of HSPA8 in normal ovarian tissue,ovarian tumour tissue and ovarian cancer cells.3.To investigate the relationship between HSPA8 and metastasis,invasion,proliferation,clonogenesis and tumorigenicity of ovarian cancer cells.4.To explore the effect of HSPA8 on the process of EMT,cGAS-STING signaling pathway and autophagy in ovarian cancer cells.Research method1.Discovery and identification of HSPA8 by co-immunoprecipitation.2.The relationship between HSPA8 and the survival and prognosis of ovarian cancer patients is mined through the Kaplan-Meier plotter public database.3.Using immunocytochemistry to detect the expression of HSPA8 in normal ovarian tissues and ovarian tumour tissues.4.Using RT-qPCR,immunocytochemistry and western blot to detect the differential expression of HSPA8 in different ovarian cancer cell lines.5.Using lentiviral transfection to increase or decrease the expression of HSPA8 in ovarian cancer cells and using RT-qPCR,immunofluorescence and western blot to validate of transfection effects.6.The ability of metastasis,invasion,proliferation,clonogenesis and tumorigenicity in ovarian cancer cells before and after HSPA8 down-expression or over-expression are detected by Transwell chamber,CCK8 assay,plate cloning and establishment of a nude mouse subcutaneous tumorigenesis model,respectively.7.Observing the morphological changes of ovarian cancer cells after HSPA8 down-expression or over-expression under transmission electron microscope.And using RT-qPCR and western blot to detect the expression changes of EMT-related genes,cGAS-STING signaling pathway target genes and autophagy-related genes in ovarian cancer cells after HSPA8 down-expression or over-expression.Research results1.HSPA8 can bind to STEAP1 in ES-2,SKOV-3 and STEAP1 overexpression cell lines OVCAR-3EX.2.Ovarian cancer patients with high HSPA8 expression have a lower survival rate than those with low HSPA8 expression.3.The expression of HSPA8 in ovarian tissues is poorly differentiated ovarian tumor tissues>highly differentiated tumor ovarian tissues>benign ovarian tumor tissues>normal ovarian tumor tissues.4.The expression of HSPA8 in the four ovarian cancer cells is ES-2>SKOV-3>CAOV-3>OVCAR-3.5.The lentiviral transfection efficiency reached more than 80%.The experiment obtained cell lines with stable low and high expression of HSPA8.6.The ability of metastasis,invasion,proliferation,clonogenesis and tumorigenicity in ovarian cancer cells with HSPA8 down-expressing are reduced,whereas these abilities are enhanced in cells with HSPA8 over-expressing.7.The expression of EMT-promoting genes(ZEB1,N-cadherin,β-catenin,Vimentin,Slug,and Snail)are reduced and the expression of EMT-inhibiting gene E-cadherin is increased in ovarian cancer cells with HSPA8 down-expressing,whereas this is opposite in cells with HSPA8 over-expressing.8.The target genes’ expression of cGAS-STING signaling pathway decrease in HSPA8 down-expressing cells,whereas this is opposite in cells with HSPA8 over-expressing.9.In ovarian cancer cells with HSPA8 down-expressing,the expression of autophagy-related genes p62,LC3,ATG5,ATG7 and WIPI2 increase,and autophagosomes increase,whereas this is opposite in cells with HSPA8 over-expressing.The expression levels of Beclinl,ULK1,and P-ULK1 don’t change significantly in cells before and after HSPA8 down-expression or over-expression.Conclusion1.HSPA8 is highly expressed in ovarian cancer,and ovarian cancer patients with high expression have poor prognosis.2.HSPA8 enhances the ability of metastasis,invasion,proliferation,clonogenesis and tumorigenicity in ovarian cancer cells.3.HSPA8 promotes EMT process,activates cGAS-STING signaling pathway and inhibits autophagy in ovarian cancer cells.