A Study Of The Role And Molecular Mechanism Of Guanylate-binding Protein 1(GBP1)in Prostate And Ovarian Cancers

Malignant tumors have become a public health problem worldwide,and the incidence rate is increasing year by year.With the development of economy and society,prostate cancer has become the seventh malignant tumor in male.Most of the patients were diagnosed with advanced prostate cancer.After treatment with androgen deprivation and radical surgical resection,the tumor often developed drug resistance and metastasis,resulting in a poor prognosis.Ovarian cancer is the most deadly malignancy in the female reproductive system.Most patients are diagnosed with local or distant metastases due to atypical early symptoms.The high recurrence rate and high mortality rate of tumor patients have not been improved after combined treatment such as surgical resection,radiotherapy and chemotherapy.Seeking more effective diagnostic markers and exploring new targeted therapy strategies have become the focus of prostate cancer and ovarian cancers research.GBP1 is a member of the guanylate-binding protein family and a kind of trigger protein,which can be induced by interferon and a variety of cytokines,and is widely involved in various biological processes such as antiviral,anti-bacterial,antigenic and inflammatory reactions.In recent years,more and more studies have found that GBP1 plays an important role in the occurrence and development of tumors and is closely related to tumor growth,tumor invasion,angiogenesis,recurrence and metastasis.However,studies on the role of GBP1 in prostate cancer and ovarian cancer are still limited,and its mechanism needs to be further explored.It was discovered from our earlier prostate cancer cell line models studies that when prostate cancer cells were treated with either ethidium bromide or a cell cycle inhibitor flavopiridol for a long-term,the treatment-survived tumor cells experienced metabolic reprogramming towards Warburg effect pathways with greater aggressive features,and one common finding from these cells was the up-regulation of GBP1.Therefore,in this study,four independent GBP1 gene knockout cell models of prostate and ovarian cancers were constructed by using the CRISPR/cas9-mediated gene editing system,and the role of GBP1 gene in biological function and characteristics of the GBP1 KO tumor cells were explored.Subsequently,the role of GBP1 gene was further verified and the relevant mechanism was discussed,combined with tumor formation experiments in nude mice.Finally,we detected the expression level of GBP1 protein in prostate cancer and ovarian cancer tissue samples by using IHC staining method,and analyzed the correlation between GBP1 expression and clinicopathological parameters and prognosis of patients.Part ?:Construction and functional analysis of GBP1 gene knockout prostate and ovarian cancer cell linesMethods1.Using the CRISPR/Cas9-mediated gene editing system,the GBP1 gene knockout was performed on prostate cancer cell lines DU 145 and PC3 and ovarian cancer cell lines SKOV3 and OVCAR3,a single clone was selected for establishing a stable GBP1 gene knockout cell line,then the expression of GBP1 protein was confirmed by immunocytochemistry(ICC)and Western blotting.2.The Incucyte ZOOM system was used to detect the cell proliferative ability.The cell cycle was examined by Flow cytometry.Transwell assay and wound healing experiment were used to determine the cell migration ability.The Incucyte ZOOM system and colony formation experiment were performed to detect the drug sensitivity of cells.Seahorse XFe96 system was used to examine energy metabolism state of cells,through which can evaluate the changes oxygen consumption and extracellular acidification rate of cancer cells after GBP1 gene knockout.Results1.Four stable GBP1 gene knockout cell line models were established in the DU 145?PC3?SKOV3 and OVCAR3 cell lines,and named DU145 GBP1 KO?PC3 GBP1 KO?SKOV3 GBP1 KO and OVCAR3 GBP1 KO,respectively.2.These GBP1 gene knockout cell line models exhibited decreased cell proliferation capacity,inhibited cell cycle,reduced migration ability,higher drug sensitivity and decreased oxidative phosphorylation and glycolysis metabolism in energy metabolism state,in comparison with the parental cells.Part ?:Effect of GBP1 on tumorigenic ability of prostate and ovarian cancer cells in vivoMethods1.Tumor formation experiments were carried out in nude mice to detect the changes in tumor formation ability of cancer cells after GBP1 gene was knockout.2.HE staining was used to observe the histomorphologic changes of tumors.3.The expression of GBP1 protein in xenograft tumor tissues was studied by immunohistochemistry.Results1.After inoculation,DU145 GBP1 KO,PC3 GBP1 KO,SKOV3 GBP1 KO and OVCAR3 GBP1 KO cells grew slower and formed smaller tumors in vivo,in compare with the control cells.SKOV3 GBP1 KO and OVCAR3 GBP1 KO cells also exhibited a lower tumor formation rate in nude mice.2.The result of HE staining exhibited less cellular atypia and nuclear pleomorphism in xenograft tumors of DU145 GBP1 KO,PC3 GBP1 KO,SKOV3 GBP1 KO and OVCAR3 GBP1 KO groups.3.The expression levels of GBP1 protein in DU145 GBP1 KO,PC3 GBP1 KO,SKOV3 GBP1 KO and OVCAR3 GBP1 KO group were significantly down-regulated in xenograft tumors of athymic mice.Part ?:Molecular mechanism of action of GBP1 in prostate and ovarian cancersMethods1.ICC,IHC and Western Blot method were used to detect the expression of EGFR protein in cancer cells and xenograft tumors of nude mice,for verifying whether GBP 1 influences the expression of EGFR protein.2.The transcriptome sequencing was performed on DU 145 GBP1 KO,PC3 GBP1 KO,SKOV3 GBP1 KO,OVCAR3 GBP1 KO cells and their parental cells,respectively.Differential gene expression and changes in differential gene-related GO and KEGG pathways had been analyzed.3.The STRING database was used to establish the protein-protein interaction network.Results1.ICC detection and Western blot experiment showed that the expression of EGFR protein decreased after GBP1 gene knockout,IHC detection also exhibited a down-regulated expression of EGFR protein after GBP1 gene knockout.The above results indicted that EGFR expression was affected by the expression of GBP1 protein in prostate and ovarian cancer cells.2.Cell transcriptome detection revealed that certain genes belong to cytokeratin(KRT)family of DU145 GBP1 KO,PC3 GBP1 KO,SKOV3 GBP1 KO,OVCAR3 GBP1 KO cells were significantly down-regulated,and the results of RT-PCR were in line with those of sequencing analysis.3.The PPI network model indicted that the protein-protein interaction between EGFR and GBP 1 was only sustained by text evidence,there was no direct interaction between GBP1 and the members of KRT family.The results reflected the innovation and truthfulness of our research.Part ?:Correlation between GBP1 expression in prostate and ovarian cancers and clinicopathological parameters and prognosisMethods1.Using IHC staining method to detect the expression level of GBP1 protein in 105 prostate cancer samples.The association between GBP1 expression and clinicopathological parameters and overall survival of patients were statistically analyzed.2.IHC staining method was used to examine the expression of GBP1 in 100 cases of ovarian cancer.The correlation between GBP1 expression and clinicopathological features was analyzed,moreover the relationship between GBP1 and the clinical and pathologic characteristics in ovarian serous carcinoma.3.SPSS software was used for statistical analysis.The correlation between expression of GBP1 protein and clinical variables were analyzed by Chi-square or Fisher's exact test.Survival analysis was evaluated by Kaplan-Meier.The differences between groups were analyzed by using Log-rank test.The COX regression was used for detecting the effect of each indicator on prognosis.Results1.The expression of GBP1 in most of the clinical samples was positive,up to 93.33 percent.The expression of GBP1 protein was positively associated with Gleason score,and negatively correlated with overall survival of patients.2.The GBP1 expression in all of the 100 ovarian cancer samples was positive.The GBP1 expression was positively correlated with FIGO stage,and negatively associated with histological grade.In the Serous carcinoma,the expression of GBP1 protein was positively correlated with FIGO stage,and negatively associated with histological grade.3.Multivariate analysis indicated that GBP1 expression,Gleason score and TNM stage could be used as predictors of overall survival rate in prostate cancer patients.Conclusions1.These prostate cancer and ovarian cancer cells with GBP1 gene knockout exhibited decreased proliferation capacity,cell cycle inhibition,decreased migration capacity,and increased sensitivity to chemotherapy.Meanwhile,mitochondrial oxidative phosphorylation and glycolysis were both inhibited in cell energy metabolism,suggesting that GBP1 was related to the malignant progression of prostate cancer and ovarian cancer.2.After the gene knockout of GBP1 in DU145,PC3,SKOV3 and OVCAR3 cell lines,the tumorigenic ability of tumor cells significantly decreased in nude mice,which verified the results of in vitro cell experiments and further demonstrated the promoting role of GBP1 in the occurrence and development of prostate cancer and ovarian cancer.3.The expression level of EGFR protein in cancer cells was down-regulated after GBP1 gene knockout,and the expression level of EGFR protein in tumor tissues of nude mice was also decreased in the GBP1 gene knockout group.After the gene knockout of GBP1,the expression of KRT family related genes was significantly down-regulated.This suggests that GBP 1 may play a role in tumors by participating in the EGFR pathway and affecting the expression of members of KRT family.4.In clinical prostate cancer tissues,GBP1 protein expression was positively correlated with Gleason score and negatively correlated with patient survival.In ovarian cancer,the expression of GBP1 protein was significantly associated with FIGO stage and the degree of tissue differentiation.This indicates that GBP1 plays an important role in promoting the occurrence and development of prostate cancer and ovarian cancer.