| BackgroundOvarian cancer(OC)is the fifth leading cause of cancer-related deaths in women.The current standard of care for OC is a combination of surgery and platinum-based chemotherapy,but there is still a high incidence of recurrence and poorly treated patients.As research progresses,targeted angiogenic and immunomodulatory therapies for OC have become a hot topic of interest.Induction of angiogenesis is a hallmark of tumors,in which there is increased secretion of vascular endothelial growth factor A(VEGFA).The molecules that mark angiogenesis are CD34 and platelet-endothelial cell adhesion molecule 1(PECAM1/CD31).Targeting VEGFA has been found to block tumor angiogenesis,slowing tumor growth and thus prolonging patient survival.However,anti-VEGFA therapy can lead to adverse effects as well as drug resistance,while the resulting hypoxic environment suppresses immune function in the tumor microenvironment(TME)and is detrimental to tumor elimination.In the TME of OC,regulatory T cells(Tregs)inhibit the recruitment and expansion of effector T cells,and are the most important immune escape cells.Forkhead box protein 3(FOXP3),a molecule specifically expressed by Tregs,has a key role in the development and immunomodulatory function of Tregs.It has been reported that Tregs can be involved in tumor angiogenesis by promoting VEGFA secretion and VEGFA receptor expression.These suggest that angiogenesis and immune escape may interact and act together in TME thereby promoting tumor growth.Vasohibin1(VAASII1)is an endogenoas angiogenesis inhibitor generated by the negative feedback regulation of VEGFA.It has been found that VASH1 is highly expressed in a variety of tumor tissues and is closely associated with tumor development and recurrence of metastasis.It was observed that VASH1 gene therapy did not affect the growth and morphological changes of normal blood vessels,which may compensate for the adverse effects caused by anti-VEGFA therapy,suggesting that VASH1 is the future direction of OC treatment,but the relationship between VASH1 and Tregs has not been clarified.The aim of this study was to initially explore the relationship between angiogenesis and immune escape in OC,in which VASH1 is a possible connecting molecule.The first part examined the expression of VEGFA and VASH1 in OC and the impact on prognosis,and analyzed the correlation between VEGFA and VASH1 and angiogenesis;the second part analyzed the correlation between Tregs and FOXP3 and angiogenesis in OC,and examined the expression of FOXP3 in OC and the impact on prognosis,and explored VEGFA,VASH1,and FOXP3 signaling pathways that act together in OC.Part Ⅰ.Relationship between VEGFA and VASH1 expression in OC tissues and the clinicopathological characteristics of OC and correlation with angiogenesisPurpose1.To detect the expression of VEGFA and VASH1 in OC tissues and to analyze the correlation between VEGFA and VASH1 and the clinicopathological characteristics and prognosis of OC patients.2.Application of bioinformatics data to explore the correlation of VEGFA and VASH1 with angiogenic marker expression and angiogenesis in OC.Materials and MethodsMaterials1.Clinical data:clinical electronic medical data were collected from 131 patients with high-grade serous ovarian cancer(HGSOC)who underwent surgical treatment at Qilu Hospital of Shandong University from 2014 to 2018,pathological sections of included patients were obtained by contacting the pathology department of Qilu Hospital of Shandong University,and prognostic information was obtained by telephone follow-up.Permission for this study was obtained from the Ethics Committee of Qilu Hospital.2.Bioinformatics data:RNA-seq data for a total of 33 tumors including OC from the TCGA database and a total of 31 solid tissues including normal ovary-fallopian tube tissue from the GTEx database were downloaded from the USCS Xena website;RNA-seq data for 380 OC patients were downloaded from the TCGA database.50 hallmark gene sets obtained from the Broad Institute’s MSigDB database.Methods1.Immunohistochemistry was performed to detect the expression of VEGFA and VASH1 in HGSOC tissues and to score the staining.Correlation analysis was used to explore the relationship between VEGFA and VASH1 expression and clinicopathological parameters and prognosis,respectively.2.To analyze the correlation between angiogenic markers(CD34 and CD31)and the expression of VEGFA and VASH1 using RNA-seq data from 33 tumor tissues in the TCGA database and 31 normal tissues in the GTEx database.3.Using RNA-seq data from 380 OC patients in the TCGA database,VEGFA and VASH1 were correlated with the remaining genes separately to obtain correlation coefficients and P values to construct gene sets.To explore the correlation between VEGFA and VASH1 and angiogenic pathways using single gene correlation GSEA.The correlation of VEGFA and VASH1 expression was analyzed in the TCGA database and clinical data.Results1.Expression of VEGFA and VASH1 in HGSOC tissues and the relationship with clinicopathological parameters and prognosis.1.1 Expression of VEGFA in HGSOC tissues and relationship with clinicopathological parameters and prognosis.Immunohistochemistry showed that VEGFA was expressed in the cytoplasm of HGSOC cells.131 patients were divided into a high VEGFA expression group(Fig.2-3-2A)and a low VEGFA expression group(Fig.2-3-2B)according to the immunostaining score.Correlation analysis showed that VEGFA expression correlated with FIGO stage(P=0.045,Fig.2-3-2E)and MVD(P=0.02,Fig.2-3-2F).KaplanMeier curves showed that patients in the VEGFA high expression group had worse overall survival(OS)(P=0.00039,Fig.2-3-2G)and progression-free survival(PFS)(P<0.0001,Fig.2-3-2H).1.2 VASH1 expression in HGSOC tissue and relationship with clinicopathological parameters and prognosis.Immunohistochemistry showed that VASH1 was expressed in the cytoplasm of HGSOC cells,and 131 patients were divided into a high VASH1 expression group(Fig.2-3-3A)and a low VASH1 expression group(Fig.2-3-3B)according to the immunostaining score.Correlation analysis showed a borderline correlation between VASH1 expression and FIGO stage(P=0.057,Fig.2-3-3D)and a correlation with MVD(P=0.001,Fig.2-3-3C).Kaplan-Meier curves showed that patients in the high VASH1 expression group had worse OS(P<0.0001,Fig.2-3-3E)and PFS(P<0.0001,Fig.2-3-3F).2.Correlation analysis of VEGFA and VASH1 with angiogenic markers in OC.2.1 Correlation of VEGFA with angiogenic markers in OC:In the OC dataset,the expression of VEGFA and CD34 were significantly positively correlated(r=0.392,P<2.2e-16,Fig.2-3-4A,B),and the expression of VEGFA and CD31 were also significantly positively correlated(r=0.401,P<2.2e-16,Fig.2-3-4C,D).In the normal ovary-fallopian tube tissue data set,there was a significant positive correlation between VEGFA and CD34(r=0.246,P=0.018,Fig.2-3-4E,F)and CD31(r=0.207,P=0.046,Fig.2-3-4G,H).2.2 Correlation of VASH1 with angiogenic markers in OC:In the OC dataset,there was a significant positive correlation between the expression of VASH1 and CD34(r=0.579,P<2.2e-16,Fig.2-3-5A,B)and between VASH1 and CD31(r=0.652,P<2.2e-16,Fig.2-3-5C,D).In the normal ovary-fallopian tube tissue dataset,VASH1 did not positively correlate with CD34(r=-0.029,P=0.78,Fig.2-3-5E,F)and CD31(r=-0.18,P=0.084,Fig.2-3-5G,H).3.Correlation analysis of VEGFA and VASH1 expression in OC.3.1 Correlation analysis of VEGFA and VASH1 with angiogenic pathways in ovarian cancer:Based on the results of the enrichment analysis,VEGFA was significantly enriched in the angiogenic pathway(P=0.016,Fig.2-3-6A).Similarly,VASH1 was significantly enriched in the angiogenic pathway(P=6.628e-06,Fig.2-3-6B).3.2 Correlation analysis of VEGFA and VASH1 expression in OC:In the OC dataset,the expression of VEGFA and VASH1 were significantly and positively correlated(r=0.364,P<0.0001,Fig.2-3-7A,B);in the normal ovary-fallopian tube tissue dataset,the expression of VEGFA and VASH1 were not correlated(r=0.168,P=0.108,Fig.2-3-7C,D);in the 131 HGSOC clinical samples,the VEGFA and VASH1 expressions were significantly correlated(r=0.45,P<0.001,Fig.2-3-7E).Conclusion1.VEGFA and VASH1 were highly expressed in HGSOC tissues and correlated with clinicopathological stage,microvessel density,and poor prognosis.2.VEGFA and VASH1 were positively correlated with the expression of angiogenic markers in the OC dataset.3.Both VEGFA and VASH1 expressions were correlated with angiogenic pathways in the OC dataset,and VEGFA and VASH1 expressions were positively correlated in the OC dataset and HGSOC tissues.Part Ⅱ Correlation study of regulatory T cells,FOXP3,and angiogenesis and the impact on the prognosis of OCPurpose1.To explore the correlation between the level of Tregs infiltration in OC and the expression of VEGFA,VASH1,and angiogenic markers.2.To explore the correlation of FOXP3 with VEGFA,VASH1,and angiogenic markers expression in OC;to detect FOXP3 expression in HGSOC tissues and analyze the correlation between FOXP3 and prognosis.3.To explore the common pathways of VEGFA,VASH1,and FOXP3 in the process of OC.Materials and MethodsMaterials1.Clinical data:clinical data and pathological tissue sections were collected from 131 patients with HGSOC in Qilu Hospital of Shandong University from 2014 to 2018.2.Bioinformatics data:RNA-seq data of 380 cases of OC were obtained from the TCGA database.Gene expression datasets of 935 OC patients were obtained from three datasets GSE9891,GSE32062,and GSE140082 in the GEO database,A marker gene set for 28 infiltrating immune cell types was obtained from Charoentong et al.RNA-seq data for a total of 33 cancers including OC from the TCGA database and a total of 31 solid tissues including normal ovary-fallopian tube tissue from the GTEx database were downloaded from the USCS Xena website.Methods1.To quantify the levels of infiltration of various immune cells in the TME of OC using the single sample gene set enrichment analysis(ssGSEA)function.The relationship between Tregs infiltration and CD34,CD31,VEGFA,and VASH1 expression was analyzed using TCGA,GEO,and the online TIMER database.2.Differential expression of FOXP3 in the OC dataset and normal ovary-fallopian tube tissue dataset was analyzed in the joint TCGA and GTEx databases;correlations between FOXP3 and CD34,CD31,VEGFA,and VASH1 expression were analyzed in the OC dataset and normal ovary-fallopian tube tissue dataset,respectively.3.Immunohistochemistry was performed to detect FOXP3 expression in HGSOC tissues and to score the staining.To analyze the relationship between FOXP3 expression and prognosis.4.To explore the potential mechanism of action of target genes VEGFA,VASH1,and FOXP3 jointly affecting the poor prognosis of OC using single gene-related GSEA.The PPI network of VEGFA,VASH1,and FOXP3 was constructed by the online database STRING and visualized using Cytoscape software.Results1.Correlation of Tregs infiltration levels with angiogenesis-related molecules in OC.1.1 TCGA database to explore the correlation between Tregs infiltration levels and angiogenesis-related molecules in the OC dataset:In the TCGA dataset,Tregs infiltration levels correlated significantly with CD34(r=0.45,P<0.0001,Fig.3-3-8B),CD31(r=0.62,P<0.0001,Fig.3-3-8D),VEGFA(r=0.36,P<0.0001,Fig.3-3-8F)and VASH1(r=0.59,P<0.0001,Fig.3-3-8H)expressions were all significantly and positively correlated.1.2 Validation of the correlation between Tregs infiltration levels and angiogenesisrelated molecules in the OC dataset in the GEO and TIMER databases:In the GEO dataset,Tregs infiltration levels were significantly correlated with CD34(r=0.27,P<0.0001,Fig.3-3-9B),CD31(r=0.61,P<0.0001,Fig.3-3-9D),VEGFA(r=0.07,P=0.043,Fig.3-3-9F)and VASH1(r=0.23,P<0.0001,Fig.3-3-9H)expression all showed significant positive correlations.In the TIMER online database,Tregs infiltration levels were significantly correlated with CD34(r=0.30,P<0.0001,Fig.3-3-91),CD31(r=0.43,P<0.0001,Fig.3-3-9J),VEGFA(r=0.25,P<0.0001,Fig.3-3-9K)and VASH1(r=0.25,P<0.0001,Fig.3-3-9L)expressions.significant positive correlations.2.Correlation between expression of FOXP3 and angiogenesis-related molecules in OC.2.1 Differential expression of FOXP3 in OC and normal tissues:FOXP3 expression was higher in a variety of tumor tissues than in the corresponding normal tissues(Fig.3-310A),and FOXP3 expression was also higher in OC than in normal tissues(P<0.001,Fig.33-10B).2.2 Correlation between FOXP3 and expression of angiogenesis-related molecules in OC:In the OC dataset,the expression of FOXP3 and CD34 were significantly positively correlated(r=0.379,P=5.11e-16,Fig.3-3-11 A,B);the expression of FOXP3 and CD31 were significantly positively correlated(r=0.527,P<2.2e-16,Fig.3-3-11C,D);the expression of FOXP3 and VEGFA were significantly positively correlated(r=0.174,P<0.001,Fig.3-3-11E,F);the expression of FOXP3 and VASH1 were significantly positively correlated(r=0.411,P<2.2e-16,Fig.3-3-11G,H).In the normal ovary-fallopian tube tissue dataset,FOXP3 correlated with CD34(r=0.096,P=0.361,Fig.3-3-111,J),CD31(r=0.054,P=0.61,Fig.3-311K,L),VEGFA(r=-0.321,P=0.002,Fig.3-3-11M,N)and VASH1(r=0.003,P=0.978,Fig.3-3-110,P)did not have significant positive correlations.3.Expression of FOXP3 in HGSOC tissues and relationship with prognosis.Immunohistochemistry showed that FOXP3 was expressed in the nucleus of OC cells,and 131 patients were divided into high and low FOXP3 expression groups based on immunostaining scores(Fig.3-3-12A,B).Kaplan-Meier curves showed that patients in the high FOXP3 expression group had worse OS(P=0.00027,Fig.3-3-12C).There was a similar trend in PFS,with patients in the FOXP3 high-expression group having more rapid disease progression(P=0.0012,Fig.3-3-12D).4.Exploring the potential common mechanism of VEGFA,VASH1,and FOXP3 in OC.4.1 Single gene-related GSEA to explore common pathways of action:GSEA was performed for VEGFA,VASH1,and FOXP3 respectively,and the top 15 gene sets in terms of enrichment scores were taken and intersected.The results showed that VEGFA(Fig.3-3-13 A),VASH1(Fig.3-3-13B),and FOXP3(Fig.3-3-13C)had five common enriched pathways,including angiogenesis,IL6/JAK/STAT3 signaling,PI3K/AKT/mTOR signaling,transforming growth factor-β(TGF-β)signaling,and the κ-light chain-enhanced signaling pathway in tumor necrosis factor-α/nuclear factor-activated B cells(TNF-α/NF-κB).4.2 Building a protein interaction network:There are 53 nodes and 998 edges in the PPI network,ranked according to the degree between each node,with importance reflected by circle size and a color shade(Fig.3-3-14A).The two most important modules identified by MCODE show that VEGFA and FOXP3 are associated with cytokines such as STAT3,IL2,IL6,IFNG,and tumor-related factors,while VASH1 is associated with microtubule activity(Fig.3-3-14B).Conclusion1.The level of Tregs infiltration in the OC tissue dataset was positively correlated with the expression of angiogenesis-related factors.2.FOXP3 expression was positively correlated with angiogenesis-related factor expression in the OC tissue dataset;FOXP3 is highly expressed in HGSOC tissue and is associated with poor prognosis.3.VEGFA,VASH1 and FOXP3 jointly mediate the development of OC through angiogenesis,IL6/JAK/STAT3 signalling,PI3K/AKT/mTOR signalling,TGF-β signalling,and TNF-α/NF-κB signalling pathways. |
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