Expression And Mechanism Of HHLA2 In Ovarian Cancer

The deadliest gynecologic cancer is ovarian cancer.Due to Chinese large population,there are many new cases and death cases of ovarian cancer in China.The early symptoms of ovarian cancer are not obvious.What’s worse,the early effective screening methods are very limited.Ovarian cancer is complex and its prognosis is poor.Traditional clinical treatments are the standard strategy for ovarian cancer,which include chemotherapy,surgery and radiotherapy.But for advanced ovarian cancer patients,these traditional treatments are rather futile.Immunotherapy is a new treatment.We believe it will play an important role in the comprehensive treatment of ovarian cancer.Immunotherapy can inhibit the occurrence and development of tumor by activating or enhancing the body’s anti-tumor immune function.It can also promote the apoptosis of tumor cells.In order to maintain physiological balance,the immune system recognizes and clears the mutant cells through T cell mediated cellular immunity.The abnormal immune monitoring function and the escape of tumor immunity lead to the occurrence and development of tumors.In the immune microenvironment of the body,costimulatory molecules are involved in the process of tumor immune escape,metastasis and migration.On the other hand,they play an important role in the occurrence and development of tumors.So costimulus molecules are an important part of the tumor microenvironment.HHLA2,also named B7-H7,is a new member of the B7 family.Many research showed that HHLA2 was broadly expressed in most kinds of human tumor tissues.It was also indicated that HHLA2 was associated with cancer progression and prognosis.HHLA2 may be a new target for tumor immunotherapy.However,the research on HHLA2 is limited up to now.The expression of HHLA2 and its mechanism are less studied.The purpose of this study was to investigate the expression,the mechanism of HHLA2 on ovarian cancer cell function.The objective is to provide a new ideas and basis for the immunotherapy of ovarian cancer.Part Ⅰ Expression characteristics and significance of HHLA2 and its receptor TMIGD2 in ovarian cancerObjective:To investigate the expression of HHLA2 and TMIGD2 in ovarian cancer.Methods:By IHC method,the expression of HHLA2 and its receptor TMIGD2 in ovarian cancer tissue microarray containing 154 points/154 cases of tissue was respectively detected.The correlation between the expression and the clinicopathological characteristics of patients was analyzed.We further analyzed the significance of the correlation between the expression and prognosis of patients.Results:Both HHLA2 and TMIGD2 expressions are related to the pathological type.Kaplan-merier survival analysis showed that the high expression levels of HHLA2 and TMIGD2 suggested a worse prognosis for type I ovarian cancer,and the difference was significant(P<0.05).Single factor Cox proportional hazards model shows that type II,FIGO Ⅲ and Ⅳ stage,lymph node metastasis,distant metastasis are associated with worse prognosis.Multivariate proportional Cox risk model showed that FIGO stage,lymphatic metastasis,distant metastasis,and TMIGD2 may be independent influencing factors for poor prognosis of ovarian cancer patients.And HHLA2 has significant positive correlation with TMIGD2 expression(r=0.6032,P<0.0001).Conclusion:The higher expression level of HHLA2 and TMIGD2 suggested a worse prognosis.Type Ⅱ patients have higher TMIGD2 or HHLA2 expression.The higher TMIGD2 expression may be an independent prognostic factor for ovarian cancer.HHLA2 and TMIGD2 expression were significantly correlated in the ovarian cancer tissues.Part Ⅱ Correlation between the expression of HHLA2,PD-1 and PD-L1 in ovarian cancerObjective:To investigate the expression of HHLA2,PD-1 and PD-L1 in EOC and their relationship.Methods:We used the mIHC to test the quantitative expression levels of HHLA2,PD-1 and PD-L1 in TMA of EOC.The relationships between the expression of various immune factors in different compartment and clinicopathological parameters were analyzed.Furthermore,the correlations between HHLA2 and other immune factors were analyzed.Results:HHLA2,PD-1 and PD-L1 are broadly expressed in EOC.The expression of HHLA2 and PD-L1 were significantly higher in the tumor compartment than in the stromal compartment(P<0.05).In tumor compartment,PD-L1 expression was significantly related with FIGO stage(P=0.006).In stromal compartment,HHLA2 expression was significantly associated with FIGO stage,lymphatic metastasis,distant metastasis and OS(P<0.05).Type II patients expressed significantly higher PD-1 and PD-L1 than type I patients(P<0.05).The Kaplan-Merier survival analysis revealed that higher HHLA2,PD-1,PD-L1 expression in stromal compartment were significantly correlated with the poor OS of EOC patients(P=0.025,P=0.022,P=0.0486).In the univariate analysis,the expression of HHLA2,PD-1,PD-L1 in stromal compartment showed significant correlation to the survival of EOC patients.Meanwhile,pathological type,FIGO stage,lymph node metastasis,distant metastasis were found to be associated with survival.The multivariate analysis indicated that distant metastasis,FIGO stage,and PD-1 expression in the stromal compartment were independent factors in OS of EOC patients.Spearman correlation analysis showed that the expression of HHLA2 in tumor compartment was positively correlated with PD-L1.In the stromal compartment,HHLA2 expression was positively correlated with the expression of PD-L1 and PD-1.Conclusion:High expression of HHLA2,PD-1,and PD-L1 in stromal compartment were significantly associated with a worse prognosis in EOC.HHLA2,PD-1 and PD-L1 might be prognostic predictors and potential therapeutic targets in EOC.Moreover,HHLA2 expression was associated with that of PD-1,PD-L1 in stromal compartment of EOC.Part Ⅲ Mechanism of HHLA2 on ovarian cancer cell functionObjective:To investigate the effect of the HHLA2 expression on growth,proliferation and invasion of ovarian cancer.Methods:Three ovarian cancer cell lines were selected according to the database.In vivo,HHLA2 down-regulated cells were constructed by lentivirus transfection with synthetic siRNA,and the HHLA2 expression was detected by qRT-PCR.CCK8,the scratch and the transwell methods were performed to test the effect of HHLA2 on the biological function of ovarian cancer cells.We used qRT-PCR and Western Blot to test the expression of IKKβ,RelA,p-IKKβ,p-RelA and CA9 in HHLA2-down-regulated cell lines.We performed luciferase activity to test CA9 promotery.We constructed CA9 overexpression cells.CCK8,the scratch and the transwell methods were also performed to test the effect of CA9 overexpression on cell function.In vitro,the volume and weight of tumor formation of ovarian cancer cells were detected and compared by tumor formation assay in mice.We used IHC to test the expression of HHLA2,CA9,IKKβ,RelA,p-IKKβ,p-RelA in tumor tissues of mice.Results:The proliferation,migration and invasion of HHLA2 down-regulated ovarian cancer cells were significantly reduced(P<0.05).The down-regulation of HHLA2 can inhibit the CA9 promoter activity,decrease CA9 expression,and increase the phosphorylation levels of NF-κB signaling molecules IKKβ and RelA.CA9 overexpression can significantly reverse the inhibitory effect of HHLA2 down-regulation on cell proliferation,migration and invasion.Tumor formation experiments in mice showed that tumors in the HHLA2-down-regulated group were significantly smaller than those in the empty vector control group(P<0.05),and tumors in the HHLA2-down-regulated-and-CA9-overexpression group were significantly larger than those in the HHLA2-down-regulated group(P<0.05).IHC showed that in HHLA2-down-regulated mice tumor tissues the expression of p-IKKβ and p-RelA were higher and the expression of CA9 was lower.Conclusion:HHLA2 down-regulation mediated the decrease of CA9 expression through the activation of NF-κB signaling pathway,further inhibited the proliferation and migration of ovarian cancer cells,and promoted cell apoptosis.