| Epithelial ovarian cancer(EOC)is one of the three major malignant tumors of the female reproductive system.Its mortality rate ranks first among gynecological malignancies and seriously affects women's health.Due to the lack of specific tumor markers and early symptoms,more than70% of EOC patients are already at an advanced stage at the time of diagnosis.At present,the main treatment for EOC is surgery +platinum-based chemotherapy.In recent years,although adjuvant treatment strategies such as PARP inhibitors have also been applied to EOC,the 5-year survival rate of patients with advanced EOC is still hovering between 30-40%,with little improvement.Therefore,it is urgent to find the deep molecular mechanism that can reveal the occurrence and development of EOC,and provide a theoretical basis for the diagnosis of EOC and the development of new treatment methods.With the development of tumor immunology,immunotherapy has become the fourth kind of treatment along with surgery,chemotherapy and radiotherapy.The use of immunotherapy has revolutionized the treatment of a number of tumors,with treatments targeting immune checkpoint molecules such as CTLA-4 and PD-1 showing exciting results in melanoma,kidney cancer,Hodgkin's disease,and lung cancer.However,immunotherapy targeting existing immune checkpoints is less effective.More seriously,a variety of tumors,including ovarian and colorectal cancer,are largely resistant to CTLA-4 or PD-1 blockers.Therefore,it is necessary to conduct detailed studies on other immune checkpoint molecules in order to develop new immune checkpoint drugs and screen effective populations for immunotherapy,so as to improve the efficacy of immunotherapy.TIM-3 protein,a member of the T cell immuno-globulin domain and mucin domain protein(TIM)family,was initially found to be specifically expressed in Th1 cells,and its encoding gene is located in human chr5q33,with a total length of 23 kb and consists of 7 exons.Subsequent studies found that TIM-3 can be expressed in a variety of immune cells,such as monocytes,DCS,NK cells and other natural immune cells,and is involved in the regulation of immune cell function.A large number of tumor immunological studies have shown that TIM-3 not only plays a negative regulatory role in tumor immunity by regulating T cell depletion and inhibiting bone marrow cell proliferation,but also regulates anti-tumor immune response by promoting the phenotypic transformation of tumor-associated macrophages(TAMs).TIM-3 plays an important role as an immune checkpoint in tumor immunity.As Dr.Romero wrote in Nature in 2016,"Immunotherapy: PD-1 Says Goodbye,TIM-3 Says Hello." Therefore,TIM-3 has assumed a prominent position as a potential drug target for tumor immunotherapy.In recent years,a number of studies have also shown TIM-3expression in tumor cells,such as melanoma,stomach,B cell lymphoma,cervical cancer,renal clear cell carcinoma,osteosarcoma,etc.,and by participating in the IL-6/STAT3 pathway,TGF-?/Smad pathway and epithelial-mesenchymal transformation(EMT)process,such as promoting tumor invasion and metastasis,and TIM-3 abnormal expression is associated with poor prognosis in patients with.Previous studies have found that the expression level of TIM-3 in peripheral blood CD4+ and CD8+ T cells of ovarian cancer patients is significantly higher than that of normal people,while the clinical stage and tumor grade of patients with high expression of TIM-3 are significantly higher than those with low expression.Further studies have shown that high expression of TIM-3 in tumor-infiltrating Tregs in ovarian cancer patients is associated with poor prognosis.Therefore,TIM-3 may play an important role in the occurrence and development of EOC and may become a potential therapeutic target for EOC.However,the expression of TIM-3 in EOC tumor cells,the role and mechanism of TIM-3 in the development and progression of EOC tumors,and its relationship with the prognosis of EOC are still unclear.To elucidate the relationship between TIM-3expression and the development of EOC tumors and the possible mechanism of action,will help deepen the understanding of the pathogenesis of EOC and the development of new therapeutic targets,and provide new molecular markers for the screening of high-risk populations and the prediction of the efficacy of immunotherapy for EOC.Therefore,in this study,we investigated the expression of Tim-3protein and mRNA at the histological level,and analyzed the relationship between TIM-3 expression and the clinicopathological characteristics and prognosis of EOC.The effect of TIM-3 on the biological behavior of EOC cells and its mechanism were studied in vitro.Through case-control study,the association between rs10053538,rs10515746 and rs1036199 SNPs and the risk and prognosis of EOC was investigated,and the relationship between SNPs and TIM-3 mRNA expression was preliminarily analyzed.This study is divided into three parts.Part one: The expression of TIM-3 in epithelial ovarian cancer and its prognostic relationshipObjective: To explore the relationship between the expression of TIM-3 in EOC tissues and the clinicopathological characteristics and prognosis of EOC patients.Methods:1.Immunohistochemistry was used to detect the expression difference of TIM-3 protein in 46 EOC tissues and 21 normal ovarian tissues.2.The expression of TIM-3 mRNA in 126 EOC tissues and 21 normal ovarian tissues was detected by RT-qPCR,and the relationship between the expression of TIM-3 mRNA and the clinicopathological characteristics and prognosis of EOC was analyzed.The expression of TIM-3 mRNA in ovarian cancer and normal ovarian tissues in TCGA database was checked by online website GEPIA2(http://gepia2.CancerPKU.cn).3.The expression of TIM-3 protein in 135 cases of EOC was detected by multicolor immunofluorescence technique,and the relationship between the expression of TIM-3 protein and the clinicopathological characteristics and prognosis of EOC patients was analyzed.Results:1.The positive rate of TIM-3 protein in EOC group was 76.1%(35/46)higher than that in normal ovarian group(23.8%,5/21),and the difference was statistically significant(P<0.001).2.The relative expression of TIM-3 mRNA in EOC tissue was higher than that in normal ovarian tissues,and the difference was statistically significant(P<0.001).The expression of TIM-3 mRNA in ovarian cancer tissues in the TCGA database was higher than that in normal ovarian tissues(P<0.05),and the risk of tumor recurrence in patients with high TIM-3 mRNA expression in EOC tissues(HR=2.43,95%CI=1.47-4.04,P=0.001)and the risk of death(HR=1.84,95% CI=1.10-3.08,P=0.021)were significantly increased compared with patients with low expression.3.The expression of TIM-3 protein in EOC tissues was closely related to the clinical stage,differentiation and surgical residue of EOC(P<0.05).The expression of TIM-3 protein in the tumor region(CK+region)was closely related to the clinical stage and differentiation of EOC(P<0.05).4.Cox multivariate analysis showed that the risk of tumor recurrence(HR=1.99,95%CI=1.29-3.08,P=0.002)and the risk of death(HR=2.13,95%CI=1.37-3.30,P=0.001)were significantly increased in patients with high expression of TIM-3 in EOC compared with those with low expression.Patients with high CK+ region TIM-3 expression had a significantly increased risk of tumor recurrence(HR=1.64,95%CI=1.09-2.46,P=0.018)and death(HR=1.81,95%CI=1.19-2.75,P=0.006)compared with those with low CK+ region TIM-3 expression.Conclusions:1.TIM-3 protein and mRNA are highly expressed in EOC tissues.2.TIM-3 expression is related to clinical pathological factors such as EOC clinical stage and differentiation.3.The high expression of TIM-3 mRNA and protein in EOC tissues is an independent risk factor for tumor recurrence and death in patients.Part two: Study on the effect and mechanism of TIM-3 on the biological behavior of ovarian cancer cellsObjective: To explore the effect and mechanism of TIM-3 gene on biological behavior of ovarian cancer cells,and to lay a theoretical foundation for targeted therapy of EOC.Methods:1.The expression of TIM-3 in human ovarian cancer cells SKOV3,A2780,OVCAR3 and human normal ovarian epithelial cells IOSE80 were detected by RT-qPCR and western-blot.2.Liposome transfection method was used to construct ovarian cancer cell lines with low expression of TIM-3.CCK-8 method and plate clone formation assay were used to detect the changes of cell proliferation activity,flow cytometry was used to detect the changes of cell apoptosis,scratch assay and transwell chamber assay were used to detect the changes of cell migration and invasion ability.3.The changes of EMT and TGF-?/Smad pathways before and after transfection were detected by western-blot.Results:1.TIM-3 mRNA and protein in SKOV3,A2780 and OVCAR3 cells were higher than those in IOSE80 cells(P<0.001),SKOV3 cells were higher than A2780 and OVCAR3 cells(P<0.05)2.After LVRU6GP-TIM-3-sh RNA was transfected into SKOV3 cells,the mRNA and protein expression levels of TIM-3 were significantly lower than those in the empty plasmid group(P<0.05).The results of CCK-8 and plate clone formation experiments showed that After knocking down the expression of TIM-3,the proliferation rate of SKOV3 cells was significantly reduced(P<0.05).The results of flow cytometry showed that after knocking down the expression of TIM-3,the apoptosis rate of SKOV3 cells was significantly increased(P<0.05).The results of the scratch test and the Transwell chamber experiment showed that the migration and invasion ability of SKOV3 cells was significantly reduced after knocking down the expression of TIM-3(P<0.05).3.The expression level of epithelial marker E-cadherin in SKOV3-sh group cells(0.67±0.11)was significantly higher than that in SKOV3-NC group cells(0.26±0.08),and the difference was statistically significant(t=-5.221,P=0.006),while the expression of mesenchymal markers N-cadherin and Vimentin in the SKOV3-sh group(0.17±0.06,0.15±0.06)was significantly lower than that of the SKOV3-NC group(0.45±0.07,0.78±0.13),The difference was statistically significant(t=4.159,P=0.014,t=7.621,P=0.002).Compared with transfected empty plasmid,the expression of Smad7 in SKOV3-sh group cells increased(0.90±0.09 vs0.20±0.05),while the expression level of phosphorylated Smad2increased(0.35±0.13 vs 0.15±0.06),the differences were statistically significant(Smad7: t=-10.844,P=0.000,p-Smad2: t=7.621,P=0.002),but there was no significant difference in the expression of Smad2 in the two groups of cells(P>0.05).Conclusion:1.TIM-3 is highly expressed in ovarian cancer cells.2.Knocking down the expression of TIM-3 can inhibit the proliferation,invasion and migration of ovarian cancer cells,and promote their apoptosis.3.Knockdown of TIM-3 expression can inhibit the EMT process of ovarian cancer cells by reducing the activation degree of TGF-?/Smad signaling pathway.Part three: Study on the association between TIM-3 gene polymorphism and epithelial ovarian cancerObjective: To explore the relationship between TIM-3 gene polymorphism and EOC genetic susceptibility and the clinical prognosis of patients.Methods:1.The peripheral blood DNA samples of 700 patients with EOC and710 healthy women in our research group were used to genotyping three SNPs(rs10053538,rs10515746 and rs1036199)on TIM-3 gene by PCR-LDR method,and the relationship between different genotypes and the risk of disease and prognosis of EOC patients was analyzed.2.RT-qPCR was used to detect the expression level of TIM-3 mRNA in EOC tumor tissues carrying two SNPs sites in the promoter region(rs10053538 and rs10515746)with different genotypes,and to analyze the influence of these two sites on the expression of TIM-3 mRNA.3.The Kaplan-Meier plotter database was used to evaluate the prognostic value of TIM-3 mRNA in EOC patients.Results:1.The genotype and allelic frequency distribution of TIM-3 genes rs10053538,rs10515746 and rs1036199 were not statistically different between the case group and the control group(P>0.05).2.Prognostic analysis showed that patients with rs10053538 CA +AA genotype had worse PFS and OS than those with CC genotype(HR=1.49,95%CI=1.05-2.09,P=0.024,HR=1.57,95%CI=1.09-2.26,P=0.017),and patients with different genotypes of rs10515746 CC and CA+AA,and patients with different genotypes of rs1036199 AA and AC+CC had no significant difference in PFS and OS(P>0.05).3.RT-qPCR results showed that the expression of TIM-3 mRNA in the EOC tissues carrying the rs10053538CA+AA genotype was significantly higher than that of the CC genotype(P=0.006),while the expression of TIM-3 mRNA in the EOC tissues carrying the rs10515746CA+AA genotype Compared with the tissues carrying CC genotype,the difference was not statistically significant(P>0.05).4.Application of Kaplan-Meier plotter database analysis showed that the high expression of TIM-3 mRNA was associated with poor PFS and OS in EOC patients(HR=1.57,95%CI=1.29-1.91,P<0.001,HR=1.31,95%CI=1.06-1.63,P=0.013).Conclusion:1.TIM-3 gene rs10053538,rs10515746,rs1036199 single nucleotide polymorphisms are not associated with the risk of EOC.2.TIM-3 gene rs10053538 single nucleotide polymorphism is related to the prognosis of EOC patients.Carrying rs10053538CA+AA genotype is an independent risk factor for tumor recurrence and death in EOC patients.3.rs10053538 may be a functional polymorphism site.4.The high expression of TIM-3 mRNA is related to the poor clinical outcome of EOC patients. |
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