The Preliminary Study On The Relationship Of Tim-3 And Polymorphisms With Breast Cancer

[Background]Breast cancer is one of the most common malignant tumors in women and the incidence of breast cancer has increased by over 2%in recent years.It has been ranked the first place in women’s malignant tumors,and has a younger trend.Although treatment effect of breast cancer is significantly improved with the improvement of etiology and pathogenesis research,treatment and therapy of breast cancer,the prognosis of breast cancer is still not optimistic.Tumor metastasis,recurrence and treatment resistance remains the principal causes of death in patients with breast cancer.The etiology and pathogenesis of breast cancer is complex accompanied with multi-factor induction,multi-gene participation and multipath.The immune system of the body is closely related to the occurrence,development and metastasis of the tumor.On the one hand,the immune surveillance and immune clearance function is used to check and eliminate tumor cells;On the other hand,under the induction of tumor microenvironment,tumor cells can abnormally express some negative regulatory molecules so that tumor cells can escape from immune surveillance and negative regulate the body’s anti-tumor immunity.The invasion and metastasis of the tumor cells are the primary factors affecting the prognosis of tumor patients and the metastasis process is an extremely complex dynamic process with the interaction with cancer cells themselves,body immunity,endocrine,metabolism,microenvironment and other factors.In addition,the incidence of breast cancer is closely related to the genetic factors.The epidemiological studies showed that 12.9%of breast cancer patients have a family history and the risk of breast cancer increases with the consanguinity.The genetic factors of breast cancer involve multiple genes,and the single nucleotide polymorphisms of these genes and their specific combinations may be the most important causes of cancer susceptibility.As an important immune regulatory molecule of antitumor immune,Tim-3 has been a hot research topic in recent years.The research on its mechanism and the development of its specific McAb or molecules are of great research values and great potential for tumor immunotherapy.Tim-3 is widely expressed in Thl cells,cytotoxic CD8 + T cells,NK cells,macrophages,monocytes,dendritic cells and other immune cells involved in both inherent and adaptive immune response in humans.It can regulate the defense function of the body by adjust the secretion of cytokines and the function of immune cells and play an important role in a variety of immune-related diseases such as inflammatory diseases and tumors.Recent studies have confirmed that Tim-3 expression is not confined to the immune cells and ectopic expression is detected in a series of normal and malignant tissues.This molecule can play an important role in promoting tumor invasion and metastasis,and its expression is associated with poor prognosis of patients with breast cancer.However,the relationship between Tim-3 and breast cancer is poorly understood.In this study,we first investigated the association between Tim-3 gene single nucleotide polymorphisms and breast cancer susceptibility and prognosis to screen polymorphism loci or alleles associated with breast cancer susceptibility,thus providing data support to explore the genetic causes of breast cancer susceptibility,revealing the pathogenesis of breast cancer,and contributing to a theoretical basis for the early diagnosis,prevention and intervention of high-risk population of breast cancer.Second,we examined the Tim-3 expression in breast cancer tissues and corresponding paracancerous tissues,and analyzed the correlation between Tim-3 expression in breast cancer tissues with clinicopathological characteristics and prognosis.We further studied the Tim-3 expression in human breast cancer cells and its effect on proliferation,migration,invasion and apoptosis by downregulation and overexpression of Tim-3 in vitro,and to investigate the effects of Tim-3 on the metastasis-related molecules of breast cancer cells and the mechanism involving in tumor development,to identify new molecular mechanisms that inhibit the proliferation and metastasis of cancer cells,and to provide new strategies and new ideas for its diagnosis and treatment.The part I The relationship of Tim-3 gene polymorphisms with the susceptibility,clinicopathological features and prognosis of breast cancerObjectives:Breast cancer is the result of the combined action of many factors,including external environment and genetic factors.The genetic factors of breast cancer involve multiple genes,and the single nucleotide polymorphisms of these genes and their specific combinations may be the most important causes of cancer susceptibility.Studies have showed the polymorphism changes in the promoter and coding regions of Tim-3 gene,which may be associated with susceptibility to multiple diseases.However,the associations between Tim-3 SNPs and breast cancer susceptibility have been poorly understood.In the present study,we aimed to investigate the associations between three Tim-3 polymorphisms:rs 10053538(G>T)and rs 10515746(G>T)SNPs in the promoter region of the Tim-3 gene at positions-1516 and-574,respectively,as well as the rs1036199(T>G)SNP mapped to exon 3(at position +4259)and breast cancer susceptibility in Han ethnicity of northern China.Meanwhile,the relationship between Tim-3 polymorphisms and clinicopathological features was compared to explore the relationship with the progression and prognosis of breast cancer.Methods:We genotyped the SNPs of Tim-3 gene in 301 invasive breast cancer patients and 151 healthy controls and analyzed the distributions of genotype frequencies to evaluate the association of the genotypes with the breast cancer susceptibility,the clinicopathological characteristics and prognosis.2-3ml venous blood was collected from the subjects,and the genomic DNA samples were extracted.The collected samples were amplified by nested PCR and sequenced.The sequence files were compared with the SNP sequences of Tim-3 gene using Sequencher 5.0 software.The results of the experiment were analyzed by SPSS 19.0 software.The Hardy-Weinberg balance analysis,haploid and linkage disequilibrium analysis were performed by SHEsis software.The observed and expected genotype frequencies in both the controls and the patients were compared using the Chi-squared test or the Fisher’s exact test with one or more variables(<5).Unconditional logistic regression calculation dominance ratio(OR)and its 95%confidence interval(CI)were used to estimate the association between risk factors and risk of breast cancer.Results:1.The distribution of genotypes and allele frequencies at-1516G/T or-574G/T loci in breast cancer patients and healthy controls did not show any significant difference,and there was no correlation between the SNPs and the risk of breast cancer(P>0.05).For the+4259T/G SNP,the prevalence of the TT and TG genotypes were 90.7%and 9.3%in cases and 98.7%and 1.3%in controls,respectively.The +4259 TG genotype revealed a significantly increased risk of breast cancer development(OR=7.641,95%CI,1.795-32.522,P=0.001).Similarly,the +4259G allele was increased in breast cancer(OR=7.317,95%CI,1.731-30.925,P=0.001).2.The gene frequencies of-1516G/T or-574G/T did not show any significant difference in breast cancer cases in terms of histological grade,tumor size,lymph node or distant metastasis(P>0.05).However,the polymorphism at + 4259T/G was not associated with histological grade and tumor size in breast cancer patients,but was associated with lymphatic metastasis or distant metastasis.The probability of breast cancer with TGgenotype associated with tumor metastasis was 3.158 times higher than that of the TT genotype(OR 3.158,95%CI:1.300-7.672,P = 0.011).3.The prevalence of the +4259T/G genotype was not significantly associated with Immunohistochemical indexes commonly used in breast cancer ER,PR and HER-2(P>0.05),but correlated with Ki-67 proliferation index(OR 2.632,95%CI:1.121-6.179,P =0.022).Conclusions:The-1516G/T and-574G/T locus SNPs in the promoter region of the Tim-3 gene are not related to the susceptibility to breast cancer,but the +4259T/G SNPs in the exon region are related to the genetic susceptibility of breast cancer patients.+ 4259T/G SNPs is associated with tumor metastasis and Ki-67 proliferation index,suggesting +4259T/G SNPs can be used as a candidate susceptibility gene for invasive breast cancer in Chinese northern Han women and an important molecular marker for predicting the progression and prognosis of breast cancer patients.The part Ⅱ The relationship of expression of Tim-3 with clinicopathological features and prognosis of breast cancerObjectives:As a negative regulator of anti-tumor immunity,Tim-3 plays an important role in tumor immunity,organ transplantation,inflammatory disease and autoimmune diseases.The mechanisms promoting tumor immune escape,invasion,metastasis and recurrence has gained much attention.Through the mechanism research and development of its specific antibody or molecule,it has important research values and great potential for tumor immunotherapy.Recent studies have confirmed that Tim-3 expression is not limited to immune cells,but also ectopic expression in a series of normal and malignant tissues,which can play an important role in promoting tumor invasion and metastasis and be associated with poor tumor prognosis.In the present study,we respectively detected the expression of Tim-3 mRNA and protein levels in breast cancer tumor tissue and corresponding adjacent normal tissue,and analyzed the correlation between the Tim-3 expression level and clinical pathological features in breast cancer to explore the role of Tim-3 in the occurrence,development and prognosis of breast cancer.Methods:We randomly selected 42 patients with invasive breast cancer in shandong university affiliated to shandong university from 2011 to 2012.The specimens of breast cancer tissues and corresponding adjacent normal tissues were embedded in paraffin,and tissue sections were used for tissue immunochemical examination.In addition,20 cases of fresh breast cancer tissues and adjacent tissues were randomly collected in 2015,which were used for qRT-PCR and Western blot.In addition,clinical information were collected,including age,tumor clinical stage,histological grade,tumor size,lymph node and distant metastasis,and PR,ER,p53,Ki-67 index and total survival time(OS).All the experimental data were analyzed using SPSS 19.0 software.Results:1.The immunohistochemical results showed that Tim-3 protein was mainly located in the cytoplasm and cell membrane of breast cancer cells.In 42 pairs of tissue specimens,positive Tim-3 staining was identified in 42.9%(18 out of 42)of breast cancer but only 18.2%(4 out of 42)of adjacent tissues.The positive rate of Tim-3 protein in breast cancer tissues was significantly higher than that in adjacent tissues(P<0.01).When compared with the semiquantitative immunoreaction score,the expression of Tim-3 protein was much higher than that of adjacent normal tissues(P<0.01).2.Fresh breast cancer and corresponding adjacent normal tissues were collected for further protein extraction and western blot to verify the Tim-3 protein expression.The Tim-3 protein bands were found in the lanes of both tissues,but the expression in breast cancer tissues was relatively stronger than adjacent tissues.The expression of Tim-3 mRNA were detected by qRT-PCR,and the relative expression levels of Tim-3 mRNA in breast cancer tissues were significantly higher than that of the corresponding normal tissues(P<0.001).3.The relationship between the expression levels of Tim-3 protein and clinicopathologic features showed that high immunoreactivity of Tim-3 was significantly correlated with clinical stage(P=0.0278),metastasis(P=0.0207)and immunohistochemistry Ki67 index(P=0.0444),while showing no significant correlation with age,histological type,tumor size and PR、ER、p53(P>0.05).4.Kaplan-Meier curve survival analysis showed that the 5 year survival rate of breast cancer patients with Tim-3 negative expression was 95.8%,which was significantly higher than that of Tim-3 positive breast cancer patients(72.2%)(log-rank χ2=5.171,P=0.023).Conclusions:1.Tim-3 protein and mRNA in human breast cancer tissues were abnormally highly expressed,suggesting Tim-3 expression may be related to the occurrence and development of breast cancer.2.Tim-3 expression level is closely related to clinical stage,tumor metastasis,Ki67 proliferation index and short survival of breast cancer,suggesting that it is closely related to tumor progression and poor prognosis of patients and is expected to become a new prognostic marker and therapeutic target.The part Ⅲ Effects and mechanisms of Tim-3 on biological behavior of breast cancer cellsObjectives:Invasion and metastasis of tumor cells in breast cancer are the primary factors that influence the prognosis of cancer patients.Metastasis is a very complex dynamic process,including the interactions between cancer cell characteristics,body immunity,endocrine,metabolism and microenvironment,and many other factors.Studies showed that EMT process plays a very important role in the occurrence and development of breast cancer,and has a correlation with high invasive and metastatic breast cancer.Studies have shown that Tim-3 can trigger invasive EMT features of osteosarcoma tumor cells and may be involved in the pathogenesis of malignant tumors.We inferred that Tim-3 promotes the development and metastasis of breast cancer,which may be related to EMT.In the present study,we studied the effect of Tim-3 on proliferation,migration,invasion and apoptosis of human breast cancer cells in vitro through overexpression and targeting silencing of Tim-3 in breast cancer cells,and further explored the mechanism of Tim-3 on breast cancer metastasis.Methods:The expression of Tim-3 protein in breast cancer cells was detected by Western blot.Silencing Tim-3 expression siRNA(siRNA-Tim-3)and Tim-3 overexpression plasmid(pcDNA3.1-Tim-3)were constructed and transfected into MCF-7 and MDA-MB-231 cells respectively.The expression level of Tim-3 mRNA in transfected cells was detected by qRT-PCR,and the transfection efficiency was verified.CCK8 assay was used to detect the proliferation of cells in each group;Cell scratch assay was used to detect the migration ability of cells in each group;Transwell chamber was used to test the invasion ability of each group;Flow cytometry was used to detect the apoptosis of cells in each group.Western blot was used to detect the effect of Tim-3 gene expression on the expression of EMT metastasis-associated molecules(MMP-9,TIMP-1,E-cadherin,N-cadherin,and Vimentin).Results:1.The expression of Tim-3 protein was found in breast cancer MDA-MB-231,MCF-7 and MCF-7/ADM cells.The results of Tim-3 mRNA in MDA-MB-231,MCF-7 and cells after transfection showed that compared with the control group,the expression of Tim-3 mRNA was significantly lower in the siTim-3 group(P<0.05),while the expression of Tim-3 mRNA was significantly higher in the Tim-3 group(P<0.05).2.The results of cell proliferation experiments showed that in MCF-7 and MDA-MB-231 cells,compared with the control group,the proliferation activity of siTim-3 cells at each time point was significantly reduced(P<0.05),while the Tim-3 group The proliferation activity of the cells at all time points was significantly higher(P<0.05).The cell scratch test results showed that in MCF-7 and MDA-MB-231 cells,compared with the control group,the migration rate of siTim-3 cells was significantly lower(P<0.05),while the migration rate was significantly increased in the Tim-3 group(P<0.05).Transwell assay results showed that in MCF-7 and MDA-MB-231 cells,compared with the control group,the number of invasive cells in the siTim-3 group was significantly reduced(P<0.05),while the invasive cells in the Tim-3 group was increased significantly(P<0.05).The results of flow cytometry showed that in MCF-7 and MDA-MB-231 cells,the apoptotic rate of siTim-3 group was significantly increased compared with the control group(P<0.05),while the apoptotic rate of Tim-3 group was significantly decreased(P<0.05).3.In MCF-7 and MDA-MB-231 cells,the expression levels of E-cadherin and TIMP-1 were significantly higher in the siTim-3 group than that in the control group(P<0.05),while the expression levels of MMP-9,N-cadherin,Vimentin protein were significantly decreased(P<0.05).Compared with control group,the expression levels of E-cadherin and TIMP-1 protein in Tim-3 group were significantly lower(P<0.05),while the expression levels of MMP-9,N-cadherin,Vimentin protein were significantly higher(P<0.05).Conclusions:1.Tim-3 is expressed on breast cancer cells and has the biological function to promote the proliferation,invasion and migration of breast cancer cells and inhibit the apoptosis of cancer cells.2.Tim-3 may promote the invasion and metastasis of breast cancer cells by inducing EMT or promoting EMT,thereby promoting the occurrence and development of breast cancer.