The Mechanism Of ANXA3 In Gastric Cancer And The Relationship Between The Distribution Of IL-35~+ B Cells In Peripheral Blood And Gastric Cancer

The incidence and mortality of gastric cancer have remained high throughout the world,especially in China and Southeast Asia.The number of incidence and deaths in China is more than half of the world every year.The pathogenesis,diagnosis and treatment of gastric cancer are always puzzled scientists all over the world.In January 2015,Obama as president of the United States of America proposed the concept of"precision medicine" in his state of the Union address,cancer as the main problem to conquest is attracting more and more attention.Different from the traditional methods of treatment,the precise treatment of gastric cancer mainly includes targeted therapy and biological immunotherapy.Targeted therapy,as the name implies,that can anchor the specific target related the progress of gastric cancer,then design and invent drugs working on these targets that could effect on the target gene exclusively in human body.Then blocking the progression of tumor through suppressing target gene function to achieve the goal of cure.The mechanism of biological immunotherapy is mainly based on immunology theory,such as building specific immune effector cells for targeted binding,specifically identifying tumor cells which in vivo can participant in the immune escape and eliminate immune cells with strong immunosuppressive effect.Under normal circumstances,body's immunity can recognize and remove alien elements or abnormal cells,but tumor cells possess abilities of escaping immune surveillance,then entering into proliferation,differentiation,formation of tumor tissue,subsequently colonization in various parts of human body via the EMT progression and walk anywhere through the blood and lymphatic system.Researches have showed that there is a large number of immunosuppressive cells in tumor microenvironment,which can release strong and effective immunosuppressive cytokines to prevent T cells and other immune cells' differentiation and proliferation,and also can promote normal immune cell differentiation as immunosuppressive cells,producing potent immune effect.The precise treatment in gastric cancer development,on the one hand is to find the specific target which can provide scientific basis for clinical early diagnosis,follow-up treatment and prognosis;on the other hand is to find key factors which interfering the body's normal immune function,then through the method of biological immunotherapy to enhance or recover the immune function,so as to achieve the purpose of cancer cure.In this paper,we have done experimental research on gastric cancer from two parts,in order to provide theoretical references and experimental basis for the clinical diagnosis and treatment of gastric cancer.Part I:The study on the expression and mechanism of ANXA3 in gastric cancerBackgroundGastric cancer(GC)is a common gastrointestinal malignant tumor which has high incidence worldwide.By 2012,the data of International Agency for Research on Cancer(IARC)showed that more than 950,000 new cases have ocurred,and more than 720,000 deaths causing of GC.Up to now,GC is becoming the fifth most common malignancy in the world after lung cancer,breast cancer,colorectal cancer,and prostate cancer.The incidence and mortality of GC is ranking the fifth and the third separately.In Asian countries,especially in China,the incidence and mortality of GC are much higher than that of other countries.Although surgery and chemoradiotherapy technology are developing constantly,the overall survival of GC patients is not improved noticeably.Patient in China who was first diagnosed usually defined advanced GC,and with the high metastatic and recurrence properties so that the 5-year survival rate of postoperative GC patients was still below 30%.So besides traditional therapy,it is extremely important for us to explore new target which can affect the development of GC.Meanwhile,it may provide theoretical evidence of early screening and subsequent targeting therapy.The epithelial-mesenchymal transition(EMT)is the essential process of embryo development,and more and more studies show that it is closely associated with the process of tumor invasion and migration.EMT showed that in early stage of tumor metastasis,the tight junction between epithelial cells was broken by tumor cells.Then tumor cells made normal epithelial cells lose the polarity and gained motional ability,subsequently migrated into the extracellular matrix via a series of roles of protease.The main feature of EMT is the reduction of epithelial cell markers like adhesion molecule(E-cadherin)and cytoskeletal protein(keratin),while mesenchymal cell markers(fibronectin,vimentin,and so on)are increasing.Moreover,EMT also involved many pathways of signal transduction,such as TGF-?,NF-?b,RTK/Ras,Wnt/?-catenin and so on,so it may play an important role in the development of cancer.Annexin A3(ANXA3),is a member of annexin family,located at chromosome 4q13-q22,which is encoding two protein hypotypes with the molecular masses of 33kDa and 36 kDa,as well as named as lipocortin3,placental anticoagulant protein3(PAP-?).ANXA3 is calcium-dependent phospholipids binding protein which can combine with the phospholipid membrane only in the presence of Ca2+.Its conserved C-terminus contains four similar annexin repeat domains(?-?)which is consisted of about 70 amino acid residues.Researchers found that ANXA3 played an important role in many malignant tumors,but there are few studies in gastric cancer.In 2014,Zhai et al found that the expression of ANXA3 was high in GC tissue,and was positively correlated with the tumor size and TNM,negatively correlated with the outcome.However,they did not do further study on the function of ANXA3 in GC.Therefore,we combined with previous studies and speculated that ANXA3 might become a marker for gastric cancer treatment and early diagnosis.Up to now,some studies found that the expression of ANXA3 in prostate cancer and thyroid cancer tissues decreased,indicating that it might act a role of suppressor gene.However,in most malignant tumors,such as lung cancer,breast cancer,ovarian cancer,liver cancer,colorectal cancer and pancreatic cancer,the expression of ANXA3 increased,meanwhile it played a role of angiogenesis,promoting metastasis,and drug resistance,it was suggested that ANXA3 might be as an oncogene.Nonetheless,the expression of ANXA3 and the mechanism in GC was reported rare.ObjectivesThis study from clinical specimens,experiments in vitro and in vivo to elaborate the role of ANXA3 in the development of GC.At the same time,we attempt to elucidate the mechanism of ANXA3 in promoting the metastasis of GC via exploring the effect of ANXA3 on EMT process.The aim is to find new targets for diagnosis and prognosis of gastric cancer.Methods1.qPCR and western blot were performed to detect mRNA and protein expression of ANXA3 in GC cells and tissues.IHC was used to detect ANXA3 expression in 183 samples,combined with clinical materials to find out the relationship between ANXA3 and tumor stage,grade.So as to provide theoretical evidence for clinical diagnosis and prognosis.2.Knocked down ANXA3 in high expression GC cell lines,and overexpressed ANXA3 in low expression GC cell lines to detect the alteration of proliferation,invasion and migration by proliferation assay,colony formation assay and transwell assay.Xenograft transfer model was infected with SGC7901 which is stably transfected with shRNA-ANXA3 to observe tumor size and weight in order to make clear the effect of ANXA3 on the progression of GC in vivo.3.Western blot was used to detect the expression of EMT marker(E-cadherin,fibronectin,Vimentin and ?-catenin)and transcription factor(snail and slug)after ANXA3 knocked down.Real-time PCR was used to find out the correlation between ANXA3 and EMT markers(E-cadherin and Vimentin)to explore the mechanism of the development of GC.Results1.The expression of ANXA3 in mRNA and protein level were remarkable increased,much higher in GC cell lines than in normal gastric epithelial cells,and elevated in GC tissues compared to normal adjacent tissues.IHC divided 183 cases into two groups,ANXA3 high expression group(105 cases)and ANXA3 low expression group(78 cases).Analyzed with relevant clinical pathological indicators found that ANXA3 high expression group was positively correlated with tumor size(P=0.006),infiltration depth(P<0.001),TNM stage(P<0.001),local lymph node metastasis(P=0.023)and distant metastasis(P=0.014).Kaplan-Meier survival curves showed that ANXA3-low patients could get higher overall survival and disease-free survival(P<0.001).Moreover,univariate and multivariate analyses showed that the expression of ANXA3 was significantly correlated with patient overall survival,tumor infiltration depth,TNM stage,local lymph node metastasis and distant metastasis,but had no correlation with patient age and sex.2.In vitro,overexpressed ANXA3 in MGC803 and HGC27 promoted invasion and migration;Knocked down ANXA3 in SGC7901 and MKN45 inhibited colony formation and proliferation.Likewise,in vivo,knocked down ANXA3 decreased the tumor size and weight markedly.3.In knocked down GC cell lines,western blot results showed that EMT markers and transcription factors,the expression of E-cadherin decreased,at the same time,the expression of Vimentin,Fibronectin,?-catenin,Snail and Slug increased.Real-time PCR results showed that the expression of ANXA3 was negatively correlated with E-cadherin,positively correlated with Vimentin.It was indicated that ANXA3 indeed promoted the metastasis of GC.Conclusions1.In GC cell lines and tissues,the expression of ANXA3 was very high,and positively correlated with tumor size,TNM stage,local lymph node metastasis and distant metastasis,negatively correlated with prognosis and survival.2.In vitro,high expression of ANXA3 could promote proliferation,invasion and migration.In vivo,low expression of ANXA3 could inhibit the growth of tumor.It was implied that ANXA3 could accelerate the development of GC and play an important role in the process.3.Knocked down ANXA3 increased the expression of E-cadherin,decreased the expression of Vimentin proved that ANXA3 could promote the EMT of GC,thus promoted the metastasis of GC.It was supposed that ANXA3 could act as a potential prognostic factor and potential therapeutic target of GC patients.Part II:The relationship between the distribution of IL-35+ B cells in peripheral blood and GCBackgroundThe high rate of recurrence and mortality is the key factor that affecting GC treatment.Mostly,people who is first diagnosed of GC is in advanced stage.Due to the limitation of traditional treatment,the new treatment as an effective supplement to conventional treatment can prolong the survival period and improve the prognosis of patients.Biological immunotherapy as an emerging therapeutic method is gradually accepted by people,however,most of the research and treatment are concentrated in T cells,NK cells,DC cells and other immune cells,the study on the role of B lymphocytes in malignant tumors is rare.B lymphocytes,as well known immune cells,when stimulated by antigens can be differentiated into plasma cells secreting antibodies and acting on antigens.Meanwhile,B cells can also serve as antigen presenting cells(APC),presenting antigens to T cells and other immune cells.In B lymphocytes,a group of cells can secrete IL-10,TGF-beta,IL-35 and other immunosuppressive cytokines,and the function is similar to regulatory T cells(Treg cells).We define this group of cells as regulatory B cells(Breg cells)temporarily.IL-35,as a member of the IL-12 family,is composed of two subunits of p35 and EBi3.It plays an immunosuppressive role in many autoimmune diseases and inflammatory diseases.However,the relationship between IL-35-producing B cells(IL-35+ B cells)and malignant tumors is rarely reported,especially in GC.ObjectivesThe relationship between IL-35+ B cells and GC are not clear yet.We detected and analyzed the distribution of IL-35+ B cells in peripheral blood of GC patients and healthy volunteers to find out the correlation of IL-35+ B cells and GC staging;meanwhile,we also detected the relationship between IL-35+ B cells and other immune cell subgroups,such as Treg cells and MDSCs.Methods1.We collected peripheral blood from GC patients(50 cases)and healthy volunteers(30 cases).Labeled monoclonal antibodies were added to peripheral blood.After incubation for 20 min in the dark at 4?,then lysed red blood cells and washed with PBS for detection.2.Peripheral blood mononuclear cells(PBMC),adding stimulating agents and Golgi blockers(CpG(ODN 2006)and GolgiPlugTM),stimulated culture at 37?,5%CO2 incubator,after washing with PBS and added labeled monoclonal antibody for detection;3.We use the method of flow cytometry to find out the relationship between IL-35+ B cells and GC stages in the peripheral blood;then we detected the distribution of myeloid derived suppressor cells(MDSCs)and Treg cells,and the relationship with IL-35+ B cell;finally,we detected the distribution of CD4,CD8 cells,activated CD4,CD8 cells,memory CD4,CD8 cells,NK cells,CD 14+monocytes and IL-10+ B cells,and analyzed the correlation with the distribution of IL-35+ B cells.Results1.In the peripheral blood of GC patients,the distribution of IL-35+ B cells was increasing according to the GC stages.The highest concentration of distribution was found in stage IV patients,and there was a significant difference among other groups(P<0.001).2.In the peripheral blood of GC patients,the distribution of MDSCs and Treg cells were increasing according to the GC stages.The highest concentration of distribution was found in stage IV patients.Meanwhile,there was a positive correlation between the distribution of IL-35+ B cells.3.Detection of CD4,CD8 cells,activated CD4,CD8 cells,memory CD4,CD8 cells,NK cells,CD14+ mononuclear cells and IL-10+ B cells.Interestingly,the frequency of CD 14+ monocytes and IL-10+ B cells were positively correlated with the frequency of IL-35+ B cells(p<0.05).There was no significant difference among other groups.ConclusionsIn the peripheral blood of GC patients,the frequency of IL-35+ B cells,MDSCs and Treg cells increased,and positively correlated with the staging of GC.While the frequency of IL-35+ B cells was also positively with the frequency of MDSCs,Treg cells,CD 14+ mononuclear cells and IL-10+B cells,suggesting that iL,35+ B cells play an important role in immune suppression of GC,at the same time via recruitment and regulation of other immune cells to play synergistic immunosuppression which accelerated the progression of GC.