The Effect Of MATK On The Proliferation,Invasion And Migration Of Diffuse Large B Lymphoma And The Preliminary Study Of Its Molecular Mechanism

Introduction:Diffuse Large B cell lymphoma(DLBCL)is a hematological malignant disease,which belongs to the most common pathological subtype of non-Hocking lymphoma(NHL).The incidence of DLBCL accounts for about 40%of all new-onset NHL patients.The disease tends to occur in the middle and old aged,and is slightly more common in males,with 50%to 60%of patients presenting with painless progressive lymphadenopathy,and 40%to 50%of patients presenting with extra-nodal disease,which may be accompanied by compression of the affected site or impairment of organ function,you may also have symptoms of fever,night sweats,and weight loss.DLBCL can be divided into two types:germinal-center type(GCB)and non-germinal-center type(non-GCB).DLBCLis heterogeneous in morphology,genetics,biology and clinical manifestations.In recent years,with the widespread application of combined treatment programs based on R-CHOP,the survival prognosis of clinical DLBCL patients has been extremely improved,with a 5-year survival rate of more than 60%.However,some(about 30%-50%)of DLBCL patients will die due to drug resistance and rapid disease recurrence.Therefore,clarifying the molecular regulation mechanism of DLBCL and finding precise and effective treatment measures have extremely important social value and clinical significance for improving the clinical prognosis of DLBCL patients.With the gradual advancement of human knowledge of molecular biology and genetics,our research on the mechanism of malignant tumors has gradually deepened.Some researchers have proposed the"seed-soil"theory,which compares cancer cells to a seed.If the seed leaves the suitable soil,it cannot germinate.Therefore,the occurrence and development of tumors not only depend on some characteristics of the tumor cells themselves,but are also affected.The influence of the soil that tumor cells are suitable for growth and development is also the tumor microenvironment(TME)that has been studied more frequently.In recent years,the research of tumor microenvironment is a hot topic in the field of tumor research.A large number of studies have confirmed that the process of tumor development is not only related to the growth and proliferation of tumor cells,but also related to the matrix,new blood vessels and a large number of infiltrating inflammatory immune cells in the tumor microenvironment.The components of the traditional tumor microenvironment include tumor cells,immune cells,fibroblasts,extracellular matrix,cytokines,and special physical and chemical environmental changes(hypoxia/low PH).The tumor microenvironment participates in the occurrence,development and metastasis of tumors and affects the biological characteristics of tumor cells.Tumor infiltrating lymphocytes(TILs)in the microenvironment are involved in regulating tumor cells,regulating tumor invasion and metastasis,which are currently the main research hotspots in the tumor microenvironment.Cytotoxic T lymphocytes(CTL),also known as killer T cells,are a type of effector T cells.CTL can play a biological function of recognizing tumor antigens and killing tumor cells.CTL is an important component of the tumor immune system.CTL can recognize tumor cells in the tumor microenvironment and mediate cell killing and apoptosis.CLTs are mainly composed of two cytotoxic T lymphocytes,CD8~+Among them,CD8~+tumor-infiltrating T cells are the main cells that perform cell killing functions.CD8~+CTL is the main effector cell of anti-tumor immunity in the body.When the co-stimulatory molecule is highly expressed by tumor cells,the antigen can be directly presented to CD8~+T cells to stimulate their synthesis of IL-2,and then proliferate and differentiate into CTL with specific killing effect on tumor cells.CD8~+CTL is activated and proliferated by double signaling.Activated cytotoxic T cells do not need the assistance of costimulatory molecules in the phase of their killing effect.CTL mainly recognizes the PMHC indicated by target cells in a way restricted by MHC class I molecules,and plays a specific role in killing target cells.The main mechanisms by which CD8~+tumor-infiltrating T cells exert cell killing include the following two:activated CD8~+T CTLs can secrete perforin and granzymes,and then form active pores in the target tumor cell membrane,changing the osmotic pressure inside the cell,leading to the inside of the cell.Dissolve;meanwhile,CD8~+tumor-infiltrating T cells can mediate apoptosis through Fas/Fas-L.Immune cell infiltration is closely related to the prognosis of tumor patients.Using immune cells infiltrating tumor tissue as a drug target can bring hope to improve the survival rate of patients.At present,there are mainly two commonly used algorithms for estimating the distribution of immune cells based on tumor tissue gene expression profiles,namely CIBERSORT and single sample gene enrichment analysis(ss GSEA).CIBERSORT is currently the most cited immune cell infiltration estimation analysis tool.It is a tool for deconvolution of the expression matrix of immune cell subtypes based on the principle of linear support vector regression.It can estimate complex tissues based on standardized gene expression data.Cell composition,this method energizes the abundance of specific cell types.Megakaryocyte-associated tyrosine kinase(MATK)gene was first confirmed to have good diagnostic performance in gastrointestinal T-cell lymphoma.However,the expression and function of MATK in DLBCL are still unclear.This topic aims to explore the expression of MATK in diffuse large B-cell lymphoma and its correlation with clinical features.And through in vivo and in vitro experiments to explore the function of MATK affecting the cell proliferation and invasion of diffuse large B-cell lymphoma and its potential mechanism,and to explore whether MATK can be used as a novel early diagnosis,prognostic judgment and treatment of diffuse large B-cell lymphoma.Markers and new therapeutic targets.In this study,we first used bioinformatics analysis tools to analyze the diffuse large B lymphoma expression profile data set in the TCGA(The Cancer Genome Atlas)and GEO(Gene Expression Omnibus)public databases,and based on the CIBERSORT immune infiltration algorithm to screen and CD8~+The most relevant gene MATK for tumor-infiltrating T cells;subsequently,the expression level of MATK in diffuse large B lymphoma tissue was detected by western lot,RT-q PCR and immunohistochemistry at the level of clinical tissue specimens and in vitro cell lines,confirming MATK Significantly high expression in diffuse large B lymphoma cell lines and tissues;at the same time,in vitro functional tests were performed to analyze the effect of silencing MATK on the proliferation and invasion of diffuse large B lymphoma cells.The results suggest that the proliferation and invasion of diffuse large B lymphoma cells after silencing MATK Invasion ability was significantly affected;at the same time,we further analyzed the functional effects of MATK gene on CD8~+tumor-infiltrating T cells,including the secretion levels of cytokines INF-γ,IL-2 and TNF-α;the immune killing function of CD8~+T lymphocytes;And the influence of IL6/JAK/STAT3.The results suggest that silencing MATK can enhance the secretion levels of INF-γ,IL-2and TNF-αof CD8~+T lymphocytes by regulating the IL6/JAK/STAT3 signaling pathway,and promote CD8~+T lymphocytes to diffuse large B lymphoma cells.Immune killing function.In conclusion,this study reveals that MATK has an important function in the occurrence and development of DLBCL,and provides a theoretical basis for its becoming a new target for early disease diagnosis,prognostic stratification and treatment.Part 1 Expression level of tumor immune infiltration-related gene MATK in DLBCLAim:Screening and DLBCL immune infiltration related genes MATK,exploring the expression of MATK in DLBCL and its clinical significanceMethods:1.Use the online tumor immune infiltration analysis tool Cibersort to analyze and screen out the MATK that is negatively correlated with the CD8~+T tumor infiltrating lymphocyte score.2.Use the m RNA chip expression profile data set of the DLBCL cohort in the TCGA database to analyze the m RNA expression of MATK in DLBCL and its correlation with the survival and prognosis of DLBCL patients.3.Analyze the protein expression level of MATK in DLBCL by immunohisto chemical technique;analyze the expression level of MATK in DLBCL cell line by western lot and RT-q PCR.Results:1.The expression level of MATK is significantly positively correlated with DLBCL CD8~+tumor infiltrating T cells.2.MATK is significantly low expressed in DLBCL tissues and cell lines.3.The low expression of MATK suggests a shorter survival time for patients with diffuse large B,and suggests that MATK may play an important role in the occurrence and development of diffuse large B.Part 2 The effect and mechanism of MATK gene overexpression on proliferation,migration and invasionof DLBCL cells in vitroitsAim:Study the effect of MATK gene overexpression on the biological behavior of DLBCL cell proliferation,migration and invasion;Analyze the effect of CD8~+T lymphocytes on the immune killing function of diffuse large B lymphoma cells and its potential mechanismMethods:1.Transfect the MATK overexpression plasmid into DLBCL cell lines SU-DHL-6 and U2932.2.Use PCR and Western Blot technology to observe the expression of MATK and identify the overexpression efficiency.3.Use cell immunofluorescence staining to further identify the overexpression efficiency.4.The CCK-8 method was used to detect the proliferation of DLBCL cell lines SU-DHL-6 and U2932 after MATK overexpression.5.Using Transwell method to detect the invasion ability of DLBCL cell lines SU-DHL-6 and U2932 after MATK overexpression.6.Co-culture CD8~+T lymphocytes and DLBCL cell line SU-DHL-6,analyze the secretion levels of INF-γ,IL-2 and TNF-αafter MATK overexpression;perform cell non-radioactive toxicity test and LDH activity test.7.Use Western Blot technology to detect the changes of IL6/JAK/STAT3signaling pathway in DLBCL cell line after MATK overexpression.Results:1.After transfection of the MATK overexpression plasmid,the protein expression level of MATK increased,and the knockdown experiment was successful.2.After MATK overexpression,the proliferation and invasion ability of DLBCL cell line was significantly weakened.3.Overexpression of MATK can increase the secretion of INF-γ,IL-2 and TNF-αof CD8~+T lymphocytes;promote the immune killing function of CD8~+T lymphocytes.4.IL6/JAK/STAT3 signaling pathway is inhibited after MATK ovexexpression.Conclusion:1.MATK is highly expressed in DLBCL,which is closely related to the clinical malignant phenotype of patients.DLBCL patients with high expression of MATK have a significantly longer survival time than those with low expression of MATK.2.After MATK overexpression,the proliferation and invasion ability of DLBCL cells was significantly weakened.3.MATK can enhance the secretion of INF-γ,IL-2 and TNF-αof CD8~+T lymphocytes by regulating the IL6/JAK/STAT3 signaling pathway,and promote the immunity of CD8~+T lymphocytes to diffuse large B lymphoma cells Killing function.