Expression And Clinical Significance Of ATF5 And BATF In Diffuse Large B-cell Lymphoma

Background and ObjectiveNon-Hodgkin lymphoma(NHL),as the most common malignant tumor of the lymphatic hematopoietic system,poses a great threat to human health once diagnosed.Among them,Diffuse Large B-Cell Lymphoma(DLBCL)is the most common NHL,accounting for 30-40% of NHL,and the incidence is also on the rise.According to the origin of tumor cells,diffuse large B-cell lymphoma is mainly divided into two subtypes,one subtype is Germinal center B cell-like DLBCL(GCB type),the other is Activated B cell-like DLBCL(ABC type).Although combined with CHOP chemotherapy regimen can significantly improve the prognosis of patients,due to the heterogeneity of DLBCL disease itself,about 30-50% of DLBCL patients will progress to refractory or recurrent,and the prognosis of patients with ABC subtype is significantly lower than that of patients with GBC subtype.Therefore,the search for new molecular markers of DLBCL can not only improve the accurate diagnosis of DLBCL patients,but also play a positive role in the clinical treatment of patients.Activating transcription factor 5(ATF5)and basic leucine zipper transcription factor ATF-like(BATF)belong to the activator protein-1(AP-1)family activating transcription factor of subfamily ATF.Previous reports of ATF5 mainly involved glioma,non-small cell lung cancer,breast cancer,etc and played an important role in the proliferation,differentiation,growth,apoptosis,migration and transformation of tumor cells.Studies have systematically analyzed the B-cell specific regulation model that exhibits the characteristics of follicular lymphoma transformation,and found that ATF5 is one of several key transcription factors that drive the transformation of follicular lymphoma into aggressive diffuse large B-cell lymphoma 1.A study of ATF5 in Burkitt’s lymphoma(BL)showed that ATF5,as one of the genes that is resistant to ascorbic acid,promotes co-operative changes that allow survival of ascorbic acid-resistant BL cells(JLPR cells).BATF is mainly expressed in hematopoietic cells,especially B cells and T cells.Studies have found that BATF and other factors can drive the overexpression of IRF4 and play a role in molecular programs that directly regulate tumor immunosuppression.It has been reported in the literature that the differential expression of IRF4 cofactors SPIB and BATF can enrich the mutation of MYD88,which helps to identify the biological and clinical heterogeneity of ABC subtype DLBCL.Among T-cell lymphomas,studies have shown that BATF and BATF3 are highly expressed in ALK anaplastic large cell lymphoma(ALCL).Both BATFs bind to the classic AP-1 protein and are involved in the dysregulated AP of ALCL.Interactions between-1 factors,specific inactivation of BATF gene or inhibition of AP-1 will lead to ALCL growth retardation and/or cell death.There are no reports on the research of ATF5 and BATF in DLBCL organization.This project is based on bioinformatics database predictions combined with basic experimental verification of clinical samples,to study the expression of ATF5 protein and BATF protein in DLBCL,and analyze the relationship between the expression of ATF5 and BATF and the clinicopathological characteristics and prognosis of DLBCL patients.The purpose is to explore the role and significance of ATF5 and BATF in DLBCL,and to find new molecular indicators related to its prognosis.Methods1.Collecting the paraffin samples of 120 patients diagnosed with diffuse large B-cell lymphoma from January 2017 to June 2020 in the Department of Pathology of the First Affiliated Hospital of Zhengzhou University as the experimental group.The criteria for inclusion:(1)DLBCL initial treatment Human,without any radiotherapy and chemotherapy;(2)The tumor tissue is sufficient and meets the conditions for RNA extraction.A total of 85 paraffin specimens met the conditions,including 55 subtypes of ABC and 30 subtypes of GBC.20 paraffin samples diagnosed with reactive lymph node hyperplasia during this time period were randomly selected as the control group.2.Using Oncomine and GEPIA databases to predict and analyze the expression levels of ATF5 and BATF m RNA in normal tissues and tumor tissues.3.The immunohistochemical method was used to detect the expression of ATF5 and BATF in 120 DLBCL paraffin samples and 20 control paraffin samples.4.The m RNA expression levels of ATF5 and BATF in 85 DLBCL paraffin samples and 20 control paraffin samples were detected by real-time quantitative PCR.5.Statistical analysis: Use SPSS 22.0 statistical analysis software to statistically analyze the expression of ATF5 and BATF protein and m RNA,and analyze the correlation between ATF5 and BATF protein and clinicopathological parameters and prognosis.Results1.Bioinformatics database prediction results: The m RNA expression levels of ATF5 and BATF in DLBCL tissues in Oncomine and GEPIA databases were significantly higher than those in normal tissues(P<0.05).2.Immunohistochemical experiment verification: The positive rate of ATF5 protein in 120 DLBCL tissues is 50%(60/120),and it is mainly located in the cytoplasm of tumor cells;in the control group of 20 cases,ATF5 is all negatively expressed;The positive rate of BATF was 44.2%(53/120)in 120 DLBCL tissues,and it was mainly localized in the cytoplasm of tumor cells.BATF protein expression was negative in all the 20 patients in the control group,and the difference of ATF5 and BATF protein expression was statistically significant compared with the control group(P<0.05).3.q RT-PCR experimental verification results: the ratio of the average relative expression of BATF m RNA in 55 cases of ABC subtype DLBCL paraffin tissues and the average relative expression of BATF m RNA in the control group was 1.797∶0.748,the difference was statistically significant(P<0.05)There was no correlation between the control group and the average relative expression of BATF m RNA in the GBC subtype DLBCL group(P>0.05).However,ATF5 m RNA expression was not detected in the control group,and ATF5 m RNA expression was also very low in the DLBCL group.4.Correlation analysis of ATF5 and BATF protein expression with clinicopathological parameters in DLBCL patients indicated that ATF5 protein expression was not correlated with clinicopathological parameters in,sex,age,Ann Arbor stage,IPI score,Hans classification,LDH Ki-67 primary lesions with or without B symptoms(P>0.05);BATF protein expression was correlated with Ki-67 proliferation index(P<0.05);ATF5 and BATF protein co-expression group was also correlated with Ki-67 proliferation index in DLBCL patients(P<0.05).5.Relationship between BATF m RNA expression and clinical parameters of DLBCL patients: Chi-square test results show that the relative expression of BATF m RNA and DLBCL patients’ clinicopathological parameters gender,age,Ann Arbor staging,IPI score,Hans classification,LDH,Ki-67.There is no significant correlation between the primary site and the presence or absence of B symptoms(P>0.05).6.Single factor analysis of ATF5 and BATF protein expression and clinicopathological parameters and prognosis of DLBCL patients: The overall survival of the BATF protein high expression group was shorter than that of the low expression group(P<0.05),suggesting that BATF is the risk of poor prognosis in DLBCL patients factor.The overall survival of DLBCL patients was significantly correlated with the patient’s age,Ann Arbor staging,IPI score,and location of the disease(P<0.05).Conclution1.The results of bioinformatics database and immunohistochemistry confirmed that ATF5 protein expression,BATF m RNA and protein levels were higher than those of the control group,suggesting that it may play a role in the occurrence and development of DLBCL.2.The expression level of BATF m RNA in ABC subtypes is significantly higher than that of GBC subtypes,which may have positive significance for the accurate typing of DLBCL.3.BATF protein expression level,ATF5 and BATF protein co-expression are positively correlated with Ki-67 proliferation index,and high BATF protein expression is associated with poor prognosis,suggesting that BATF may be a new indicator related to prognosis.