Involvement Of SMURF2 Gene In The Regulation Of LAG-3 And PD-1 Expression During The Treatment Of Diffuse Large B-cell Lymphoma

Objective: Immunochemotherapy has become a standard of care for diffuse large B-cell lymphoma(DLBCL).In the course of immunochemotherapy,the features of T cell immunity and the mechanisms of action of immune checkpoints are incompletely defined.The aim of this study was to investigate the potential influence of programmed cell death protein 1(PD-1)and lymphocyte activation gene 3(LAG-3)in CD8+T cells in response to immunochemotherapy of DLBCL and to screen key genes involved in the prognosis of DLBCL related to immunotherapy.Offers more possibilities for DLBCL immunotherapy.Methods: Part I: clinical data of 39 patients with DLBCL after treatment with first-line chemotherapeutic agents were collated.The contents of CD4+T cells and CD8+T cells,the levels of cytokines,and PD-1 and LAG-3 expression in venous blood samples of DLBCL patients before and after treatment and 10 healthy controls were detected by flow cytometry.The levels of cytokine production were examined by sorting CD8+T cells from the venous blood of DLBCL patients before and after treatment with magnetic beads and blocking PD-1 and LAG-3 expression.Part II: The DLBCL associated datasets GSE83632,GSE31312 and GSE87371 were downloaded from the gene expression database(GEO)for gene expression profiling data.Differentially expressed genes between DLBCL and controls in GSE83632 were identified by differential expression analysis.Genes in GSE31312 and GSE87371 that significantly influenced the prognosis of DLBCL patients were identified by Cox regression analysis.The intersection of three groups of genes,that is,differentially expressed genes affecting the prognosis of DLBCL patients,was acquired by intersection analysis.Enrichment analysis identifies biological functions and signaling pathways of intersection genes.The protein-protein interaction(PPI)network identified the top 10 genes with the largest connectivity as core genes,and the prognostic predictive role of core genes was evaluated using Cox regression analysis.Differentially infiltrated immune cells between DLBCL and controls were identified by cibersort algorithm and aberrant expression of PD-1 and LAG-3 in DLBCL was analyzed in TCGA database.Correlation analysis was used to identify the core genes most relevant to immune cells and immune checkpoints as key genes.Part III: The experimental methods of q RT-PCT were used to detect the differential expression of core genes between DLBCL patients and healthy people before and after treatment,and the experimental methods of flow cytometry were used to detect the differences of immune cells in the three groups.The protein expressions of SMURF2,LAG-3 and PD-1 in the three populations were detected by Western blot.Pc DNA3.1-empty plasmids and pc DNA3.1-SMURF2 was transfected using Lipofectamine 2000 transfection reagent were cultured after transfection into SU-DHL-8 cells.The proliferation of blank cells,empty cells and cells overexpressing SMURF2 was detected by CCK8 kit,and the apoptosis of the three groups was detected by flow cytometry.CD4+T cells and CD8+T cells from DLBCL patients after treatment were sorted by magnetic beads and cocultured with the three groups of cells.Detection of PD-1 and LAG-3 expression in the coculture system.Results: Part I:(1)The mean age of DLBCL patients in this study was 59.8 years,and 69.2% of DLBCL patients were in stage IV.The proportion of patients who achieved complete remission(CR)after three courses of chemotherapeutic agents was 51.3%,and the proportion of CR after six courses was64.1%.A comparison of T-cell proportion changes before and after treatment in patients with DLBCL revealed that the proportions of CD4+T cells and CD8+T cells gradually increased in patients with DLBCL after treatment compared with patients with untreated DLBCL.With the extension of treatment time,the Th2 and Th17 cells gradually increased in the peripheral blood of DLBCL patients,the proportion of Th1 cells gradually decreased,and Treg cells also gradually increased.Corresponding cytokine levels were also significantly altered.(2)Levels of PD-1(+),LAG-3(+),or PD-1(+)LAG-3(+)on both CD4+T cells and CD8+T cells were significantly lower after first-line therapy than in untreated DLBCL patients.Blockade of PD-1 and LAG-3 in sorted CD8+T cells significantly inhibited cytokine production in response to treatment.Part II:(1)A total of1515 intersection genes were identified among 11330 differentially expressed genes to significantly affect the prognosis of DLBCL patients.Enrichment analysis found that the intersection genes were significantly enriched in protein ubiquitination modification and toll like receptor signaling pathways.Ten core genes were identified through the PPI network,including UBE2E1,SKP1,UBE2D1 and UBC,which were upregulated in DLBCL,and DYNC1H1,UBR4,EP300,TP53,SMURF2 and WWP1,which were downregulated.Based on core genes,Cox regression analysis stratified the DLBCL samples into high-risk and low-risk groups with the median of risk scores.The overall survival rate in the high-risk group was significantly poorer than that in the low-risk group,and the median risk score had a better predictive ability for the prognostic diagnosis of patients with DLBCL.(2)Immune cells differentially infiltrated between DLBCL and controls were identified by cibersort,while PD-1 and LAG-3 were highly expressed in DLBCL.The results of correlation analysis showed that SMURF2 was most significantly negatively correlated with LAG-3 and PD-1 and was identified as the key gene.Patients with DLBCL had poor OS when SMURF2 was lowly expressed.Part III:(1)q RT-PCT results showed that core genes were significantly differentially expressed between DLBCL and controls,and their expression changed after patients received treatment.Flow cytometry verified the differential ah infiltration of neutrophils,NK cells as well as Treg cells between DLBCL and controls and correlated with treatment.The results of Western blot assay showed the low expression of SMURF2 in DLBCL patients,as well as the high expression of LAG-3 and PD-1,all three of which were changed after treatment.(2)Empty plasmid as well as SMURF2 overexpression plasmid were successfully transfected into SU-DHL-8 cells with smooth overexpression of SMURF2 protein by transfection reagent.Following SMURF2 overexpression,SU-DHL-8 cells exhibited decreased levels of proliferation and enhanced levels of apoptosis,as well as decreased levels of LAG-3and PD-1 expression.The decreased levels of LAG-3 and PD-1 were more pronounced after coculture with CD4 + T cells or CD8 + T cells.Conclusions:(1)first line treatment options have a positive effect on the overall recovery of patients with DLBCL.The proportion of CD4+T and CD8+T cells increased in DLBCL patients after treatment,accompanied by Th2,Th17 as well as Treg cell subtype predominance.In addition,PD-1and LAG-3 expression on the surface of CD4+T and CD8+T cells is increased,suggesting that the immune efficacy of first-line regimens may be mediated by immune checkpoints.(2)Through sequencing data of DLBCL samples in public databases,combined with bioinformatics analysis methods,the identification of ubiquitination modification and Toll-like receptor signaling pathways may be DLBCL related molecular deregulation mechanisms and lay a theoretical foundation for exploring the biological function changes of DLBCL.In addition,a large number of abnormal immune cell infiltration was found in DLBCL patients,with the highest negative correlation of SMURF2 with immune checkpoints LAG-3 and PD-1 identified among the core genes identified.With more in-depth studies,SMURF2 will become a new predictive marker for DLBCL patient prognosis.(3)Differential expression of core genes as well as differential immune infiltration of immune cells in DLBCL patient pre-and post-treatment and healthy control samples were validated by molecular experiments,while differential expression of SMURF2,LAG-3 and PD-1 was validated.The expression of SMURF2 was increased in SU-DHL-8 cells after transfection of SMURF2 overexpression plasmid.Overexpression of SMURF2 obviously inhibited the proliferation,promoted the apoptosis of SU-DHL-8cells and decreased the expression of LAG-3 and PD-1.And provides an auxiliary role for the immune function of CD4+T cells or CD8+T cells.High expression SMURF2 acts as a tumor suppressor gene in DLBCL.