The Study On Functions And Molecular Mechanisms Of RDM1 In ABC-subtype Diffuse Large B-cell Lymphoma

Objective: Diffuse large B-cell lymphoma(DLBCL)is a common subtype of invasive lymphoma.DLBCL is heterogeneous in histology,immunology,genetics and other aspects,so 40-50% patients suffered from treatment failure,such as recurrence and refractory.ABC subtype DLBCL(ABC-DLBCL)has worse prognosis than others.Since potential mechanisms of development of relapse and refractory ABC-DLBCL has not been clarified,there was lack of specific and efficient interventions for these patients in clinical practice.RDM1(Rad52 motif-containing protein 1)is a molecular with functions of transcription factor and damaged DNA repair.It has been reported that abnormal expression of RDM1 was found in different kinds of tumors.RDM1 functions as oncogene or tumor suppressor gene,which is involved in the occurrence and development of various kinds of tumors.However,the function and molecular mechanism of RDM1 in ABC-DLBCL remain unclear.In this study,the whole transcriptome sequencing was performed to obtain the RDM1 gene,which is related to development of relapse and refractory ABC-DLBCL.The correlation between expression level of RDM1 and clinicopathological characteristics and prognosis of ABC-DLBC patients was analyzed.Then,the regulatory role of RDM1 in the growth of ABC-DLBCL cells was studied by in vivo and in vitro.Finally,the potential regulatory mechanisms of RDM1 on downstream molecules and its’ potential functions were further revealed.This study aims to clarify the role and mechanism of RDM1 in the occurrence and development of ABC-DLBCL,and provide new targets and theoretical basis for the treatment of ABC-DLBCL.Methods: 1.The diagnosis and treatment data of DLBCL patients treated in our hospital from 2011 to 2018 were collected,patients were followed up,and the incidence of relapse and refractory in DLBCL patients was analyzed.2.Histopathological diagnostic tissue samples were collected from ABC-DLBCL patients with relapse/refractory and good prognosis.The whole transcriptional sequencing was performed on these samples to screen the differential expression genes related to the formation of relapsed and refractory ABC-DLBCL.3.RDM1 gene with the most significant difference was selected.The clinical significance of RDM1 expression in ABC-DLBCL patients was analyzed using patients’ data of diagnosis,treatment and follow-up.4.ABC-DLBCL cell lines HBL1 and U2932 with stable overexpression and stable knockdown of RDM1 were constructed.The effects of overexpression or knockdown of RDM1 on the proliferation,apoptosis and cell cycle of ABC-DLBCL cells were detected by CCK-8,clone formation assay and flow cytometry in vitro,respectively.5.Subcutaneous xenograft model of mice was constructed to study the effect of RDM1 on the growth of HBL1 cells in vivo.6.High-throughput RNA sequencing analysis,dual luciferase reporter gene assay,chromatin immunoprecipitation and functional recovery assay were used to screen and verify the possible regulatory mechanism of RDM1 on potential downstream target genes and its’ potential functions.Results: 1.Nearly 30% of DLBCL patients suffered from relapse and refractory.2.Compared with patients with good prognosis,a total of 558 differentially expressed genes existed in relapsed and refractory ABC-DLBCL patients,including 223 coding genes and 335 non-coding genes.3.High expression of RDM1 was associated with shorter OS and PFS in ABC-DLBCL patients,and was an independent poor prognostic factor for PFS.4.In vitro,overexpression of RDM1 promoted proliferation and clone formation of ABC-DLBCL cells,decreased apoptosis and affected cell cycle distribution,while knockdown of RDM1 inhibited proliferation and clone formation of ABC-DLBCL cells,increased apoptosis and affected cell cycle distribution.5.In vivo,RDM1 promoted the growth of subcutaneous xenograft of HBL1 cell in mice.6.Results of high-throughput RNA sequencing were confirmed that HSP27 was a downstream target gene regulated by RDM1.7.RDM1 could bind to the promoter of HSP27 gene,then regulate its’ expression of m RNA and protein,thus regulate the growth of ABC-DLBCL cells and its’ sensitivity to adriamycin.Conclusions: 1.Relapse and refractory DLBCL is a common problems in the clinical practice.2.High expression of RDM1 is associated with poor prognosis in ABC-DLBCL patients,and is expected to be a new target for prognosis prediction in ABC-DLBCL patients.3.RDM1 can promote proliferation of ABC-DLBCL cell,inhibit its’ apoptosis,and affect its’ cell cycle distribution.4.As a transcription factor,RDM1 can bind to the promoter of HSP27 gene and promote its’ expression,thereby regulating the growth of ABC-DLBCL cells and its’ sensitivity to Adriamycin,thus playing the role of oncogene.