| Background: Diffuse large B cell lymphoma(DLBCL)is one of the most common non-Hodgkin's lymphomas.Diffuse large B-cell lymphoma progresses rapidly,and traditional chemotherapy is not effective.Since the CD20 monoclonal antibody(rituximab)is applicated in clinical practice,Rituximab combined with chemotherapy which is called R-CHOP(rituximab,cyclophosphamide,doxorubicin,vincristine,prednisone)increases five-year survival of DLBCL from 32% to 68%.However,some patients still fail to R-CHOP treatment,and the disease enters the relapsed/refractory stage.It is difficult for patients to obtain satisfactory therapeutic effect with anti-CD20 monoclonal antibodies again.The therapeutic effect of rituximab on DLBCL depends on the expression level of CD20 in tumor cells,while the CD20 expression of DLBCL is heterogeneous.Patients with low CD20 expression are not sensitive to rituximab and have innate resistance;Patients with high expression of CD20,which is sensitive to rituximab,have a decreased or even negative expression of CD20 when accepting rituximab,resulting in acquired resistance.Undoubtedly,the important reason for the failure of DLBCL treatment is that tumor cells are resistant to rituximab,and CD20 plays an important role here.Our study found that PINK1 has a function of regulating CD20 expression in DLBCL cells.The PTEN-induced putative kinase 1(PINK1)gene is a key gene in Parkinson's disease,and PINK1 is also closely related to tumor development.In tumor cells,PINK1 regulates the biological function of tumor cells by regulating protein expression by ubiquitination of proteins.There is a significant difference in the expression of PINK1 in different tumor types,and PINK1 is significantly up-regulated in hematopoietic tumors,but the role of PINK1 in DLBCL is rare,especially in the study of the mechanism by which PINK1 regulates CD20 expression.Although it has long been known that an important cause of R-CHOP failure is that DLBCL cells are resistant to rituximab.Understanding the mechanism of rituximab resistance and reversing the resistance of rituximab to improve the efficacy of relapsed/refractory DLBCL is an urgent problem to be solved.Purposes: This study focused on the correlation between the PINK1 gene and the clinical prognosis of patients with DLBCL,the mechanism by which PINK1 regulates CD20 expression in DLBCL,and the in-depth exploration of the synergistic effect between Chinese-developed HDAC inhibitor Chidamide and anti-CD20 monoclonal antibody rituximab.Method: 1.Collect clinical data and gene expression data of DLBCL patients in GEO database and analyze the correlation between mRNA expression of PINK1 and other Parkinson's disease-related genes and the overall survival rate of patients with DLBCL;Comparison of mRNA expression differences between PINK1 gene in tumor tissues and normal tissues;analyze the correlation between mRNA expression of PINK1 and Overall survival of DLBCL patients treated with R-CHOP regimen or CHOP regimen by Kaplan-Meier Overall survival;correlation analysis of PINK1 expression and Rituximab resistance by Western Blot assay and MTT;correlation analysis of PINK1 and CD20 mRNA expression in GEO database.2,DLBCL cells were infected by shRNA lentivirus,and cells with low expression of PINK1 were constructed.The regulation of CD20 by PINK1 was verified by RT-PCR,Western Blot and flow cytometry.In vitro and in vivo experiments were used to explore the correlation between PINK1 expression and the sensitivity of rituximab;To investigate the cellular localization and expression of PINK1 by immunofluorescence and immunohistochemistry,and to investigate whether PINK1 regulates phosphorylation of nuclear histone deacetylase 3(HDAC3)by Western Blot and cytoplasmic/nuclear separation techniques.;exploration of the regulation of CD20 and rituximab sensitivity by HDAC3 in DLBCL cells by flow cytometry and MTT assay.3.Flow cytometry,Western Blot assay,RT-PCR and immunofluorescence techniques were used to investigate the regulation of CD20 expression by rituximab in DLBCL cells.The RNA seq technique was used to discover and explore the effects of Chidamide on mRNA expression and signaling pathways in DLBCL cells;Western Blot assay and RT-PCR demonstrated the regulation of CD20 expression in DLBCL cells and the inhibition of rituximab on CD20 expression in DLBCL cells;MTT assay and xenograft model validated the synergistic effect of Chidemide and rituximab in DLBCL;the proliferation and cell cycle regulation of DLBCL cells by Chidemide monotherapy was investigated by MTT assay and flow cytometry,respectively.Result: 1.Data analysis found that PINK1 can be used as a poor prognostic indicator in R-CHOP-treated patients with DLBCL;patients with high expression of PINK1 were found to have the same clinical prognosis using R-CHOP and CHOP regimens;expression of PINK1 in DLBCL cells and Rituximab resistance were significantly correlated;a significant negative correlation was found between mRNA expression of PINK1 and CD20.2,by constructing cells that knockdown of PINK1,PINK1 negatively regulated the expression of CD20 which was proved by RT-PCR,Western Blot and flow cytometry;in vitro and in vivo experiments proved that the knockdown of PINK1 is helpful to enhance effect of rituximab in DLBCL;By immunofluorescence and immunohistochemistry techniques prove PINK1 cell positioning is the nucleus and cytoplasm;PINK1 could phosphorylate S424 site of HDAC3 which was proved by Western blot and cytoplasmic/nuclear separation techniques;flow cytometry and MTT assay demonstrate that HDAC3 could inhibit CD20 expression and regulation of the sensitivity of DLBCL cells to rituximab.3,Rituximab down-regulated CD20 expression in DLBCL cells which was demonstrated by flow cytometry,Western Blot,RT-PCR and immunofluorescence techniques;Chidemide was found to significantly regulate the mRNA expression of DLBCL cells which was demonstrated by RNA seq technology.By differential gene enrichment analysis,we found that Chidamide mainly regulates the mRNA expression of cell membrane proteins,which has a significant regulatory effect on the mRNA expression of hematopoietic signaling pathways,including the expression of CD20,Western Blot assay and RT-PCR technique demonstrated that Chidemide up-regulated CD20 expression in DLBCL cells by CD20 transcription and reversed the down-regulation of CD20 expression in DLBCL cells induced by rituximab;Chidemide and rituximab have synergistic effects in DLBCL was demonstrated by in vivo and in vitro experiments,respectively;the proliferation and cell cycle regulation of DLBCL cells were regulated by Chidemide alone is demonstrated by MTT assay and flow cytometry,respectively.Conclusion: 1,Expression of PINK1 is associated with Rituximab-resistant in DLBCL 2,PINK1 regulates CD20 expression of DLBCL by phosphorylating nuclear HDAC3 3.Chidemide,a kind of HDAC inhibitor,sensitizde the efficacy of rituximab by up-regulating the expression of CD20 expression in DLBCL... |
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