Gene Expression Profiling Of Diffuse Large B-cell Lymphoma Drug-resistant Cell Lines And Discussion Of The Mechanism Of Paclitaxel’s Resistance To Lymphoma

Background:Diffuse large B-cell lymphoma(DLBCL)is the most common histological subtype in non-Hodgkin’s lymphoma.Although the current DLBCL chemotherapy regimen is maturing,about 40%of patients relapse after first-line therapy and become resistant to initially treated chemotherapy drugs,leading to disease progression and poor prognosis.It is urgent to explore the occurrence and development of drug resistance of DLBCL from the molecular mechanism,and to screen the potential targets of drug resistance,which will help clinically precise treatment of patients with DLBCL resistance.Our previous study found that paclitaxel can be used as a potential drug for the treatment of DLBCL,and it has a good inhibitory effect on DLBCL resistant cells.However,the mechanism of the effect of paclitaxel on DLBCL resistant cells is unknown,and further study and analysis are still needed.Objective:The purpose of this study was to analyze the gene expression profiles of DLBCL-resistant cell lines and paclitaxel intervention in DLBCL-resistant cell lines using transcriptome sequencing technology(mRNA-seq),and to discuss the occurrence and development of DLBCL resistance and the potential molecular mechanism of paclitaxel against DLBCL resistance at the genetic level.Methods:1.The donated DLBCL adriamycin resistant Ramos/ADM and DLBCL cell line Ramos were identified,and the drug maintenance culture of Ramos/ADM was conducted by gradient increase of adriamycin concentration.2.The effects of adriamycin and paclitaxel on the biological characteristics of Ramos/ADM and Ramos cell lines were detected by CCK-8 assay.3.Ramos/ADM and Ramos cell lines were sequenced by mRNA-seq,and the mechanisms of acquired drug resistance of DLBCL were analyzed from the level of gene expression.4.The sequencing data of mRNA-seq is processed and analyzed by computational biology.Use DESeq2 to analyze gene expression levels and screen for differentially expressed genes(DEGs);use ClusterProfiler to perform functional annotation and pathway enrichment analysis of DEGs to further understand the DEGs involved in biological functions and possible molecular mechanism.5.The protein-protein interaction(PPI)network of DEGs was constructed through the STRING database,and the cytoHubba was used to screen the network to find the hub genes that play a role in DLBCL resistance.Use ClusterProfiler to analyze hub gene function annotation and pathway enrichment;use cBioPortal hub gene for mutation analysis,GEPIA2 database for hub gene mRNA expression level analysis,HPA database for hub gene protein expression level analysis,and UALCAN database for clinical staging and prognosis analysis,the DGIdb database constructs gene targeting drug networks for hub genes.6.Through the MCODE,cluster the PPI network to construct functional modules,and analyze the gene function annotation and pathway enrichment in the module.7.The expression of hub genes was verified by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR),and the reliability of RNA-seq results was verifiedResults:1.Biological characteristics of DLBCL drug-resistant cell linesThe results of CCK-8 assay showed that Ramos/ADM had significant resistance to adriamycin in the three time periods of 24h,48h and 72h.The IC50 values of Ramos/ADM and Ramos for adriamycin were 1.3256±0.0328μmol/L and 0.1098 ± 0.0017μmol/L,respectively.Resistance Index was 12.0724.Ramos/ADM cell lines also showed paclitaxel cross-resistance.The IC50 values of Ramos/ADM and Ramos to paclitaxel were 0.4912 ±0.0230μmol/L and 0.0080 ±0.0001μmol/L,respectively.Resistance Index was 61.2862.The inhibitory effect of paclitaxel on Ramos and Ramos/ADM is significantly higher than that of adriamycin(P<0.001).2.Ramos and Ramos/ADM blank group RNA-seq data over-characterization analysis2.1 The RNA-seq data was over-characterization analysis,and a total of 1,234 DEGs were obtained,including 828 up-regulated genes and 406 down-regulated genes.The ATP-binding cassette transporters family ABCB1,ABCB4,ABCB9,ABCA2,ABCA6 and ABCD3 were highly expressed in Ramos/ADM,which proved the successful construction of drug-resistant cell lines.2.2 The results of functional annotation of DEGs showed that cell adhesion,migration and movement were significantly related to the occurrence and development of DLBCL resistance.KEGG analysis found that Adherens junction signaling pathway also played a vital role in the occurrence of resistance.2.3 The topology algorithm was used to screen out 10 hub genes:EGFR,SRC,ITCH,APP,CDC27,ITGB1,UBC,CUL1,GNG2 and TNF,among which UBC ranked first among the six topological algorithms.Functional annotation results found that the hub genes are mainly involved in stress activated protein kinase,MAP kinase,ubiquitination and ubiquitin-like protein ligase binding;KEGG pathway analysis is enriched in Proteoglycans in cancer signaling pathway.2.4 The cBioPortal database found that EGFR(13%),TNF(10%)and CUL1(10%)are the three genes with the most genetic changes in DLBCL;In the GEPIA2 database,it was found that the mRNA expression level of SRC,CUL1,APP,CDC27,ITGB1,and TNF in DLBCL was significantly higher than that in normal tissues(P<0.05);The protein expression levels of SRC,CUL1,TGB1,ITCH and EGFR in lymphoma were found to be higher than those in normal lymphoid tissues in the HPA database.The protein level of UBC in lymphoma was lower than that in normal lymphoid tissue;Using the UALCAN database,it was found that the mRNA expression level of the hub gene SRC was significantly related to the clinical stage 2 of DLBCL patients(P=0.043),the expression level of CUL1 was significantly related to Stage 4(P=0.045),and the expression level of APP was significantly related to Stage 4(P=0.018);The expression level of CDC27,ITCH and TNF increased gradually with the increase of stage,but it was not statistically significant(P>0.05),and the high expression of CDC27 was significantly associated with the poor prognosis of DLBCL patients(P<0.05).Compared with the RNA-seq results of this cell lines,it was found that the genes that were not significantly expressed in DLBCL(UBC,GNG2)or even high expression(CUL1,TNF)became low expression in drug-resistant cell line.We speculate that the down-regulated expression of these genes may be related to the occurrence and development of drug resistance.Combined with the database analysis of hub genes,we can preliminarily consider that the changes in the expression of the three hub genes of UBC,CUL1 and TNF play an important role in the occurrence and development of DLBCL resistance;There are 36 drugs targeted by drug-gene network,among which ibrutinib and others have known effects of treating DLBCL and drug resistance.Whether the remaining drugs can be used as a potential treatment for DLBCL resistance needs further study.2.5 The network function module analysis results showed that UBC,CUL1,CDC27 and ITCH four hub genes were found to be included in the top-ranked module.Functional annotation of genes in the module revealed that the functions involved were mainly related to ubiquitination.Combining the results of hub genes,it can be seen that ubiquitination is closely related to DLBCL resistance;KEGG pathway analysis is significantly enriched in the Ubiquitin mediated proteolysis signaling pathway.3.Ramos/ADM paclitaxel group RNA-seq data over-characterization analysis3.1 The RNA-seq was performed after paclitaxel intervention in Ramos/ADM cells,followed by over-characterization analysis.A total of 971 differentially expressed genes were obtained,including 519 up-regulated genes and 452 down-regulated genes.3.2 The results of functional annotation of DEGs showed that the effect of paclitaxel on DLBCL-resistant cells may be closely related to functions such as protein folding,ribosome biosynthesis,RNA polymerase III and filopodium;KEGG pathway analysis was significantly enriched in the Protein processing in endoplasmic reticulum signaling pathway.3.3 Five hub genes were screened out by using 6 topological algorithms:UBC,TSR1,WDR46,HSP90AA1 and NOP56.The five hub genes were all up-regulated genes in the RNA-seq results.3.4 Through the cBioPortal database,it was found that WDR46(6%)and HSP90AA1(6%)are the two genes with the most genetic changes in the hub genes;GEPIA2 and HPA databases found that the expressions of TSR1,WDR46,HSP90AA1 and NOP56 in DLBCL were significantly higher than normal tissues(P<0.05);The expression of the five hub genes and the clinical stage of DLBCL patients were not statistically significant(P>0.05),but it can be seen that the expression of TSR1,HSP90AA1,and NOP56 gradually increased with the increase of stage.Comparing the RNA-seq results,it was found that UBC,TSR1,WDR46,HSP90AA1 and NOP56 were significantly up-regulated after the effect of paclitaxel on Ramos/ADM.We suspect that paclitaxel may play a role by targeting these five hub genes.3.5 The network function module analysis results showed that TSR1,WDR46 and NOP56 three hub genes were found to be included in the top-ranked module.Functional annotation from genes in the module revealed that the functions involved were mainly related to ribosomes.KEGG pathway analysis significantly enriched the Ribosome biogenesis in eukaryotes signaling pathway.Combining functional annotation and KEGG pathway analysis results,it was found that ribosomal biosynthesis plays an important role in paclitaxel action of DLBCL resistance.4.The mRNA expression of the gene was detected by RT-qPCRPCR results showed that compared with Ramos,ABCB1,ABCB4 and ABCA2 three drug-resistance genes were significantly over-expressed in Ramos/ADM(P<0.05);UBC and CUL1 showed low expression in Ramos/ADM,EGFR and SRC showed high expression in Ramos/ADM(P<0.05).After paclitaxel intervention Ramos/ADM,compared with the negative control,the mRNA levels of the five hub genes were significantly higher in the paclitaxel group,the difference was statistically significant(P<0.05).The RT-qPCR results were consistent with the RNA-seq results,and the reliability of the RNA-seq data could be determined.Conclusions:1.Compared with Ramos cells,Ramos/ADM is resistant to adriamycin,and is found to be cross-resistant to paclitaxel.However,paclitaxel is more sensitive to DLBCL and DLBCL drug-resistant cell lines than the conventional drug adriamycin.2.Gene function annotation and pathway analysis of Ramos/ADM cells,it was found that cell adhesion,migration and movement and Adherens junction signaling pathways play a role in the development of DLBCL resistance.Ubiquitination-related functions and Ubiquitin mediated proteolysis signaling pathway play a major role in the development of DLBCL resistance.3.Combined with the analysis of public databases,it is found that the three hub genes of UBC,CUL1 and TNF may play an important role in the occurrence and development of DLBCL resistance.4.Gene function annotation and pathway analysis of paclitaxel intervention in Ramos/ADM cells,it was found that ribosome biosynthesis and Protein processing in endoplasmic reticulum signaling pathways played a major role in paclitaxel action;UBC became highly expressed after paclitaxel intervention in Ramos/ADM.It is speculated that paclitaxel may play an anti-DLBCL resistance role by up-regulating UBC expression level.Ribosomal-related functions and ribosomal biosynthesis signaling pathways may play an important role in the mechanism of paclitaxel’s action to DLBCL resistance.5.The results of the computational biological analysis used in this study provide some support for exploring the basic experimental research on the molecular mechanism of DLBCL resistance and paclitaxel anti-DLBCL resistance,as well as searching for potential therapeutic targets.The choice of potential drugs provides a theoretical basis.However,this study is still in the stage of data analysis,and further molecular experiments are still needed for the relevant molecular mechanisms to confirm the hub genes and related signaling pathways screened in our study.