Preliminary Study On Transcriptome Diagnostic Biomarkers Of Plasma Exosomes In DLBCL

Objective:Exploring microRNAs of plasma exosomes differentially expressed in patients with Diffuse large B-cell lymphoma versus healthy individuals for potential diagnostic markers of DLBCL.Methods:From September 2021 to November 2022,twenty-four patients with DLBCL diagnosed by the Department of Pathology of The First Affiliated Hospital of Gannan Medical University were the case group,and twelve healthy volunteers were the control group.Inclusion criteria:(1)According to the 2016 World Health Organization(WHO)classification criteria for hematopoiesis and lymphoid tissue tumors,diffuse large B-cell lymphoma was confirmed by pathological biopsy;(2)The patient was diagnosed as DLBCL for the first time without treatment;(3)The clinical and pathological data are complete,and all patients do not have other blood system diseases.All participants were reserved for a fasting peripheral blood plasma sample of 3-5 m L for plasma exosome extraction.Application of kits to extract plasma exosomes.Transmission electron microscopy was applied to identify exosome morphology,Western blot to identify protein markers of plasma exosomes,and Nanoparticle tracking analysis to identify exosome size.Combining high-throughput sequencing with bioinformatics analysis to screen for differentially expressed miRNAs in plasma exosomes between twelve patients with DLBCL and six healthy controls.Application of RT-q PCR to validate the screened miRNAs.The application of the receiver operating characteristic curve to analyze the diagnostic value of differentially expressed miRNAs for DLBCL and to validate the potential of miRNAs as diagnostic markers.Further,GO analysis and KEGG analysis were performed on the target genes of the differential miRNAs.Results:1.Plasma exosomes were extracted from the kit,and their morphological structure and integrity were observed by transmission electron microscopy,with a teatro-like appearance and intact bilayer membrane structure.Western blot detected the expression of its marker proteins CD63,TSG101,and HSP70.Nanoparticle tracking analysis determined the particle size to be between 40-111.8 nm,in the range of exosomes reported in the literature.This proved that via the kit successful extraction of plasma exosomes.2.Second-generation high-throughput sequencing to identify miRNAs carried by plasma exosomes and applied bioinformatics to calculate the expression of miRNAs.Using q-value <= 0.05 and Fold-change >= 2 as screening criteria,67 miRNAs differentially expressed in the DLBCL group and healthy control group were selected.Among them,the expression of has-miR-106b-5p was lower in the DLBCL group than in the healthy control group,the expressions of has-miR-28-3p and has-miR-184 were higher in the DLBCL group than in the healthy control group,and the differences were statistically significant.3.Go analysis of the differentially expressed miRNAs showed that the go functional enrichment analysis of the up-regulated and down-regulated differential miRNAs target genes was mainly significantly enriched to cellular process,single-organism process,and metabolic process at the biological process;At the cellular component level,it was mainly enriched in cell part,cell,and organelle;At the molecular function level,it was mainly enriched in binding and catalytic activity.4.Further biological pathway enrichment analysis of differential miRNAs target genes was performed according to the KEGG database to extract pathways significantly enriched in differential miRNAs target genes.Upregulated differential miRNAs target genes functioned in serval signaling pathways,such as Cholinergic synapse,Neurotrophin signaling pathway,and Erb B signaling pathway.Downregulated differential miRNAs target genes functioned in serval signaling pathways,such as the Oxytocin signaling pathway,AMPK signaling pathway,and Circadian entrainment.5.RT-q PCR was performed to quantify and validate has-miR-106b-5p,has-miR-28-3p,and has-miR-184,and the results showed that the expression of has-miR-106b-5p was downregulated and the expression of has-miR-28-3p was upregulated in the DLBCL group,the differences were statistically significant,and the results were consistent with the sequencing results consistent.However,the expression of has-miR-184 was comparable in DLBCL and healthy controls,and the difference was not statistically significant.6.The ROC curve was used to evaluate the values of exosomal miRNAs for the diagnosis of DLBCL patients,and the results showed that has-miR-106b-5p has a moderate diagnostic value for DLBCL(AUC=0.806,CI 95%=(0.595-1.00)),has-miR-28-3p has significant diagnostic value for DLBCL(AUC=0.889,CI 95%=(0.72-1.00)).The combination of has-miR-106b-5p and has-miR-28-3p improved the diagnostic values of DLBCL(AUC=0.944,CI 95%=(0.826-1.00)).The mentioned results showed that has-miR-106b-5p and has-miR-28-3p have potential diagnostic values in DLBCL.The combination with has-miR-106b-5p and has-miR-28-3p notably improved the diagnostic accuracy of DLBCL.Conclusion:This study found that 67 plasma exosomal miRNAs were differentially expressed in DLBCL,and after screening and validation,plasma exosomal has-miR-106b-5p and has-miR-28-3p were found to have potential diagnostic value in predicting DLBCL.