Differentially Expressed TRFs In CD5 Positive Relapsed&Refractory Diffuse Large B Cell Lymphoma And The Bioinformatic Analysis

Background:Diffuse large B-cell lymphoma(DLBCL)is one of the most common lymphomas in adults which accounting for 38%of non-Hodgkin's lymphoma(NHL)in China.A sub-group of DLBCL patients with CD5 positive exhibit more aggressive course of the disease and a considerable number of them will develop into relapsed&refractory(R/R)DLBCL.Furthermore,patients with CD5+DLBCL in Asian countries showed a female predominance.The exact mechanism of the pathogenesis,progression,and chemotherapy-resistance of these patients is still unknown and it may be related to multiple oncogene mutations and abnormal signal pathways.Therefore,investigating the accurate molecular mechanisms of the pathogenesis and progression of CD5+R/R DLBCL is extremely meaningful.It has been confirmed that small non-coding RNAs play important roles in the classification,diagnosis,treatment selection,and prognosis of DLBCL.The tRFs,which derived from tRNA cleavage in a specific manner,are groups of abundant non-coding RNAs only less than miRNAs and were proved can play roles similar to miRNA in some biological processes.Different from miRNA,tRFs are highly conserved,tissue-specific and time-specific and are reported to express stability in almost all types of cells and organisms and can be detected in body fluid.Compared with miRNAs,tRFs have their advantages to be used as candidate fluid-based biomarkers.Objective:This study was designed to demonstrate the differentially expressed tRFs in patients withCD5 positive relapsed&refractory diffuse large B cell lymphoma and the relationship between our results and data from tRF2Cancer.After that,we select a few of tRF sequences for further study and predict their potential clinical functions.Materials and Methods:1.Patients and normal specimen selection and collection,2.Total RNA Isolation and RNA Sequencing3.Data Analysis of tRFs&tiRNAs4.Data collection and analyzation5.RNA extraction and Real-time quantitative polymerase chain reaction,6.Bioinformatic analysis for the potential clinical use of the tRFs.Results:1.Three female patients were enrolled in this study at Shandong Provincial Hospital(Shandong,China).They were diagnosed as typical R/R DLBCL with CD5+according to the entries formulated by ESMO Clinical Practice Guideline.All three patients received at least three cycles of R-CHOP regimen before the disease progression or relapse.2.Totally,308 tRFs&tiRN As were identified to expressed specificly in patients with CD5+R/R DLBCL and 406 tRFs&tiRNAs were identified to expressed specificly in the control group.Among subtypes of tRFs&tiRNAs,tRF-5 has the highest expression level which accounts 49 percent and the lowest part is tiRNA-3.3.From the perspective of tRFs and tiRNA classification,tRF-3 and tRF-5 are highly expressed tRFs in DLBCL4.Four sequences were significantly upregulated and six were significantly downregulated in patients with CD5+R/R DLBCL.To confirm the content of the differentially expressed tRFs in the patient's group,we performed quantitative real-time PCR.Finally,we verified the three tRFs including AS-tDR-011395,AS-tDR-008946,and AS-tDR-013492.The results of RT-PCR were consistent with the sequencing analysis.5.The sequences were loaded to a homemade software based on TargetScan and miRanda,we identified the associated target genes for the three tRFs(AS-tDR-013492,AS-tDR-011395,and AS-tDR-008946).The number of target genes for AS-tDR-013492,AS-tDR-011395,and AS-tDR-008946 is 497,9114,and 730,respectively.6.Because sequence AS-tDR-011395 has a large number of potential regulatory target genes(9114).It is commonly not reasonable in scientific significance,so we made a functional enrichment analysis for the other two tRFs to get their potential regulatory genes.Biological annotation of the performed using the DAVID online analysis tool,and GO functional enrichment of up-regulated genes were obtained.Based on the KEGG database,we analyzed the pathways in which the differentially expressed target genes were involved.The PPI network of the target genes number of AS-tDR-008946 comprised of 227 nodes and 480 edges.The PPI network of the target genes number of AS-tDR-013492 comprised of 289 nodes and 572 edges.NEDD4L,UBA52 and PDGFB were identified by the overlap of the top 20 genes according to four ranked methods in cytoHubba.7.UBA52 is significantly underexpressed in DLBCL cell lines,suggesting that UBA52 may be involved in the pathogenesis of DLBCL.AS-tDR-013492 may regulate UBA52 expression in a miRNA-like manner.8.NEDD4L,PDGFB and UBA52 may play important roles in signal transmission and RNA synthesis,DNA methylation and other biological process.The drugs target these genes may be beneficial to the treatment of CD5+R/R DLBCL.Conclusions:This study indicated that 308 tRFs&tiRNAs expressed specificly in patients with CD5+R/R DLBCL and 406 tRFs&tiRNAs were identified to expressed specificly in the control group.tRF-3 and tRF-5 are the highly expressed tRFs types in DLBCL.The relative expression of AS-tDR-008946 and AS-tDR-013492 are higher than other fragments.So that these two fragments have the potential to be the specific biomarkers for the diagnosis of CD5+R/R DLBCL.PI3K/AKT and MAPK/ERK are the pathways that influenced most frequently by tRFs associated genes.As a result,NEDD4L and UBA52 were identified and may play important roles in signal transmission and RNA synthesis.respectively.The drugs target these genes may be beneficial to the treatment of CD5+R/R DLBCL.