Differentially Expressed Trfs in Relapsed & Refractory Diffuse Large B Cell Lymphoma and the Bioinformatic Analysis for Their Potential Clinical Use

Introduction

Diffuse large B-cell lymphoma (DLBCL) is an aggressive but potentially curable malignancy in some low-risk patients. However, patients with high-risk factors or diagnosed as some special subtypes often have a poor prognosis although they received intensive combine chemotherapy or bio-target treatment. The exact mechanism of the anti-chemotherapy is unknown and it may be related to multiple oncogene mutations and signal pathways. tRFs is reported stability in almost all types of cells and organisms ranging from humans to bacteria. They are highly conserved, tissue-specific and time-specific tRNA fragments and can be detected in body fluid.

Their precise sequence structure located at the end of tRNA or nearby. The length of them has a close relationship with the nucleotide composition at cleavage junctions, indicating that the tRFs are derived from tRNA cleavage in a specific manner. tRFs is a group of abundant non-coding RNAs secondly only to miRNAs. It has been proved that tRFs participate in many biological processes including cell proliferation, viral reverse transcriptases activation, gene expression, RNA procession regulation, DNA damage response modulation and tumor suppression. In this study, we aimed to investigate the potential function of associated tRFs in patients with relapsed & refractory (R/R) DLBCL.

Materials and Methods

Peripheral blood mononuclear cells (PBMCs) were separated from patients with R/R DLBCL and control groups, respectively. Next-generation sequencing and quantitative real-time reverse transcription-PCR were used to determine the expression profiles of tRFs in the two groups. Two databases of tRFs, tRFs2Cancer and tRFdb were utilized to analyse the similarity and dissimilarity of homologous tRNA fragment.

Results

1. Among the sequences with significant difference value, ten tRFs were picked, four (sequence 1- sequence 4) were up-regulated and six (sequence 5- sequence 10) were down-regulated (Figure1). To investigate the content of the differentially expressed tRF in patients group, we performed quantitative PCR to verify three tRFs. The PCR result was consistent with the sequencing analysis.

2. Combined with the results of qRT-PCR and database searching (Figure2,3), we finally chose three sequences (sequence 2, sequence 4 and sequence 10) for further study.

3. With the help of TargetScan, miRanda and other target gene predicting tools, we identified the associated target genes for the three tRFs (sequence 2:497, sequence 10: 9114, sequence 4: 730). The regulatory relationship of sequence 4 is shown in figure 4, the color of each line corresponds to the chromosome which the tRNA comes from.

4. Because sequence 10 has a large number of potential regulatory target genes and it is difficult to obtain satisfying results, we made a functional enrichment analysis for the other tRFs to get their potential regulatory genes. The results showed that the potential target genes of sequence 4 tend to be enriched in multiple ways, including plasma membrane-bounded cell projection morphogenesis, nervous system development, and sensory organ development, etc. The potential target genes of sequence 2 tend to be enriched in FCGR-dependent phagocytosis, Ras signaling pathway, and cellular component regulation(Figure 5).

5. The results of protein interactions analysis showed that the potential regulatory target genes of sequence 4 and sequence 2 can be constructed to gene-protein interaction networks. In this network, several biological processes such as alternative splicing, endosomal transport, and endocytosis might play important roles in tumorigenesis(Figure 6).

Conclusion

In this study, we analyzed the differently expressed tRFs profiles in patients with R/R DLBCL and the results are consistent with the database data analysis. We also predicted the possible mechanism of the up- or down-regulated tRFs which might be used in clinical although they are unexploited yet. Up or down-regulated tRFs is common in many types of tumors. It is well known that knockdown of the overexpressed tRF may inhibit tumor cell proliferation and the recovery of the downregulated tRFs may reverse the sensitivity to chemotherapy. We hyperthesis that the associated tRFs can be used as fluid-based biomarkers for diagnosis and prognosis assessment. Future investigation on them may help to develop new methods to repair the disordered biological signaling pathways associated with the DLBCL.

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