Plasma EBV miRNAs Profiles Reveal Potential Biomarkers for clinical prognosis of Acquired Immune Deficiency Syndrome-related Lymphoma

Background : Acquired immune deficiency syndrome-related lymphoma (ARL) is closely related to Epstein-Barr virus (EBV) infection. However, there are few studies on the occurrence and development of EBV microRNAs (miRNAs) in ARL patients. Methods : The plasma of 5 EBV-infected ARL patients and 8 EBV-infected HIV patients were screened for EBV miRNAs differentially expressed between the two groups through a customized EBV microRNA quantification chip. The plasma of 35 EBV-infected ARL patients and 20 EBV-infected HIV patients was verified by qRT-PCR expanded samples. And we gave a further analysis of the correlation between differentially expressed EBV miRNAs and clinical indicators in ARL patients. Results : 1. There were differential expressions of EBV miRNAs in the plasma of EBV-infected ARL patients and EBV-infected HIV patients. It was found that ebv-miR-BART2-5p, expression was significantly up-regulated and ebv-miR-BART9-5p expression was significantly down-regulated; 2. EBV miRNAs with significantly different expressions in the screening results were expanded and verified by qRT-PCR, and the differential expression was found to be consistent with the screening results; 3. The relative expression levels of ebv-miR-BART8-3p,ebv-miR-BART19-5p and ebv-miR-BART9-5p in plasma were positive correlated with the international prognostic index (IPI) score of Lymphoma, eastern cooperative oncology group (ECOG) scores are the incidence of lymphoma B symptoms ( P = 0.03, 0.01, and 0.04, respectively);4. EBV miRNAs could be used as biomarkers for ARL prognosis evaluation. Conclusions : The expression of ebv-miR-BART2-5p, ebv-miR-BART8-3p, ebv-miR-BART15,ebv-miR-BART19-5p,ebv-miR-BART9-5p and ebv-miR-BART6-3p in ARL patients were highly different; and ebv-miR-BART8-3p, ebv-miR-BART19-5p, ebv-miR-BART9-5p could be used as the biomarkers of the prognosis of ARL evaluation.


Background
Epstein-Barr virus (EBV) is the first human tumor virus to be discovered [1]. EBV mainly establishes infection in two types of cells, lymphocytes and epithelial cells, and exists for a long time in the host，which is the cause of many malignancies, including nasopharyngeal carcinoma (NPC), EBV-associated gastric cancer (EBVaGC) as well as acquired immune deficiency syndrome-related lymphoma (ARL) and post-transplant lymphoproliferative disease (PTLD) [1，2]. EBV has two infection states in the human body, namely latent infection and lytic infection. The latent infection period can be divided into 4 types: latent period 0, I, II and III [3]. EBV infections in different periods have been proved to promote the occurrence and development of EBV-related tumors [1， 4]. MicroRNAs (miRNAs) are a group of non-coding small RNAs, consisting of [19][20][21][22][23][24][25] nucleotides, which directly bind to the 3'-untranslated region (3'-UTR) of mRNA, promote mRNA degradation and inhibit its translation gene regulation [2]. EBV was the first virus found to encode miRNAs [5]. So far, previous studies have found that miR-BHRF1s is higher in patients with AIDS and DLBCL [6].
In this study, we firstly performed differential expression analysis of EBV miRNAs in Whether these EBV miRNAs could be the biomaker for clinical prognosis were be tested by ROC analysis.

RNA extraction
Remove the plasma sample, centrifuge at 3000G for 5 min after thawing, add 750 ul TRIZOL-LS (Invitrogen, NO. MAN0000806) to the supernatant, vortex for 5 s, and incubate at room temperature for 5 min. Add 0.2ml of chloroform per 1ml of homogenized sample and vortex for 15s, then incubate for 3min. Centrifuge at 13000G for 15 minutes at 4 ° C. Isopropanol was added to the aqueous phase layer, and after standing at 4 ° C for 30 minutes, it was centrifuged at 13000 G at 4 ° C for 15 minutes.
Remove the supernatant and add 1ml of 75% ethanol to each 1ml of homogenized sample to wash the RNA pellet. Let stand for 10min, then centrifuge at 10000G for 5min at 4 ° C. Precipitate the RNA in the air for 5-10 min, add RNA-free water to elute the RNA, and measure the optical density (OD) at 260 nm, 260/280 ratio using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) To assess the concentration and purity of RNA, and finally stored at -80 ℃.

Synthesis of complementary DNA (cDNA)
Add 1μl of ATP (10mM) to the reaction system, 1μl of 10X A-Plus Reaction Buffer, 700ng of RNA sample, 0.5μl of A-Plus Poly (A) Polymerase (4U / μl), 0.2μl of RNase inhibitor (40u / μl), Add RNase-free water to a total volume of 10 μl. After incubating at 37 ° C for 10 minutes, add oligo dT linker primer miR-RT qRT-PCR verification of differentially expressed EBV miRNAs qRT PCR reaction system is 2 × Master Mix 5μl, forward and reverse miRNA primers (see Table 2) 0.5μl, add water to a total volume of 8μl, centrifuge briefly at 5000 rpm, and sequentially add to the 384-PCR well plate, each add 2 μl of cDNA to the well, cover with sealing film and centrifuge briefly. The specific PCR reaction program is 95 ℃, 10min; 40 PCR cycles (95 ℃, 10 seconds; 62 ℃, 60 seconds).

Data analysis
The results of the differential expression levels of EBV miRNAs are expressed in standard errors.

Differential expression of EBV miRNAs in ARL patients
In this study, 2-ΔΔCt was used to calculate the differential expression multiples of EBV miRNAs between ARL and the control group, and the P values were plotted as scatter plots, volcano plots, and histograms ( Figure 1 (P value <0.05), and then screen based on the condition that the CT value is less than 35, and finally screen out ebv-miR-BART2-5p, ebv-miR-BART8-3p, ebv-miR-BART15, ebv-miR-BART19-5p ebv-miR-BART9-5p 5 EBV miRNAs. Compared with the control group, the relative expression of ebv-miR-BART19-5p in ARL was down-regulated, and the rest were up-regulated.

Expanded sample qRT-PCR verification
Further expanded sample qRT-PCR verification found that the results are consistent with the screening results (see Figure 2).

miR-BART9-5p in patients with ARL is related to clinical prognosis
We used the Spearman rank correlation coefficient for analysis and found that in ARL patients, the expressions of ebv-miR-BART8-3p, ebv-miR-BART19-5p, ebv-miR-BART9-5p were associated with IPI score, ECOG score There is a correlation between the occurrence of B symptoms (Figure 3). We used the clinical outcome of ARL patients to achieve complete remission after clinical chemotherapy as a clinical outcome, and found that EBV miRNAs were superior to IPI scores in ARL prognosis judgment ( Figure 4).

Discussion
EBV expresses high levels of miRNAs at all stages of its life cycle, indicating that these miRNAs may be involved in the interaction between EBV and the host immune system [9]. More and more studies have found that EBV miRNAs can promote cell proliferation and transformation by targeting host mRNA and inhibit cell apoptosis [10].
In addition, EBV miRNAs can also suppress the expression of viral antigens, thereby allowing infected cells to escape immune recognition [11]. More interestingly, EBV miRNAs can directly suppress the host's antiviral immunity by interfering with antigen presentation and immune cell activation [12]. In ARL patients, there are few studies on the expression characteristics and related clinical significance of EBV miRNAs.
Currently, the IPI score is the most commonly used prognostic evaluation system for patients with lymphoma [13]. Clinicians evaluate the prognosis of patients by scoring five items: patient age, lymphoma stage, ECOG score, extranodal lesions, and LDH.
These projects involve a variety of blood, ultrasound, PET-CT and other tests, which consume a lot of medical resources. Our research and further analysis found that the relative expression of ebv-miR-BART8-3p, ebv-miR-BART19-5p and ebv-miR-BART9-5p in plasma is correlated with ARL's IPI score, ECOG score and the occurrence of B symptoms -miR-BART9-5p, which can be used as a biomarker for prognosis evaluation, can more easily achieve prognosis evaluation, and is better than IPI score in prognosis judgment. ECOG as a system for evaluating the physical status of patients with lymphoma can better assess the tolerance of patients to treatment. Our research results found that the expression level of ebv-miR-BART19-5p can also be used as a reference marker for this evaluation.
Related research further studies the mechanism of EBV miRNAs in promoting tumorigenesis and development. RND3 is a miR-BART2-5p targeting gene. RND3 is related to apoptosis, cell cycle arrest and cell differentiation [14]. According to reports, in nasopharyngeal carcinoma, miR-BART2-5p has potential value in promoting nasopharyngeal carcinoma tumor metastasis and its use as a prognostic indicator or therapeutic target [15]. There are also reports that miR-BART2-5p can be used as an early detection indicator for patients with nasopharyngeal carcinoma [16]. Experiments have shown that miR-BART8-3p can regulate ataxia telangiectasia mutations / ataxia telangiectasia mutations (ATM / ATR) and Rad3 The activity of related signaling pathways promotes NPC's radiation resistance [17]. MiR-BART15 is capable of targeting nucleotide-bound oligomerization domain-like receptor family pyridine domain-containing 3 (NLRP3) with less inflammation to limit inflammation and promote EBV infection [18]. LMP1 is a transmembrane latent protein encoded by EBV, which is essential for cell proliferation and transformation [19], but overexpression of LMP1 will inhibite cell proliferation and increase the sensitivity of cells to proapoptotic stress [20]. According to previous reports, miR-BART19-5p can inhibit the expression of LMP1, thereby maintaining a balance between the growth-promoting effect of LMP1 and its pro-apoptotic function [10]. We infer that the EBV miRNAs found in our research may also be through similar molecular mechanisms It led to the occurrence and development of ARL and led to different clinical outcomes.
In the next stage of research, we will predict the target genes of differentially expressed EBV miRNAs and obtain relevant cell signaling pathways through further analysis to further reveal and clarify the role and mechanism of EBV miRNAs in the development and development of ARL for the prognosis of ARL Provide a basis for judging and finding new therapeutic targets.

Conclusion
This study found that the differentially expressed EBV miRNAs in ARL patients are closely related to the clinical prognostic indicators (IPI score, ECOG score, and lymphoma B symptoms) of ARL patients, and can be used as biomarkers for ARL prognosis assessment. The study has obtained informed consent from all study participants.

Consent for publication
Not applicable

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.