EBV-miR-BART5-3p dysregulates the cell cycle induced proliferation of Burkitt Lymphoma cells through downregulating TP53 (Preprint)

Abstract

Burkitt’s lymphoma (BL) is a highly aggressive form of B-cell lymphoma that primarily arises within or after the germinal center[1]. While Burkitt’s lymphoma is considered rare in China, it constitutes 30%-50% of childhood tumors in Africa, as reported by the World Health Organization (WHO)[2, 3]. Unfortunately, the incidence of this condition has been steadily increasing, posing a significant threat to human health[4]. It should be emphasized that a significant number of individuals diagnosed with Burkitt’s lymphoma are already in the advanced stages of the disease (stages III to IV) when they seek medical attention[5]. Although chemotherapy remains the cornerstone of treatment, the effectiveness of medical treatments and predicted outcomes for individuals with advanced-stage illnesses and refractory recurrence are far from satisfactory[6, 7]. Therefore, there is an urgent need for further research on the pathogenesis of Burkitt’s lymphoma, with a focus on identifying potential therapeutic targets via gateway molecules. EBV infection plays a pivotal role in the pathogenesis of BL, exerting a profound influence on the development of B-cell malignancy[8, 9]. Based on clinical manifestations, geographical distribution, infection patterns, and immune status, BL has been categorized into three distinct subtypes by WHO: endemic (Burkitt’s lymphoma), sporadic (Burkitt’s lymphoma), and immunodeficient-associated (ID-Burkitt’s lymphoma)[10]. EBV is found in approximately 95% of eBurkitt lymphoma cases, 20%-30% of sBurkitt lymphoma cases, and 25%-40% of ID-Burkitt lymphoma cases[11, 12]. Notably, nearly 100% of children with endemic BL demonstrate the existence of EBV infection[13, 14]. Nevertheless, the exact function of EBV infection in the cause of BL is still not be fully elucidated. EBV is the first virus to possess microRNAs (miRNAs) in its genome. These mature miRNAs are primarily encoded by two gene groups, namely, BamHI Fragment H Rightward Open Reading Frame 1 (BHRF1) and BamHI-A Region Rightward Transcript (BART). BHRF1 encodes only four miRNAs, whereas the remaining miRNAs are encoded by BARTs[15, 16]. The BART gene contains 22 precursor miRNAs (miR-BART 1-22), which can generate 44 mature miRNAs, all of which are highly expressed in EBV-associated lymphoma[17]. Studies have shown that in Akata cells, miR-BART10-3p[18] can target BHRF1, inhibit EBV replication and B lymphocytes, and reduce immune clearance. Additionally, EBV can directly or indirectly affect various tumor-related genes, such as the BIM tumor suppressor gene and the TCL-1 oncogene, in B lymphocytes, contributing to its carcinogenic role [19-22]. Zhang et al. found that EBV-miR-BART6-3p and miR-197 had a synergistic effect on reducing IL-6R in Burkitt lymphoma[23, 24]. Another study discovered that miR-BART6-3p can downregulate PTEN to encourage the growth of EBV-positive Burkitt lymphoma cells [25]. Furthermore, in Burkitt lymphoma cells (Raji), miR-BART20-5P can silence the BCL-2 related death promoter BAD gene, leading to anti-apoptotic effects [26]. The abnormal expression of EBV-miRNA-BARTs in Burkitt lymphoma suggests their crucial role in tumorigenesis and development[27-29]. Yet, in regards to Burkitt lymphoma, the specific mechanisms of action and target genes controlled by these miRNAs have not been thoroughly investigated. The cell cycle is the basic process of cell life activities, which includes two stages, cell division and cell growth, and is crucial for cell growth, division, and repair[30]. The regulatory mechanisms of the cell cycle are complex and involve the interaction of multiple genes and proteins, including CDK1 and cyclinD1[31, 32]. EBV is closely associated with the cell cycle[33]. Some studies have pointed out that EBV can also regulate the latent genes required for B-cell immortalization, thus affecting the cell cycle[34]. Therefore, an in-depth study of the relationship between EBV and the cell cycle is helpful to explore the pathogenesis of EBV infection-related diseases and to provide new ideas for treatment.

Background: EB virus infection runs through the entire process of B-cell malignant Burkitt lymphoma. The role of EBV-miRNA-BART5-3p has been investigated in nasopharyngeal carcinoma and gastric cancer, but has not been studied in Burkitt lymphoma. The primary aim of this study was to explore the impact of EBV-miRNA-BART5-3p on the progression of Burkitt lymphoma. Methods: We observed the upregulation of EBV-miR-BART5-3p in EBV-positive Burkitt's lymphoma cells using a range of methodologies, including the use of a bioinformatics tool, real-time PCR, western blot analysis, and flow cytometry analysis. The effects and underlying mechanisms of BART5-3p in Burkitt’s lymphoma were further investigated through exogenous overexpression or inhibition in cells. Results: In this study, we detected BART5-3p was highly expressed and decrease P53 levels in Epstein-Barr virus-positive Burkitt lymphoma cells. A higher level of BART5-3p can downregulate P53 and is significantly associated with cell proliferation. In addition, flow cytometry analysis revealed that BART5-3p may regulate the cell cycle of Burkitt lymphoma cells by downregulating P53 through the flow cytometry analysis. Conclusions: Our findings suggest that EBV-miR-BART5-3p contributes to dysregulation of the cell cycle and proliferation in Burkitt’s lymphoma cells by directly downregulating P53, thereby providing a potential prognostic target for EBV-positive Burkitt's lymphoma patients.

Cell line and cell culture Two EBV-negative BL cell lines were obtained from Shanghai Fuheng Biotechnology Co., Ltd. and Henan Engineering Research Center of Industrial Microbiology, respectively, and named Ramos and CA46. The EBV-positive BL cell line Raji was purchased from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). HEK293T cells were originally obtained from Guangzhou Cellcook Biotech Co., Ltd. and were grown in a medium supplemented with 10% fetal bovine serum (Every Green, Zhejiang, China) and Dulbecco's modified Eagle medium (DMEM; BasalMedia). The other cells were maintained at 37°C and 5% CO2 in RPMI-1640 (BasalMedia, Shanghai, China) supplemented with 10% fetal bovine serum. Target estimation Employing accession number NM_000546.6, the TP53 sequence utilized to predict miRNA targets was obtained from the National Center for Biotechnology Information (NCBI). EBV-miRNA-BART5-3p sequence was obtained from miRBase (MIMAT0009205). The RNAhybrid program (http://bibiserv. techfak. uni-biele. org. de/rnahybrid/) was used to estimate the relationship and minimum energy-free hybridization of miRNAs and target RNAs. Lentivirus transductions Lentiviral particles containing the BART5-3p precursor and related control were provided by Prof. Wenqiang Yu from Fudan University in Shanghai, China. Lentiviral particles were generated using the GV209 plasmid containing the H1-MCS-CMV-EGFP construct. Lentiviral particles were transduced into Burkitt lymphoma (BL) cells according to the manufacturer’s instructions. Plasmid preparation and cell transfection A TIANprep Midi Plasmid Kit (TIANGEN, China) was used to purify plasmid DNAs. Plasmid DNA (200 ng of plasmid DNA was transfected using Invitrogen's Lipofectamine 2000 reagent in Ramos-BART5-3p and CA46-BART5-3p cells. Western blotting and RT-qPCR were performed to verify the levels of BART5-3p 48 hours after transfection. For the transfection of the BART5-3p inhibitor, P53 inhibitor, and their corresponding negative controls (NCs), cells were transfected with Lipofectamine 2000 (Invitrogen) at an ultimate dosage of 50 nmol/L in serum-free conditions. The medium was replaced with freshly prepared RPMI-1640 containing 10% fetal bovine serum four hours later. The BART5-3p inhibitor and its control, as well as the P53 inhibitor and its control inhibitor (TargetMoI), were purchased from RIBOBIO (Guangzhou, China). All cells were maintained at 37 °C in a humidified environment with 95% air and 5% CO2. RNA extraction and RT-qPCR Total Ambion's harvested USA the TRIzol reagent (Ambion, total RNA was harvested. MonScriptTM RTLLL ALL-in-One Mix with dsDNase (Monad Biotech Co., Ltd.) was used to synthesize cDNA. MonAmpTM SYBR Green qPCR Mix (Monad Biotech Co., Ltd.) was used for RT-qPCR analysis. Supplementary Table 1 includes a list of the primers used for PCR. Except if otherwise noted, the data were first normalized to the level of GAPDH and then to the negative control group. Each reaction was performed three times and again in separate experiments. Relative quantification (2^(− ΔΔCt)) was performed to compute fold-changes. Dual-luciferase reporter assay The day before transfection, the cells were spread in 96-well plates, and on the second day, the cell confluence was approximately 70–80%. The following day, Hieff TransTM, reporter vector, miRNA, and serum-free medium were successively added to 96-well plates; 37℃, 5% CO2 incubator for 6h and replaced with complete medium. The firefly luciferase reaction working solution and arrilla luciferase reaction solution were prepared to detect the viability of firefly luciferase and arrilla luciferase. CCK-8 assay and EdU proliferation assay Following transfection, plates containing 96 wells were seeded at 1x104 cells per well of Ramos, CA46, and Raji cells per well. The Yeasen Biotechnology (Shan) cell Counting Kit 8 (CCK-8) was used to measure cell proliferation. After seeding, 10 mL of CCK-8 solution was added to each well at 24, 48, 72, and 96 h, the plates were incubated at 37°C for 4 h. After incubation, a microplate reader (Thermo MULTISKAN FC) was used to determine the absorbance at 450 nm. Proliferating BL cells were analyzed for EdU incorporation using the C0071 BeyoClickTM EDU-488 Cell Proliferation Assay Kit in accordance with the manufacturer's instructions. Transwell assay Transwell chambers with 8 mm pore filters (Corning Incorporated, Corning, NY, USA) were used to evaluate the migratory ability of cells. BL cells were cultured for 48 h after transfection. Following dissociation, 1×105 cells were reconstituted in serum-free medium and inserted into Transwell chamber inserts, which had 8 µm pores. The testing chambers were then placed in wells filled with media containing 10% FBS. The cells located on the upper surface of the membrane were removed after incubation for 24 h. Cells that had moved to the lower surface of the membrane were fixed with 100% methanol and stained with 0.5% crystal violet Ammonium Oxalate Solution and 1% (Solarbio). Under various microscopic conditions, the moved cells were analyzed in six randomly selected optical fields. Three independent runs were performed for each assay. Cell cycle assay A Cell Cycle Staining kit from MULTI SCIENCES was used for cell cycle detection. Initially, the cells were transfected with miRNA plasmid DNA for 48 h after they were placed into 6-well plates. Following the harvesting of 5x104 cells, 1 ml of 1x Staining buffer was added to the cells. After resuspending the cells, 4 μl of the RedNucleus I staining solution was added to the sample. The samples were incubated for 20 min at room temperature. Flow cytometry was used to evaluate and measure the stained cells following the incubation period. Western blotting Western blotting was performed according to the manufacturer’s protocol. Protease inhibitors from AbMole BioScience were added to RIPA buffer (high, Solarbio) before cells were harvested and lysed. Next, the proteins were separated using Servicebio SDS-polyacrylamide gel electrophoresis gels. Once the proteins were separated, they were placed on a PVDF membrane (Pore Size: 0.45um,). Subsequently, the PVDF membrane was examined using an initial antibody and probed with antibodies against p53 (1:10,000; 60283-2-Ig; Proteintech, China), Cyclin D1 (1:10,000; 26939-1-AP; Proteintech, China), GAPDH (1:10,000; 60004-1-Ig; Proteintech, China), and β-actin (1:50,000; 66009-1-Ig; Proteintech, China). A secondary antibody conjugated with peroxidase was then used to probe it (SA00001-1; Proteintech, China), and GAPDH and β-actin served as controls for protein loading. To visualize the western blotting bands, a Servicebio Hypersensitive ECL chemiluminescence kit was used. Bands were captured using a ChemiDocTM CRS þ Molecular Imager (Bio-Rad, USA). All blots in the figures were labeled with the locations of the molecular weight/size markers. Statistical analysis. Except as noted otherwise, every experiment was run in triplicate, and the results are displayed as the mean ± s.e.m (standard error of the mean). GraphPad Prism (GraphPad Software, San Diego, CA) was used for curve fitting and assessment. A significance threshold of P<0.05 was used to assess the probability of significance. Statistical significance was set at p < 0.05. One-way analysis of variance (ANOVA) was performed for all groups. Single, double, and triple asterisks, which represent p-values less than 0.05, 0.01, and 0.001, respectively, were used to indicate the level of statistical significance.

The expression of BART5-3p is upregulated in EBV associated BL cells To investigate the expression level of BART5-3p in BL, the experimental design was determined by performing RT-qPCR analysis of BART5-3p expression in Epstein-Barr virus-negative BL cell lines (Ramos and CA46) and Epstein-Barr virus-positive BL cells (Raji). In contrast to the other two cell lines, Raji cells showed substantially higher levels of BART5-3p expression (Figure 1a). Next, we aimed to upregulate BART5-3p levels in Ramos and CA46 cell lines using liposome-mediated transduction. We successfully generated two stable EBV-negative BL cell lines expressing BART5-3p. The transfection efficiency of BART5-3p in these two cell lines was compared to the control vector using RT-qPCR (Figure 1b), The fluorography after the transfection of BART5-3p was visualized by inverted fluorescence microscopy as shown in Figure 1c. Figure 1 | EBV-miR-BART5-3p was upregulated in EBV-positive Burkitt's lymphoma cells. (a) analysis of miR-BART5-3p levels in three cells by RT-qPCR. GAPDH was used as an internal control. (b) statistical analysis of RT-qPCR results showed significantly increased BART5-3p expression in CA46 and Ramos cells after plasmid transfection. (c) the fluorography after the transfection of BART5-3p was visualized by inverted fluorescence microscopy. Values are mean ± SD; * * *: P <0.001. Highly expressed BART5-3p dysregulates the cell cycle enhanced the proliferation of BL cells To assess the effect of BART5-3p on the growth of BL cells, we conducted CCK8 cell proliferation and EdU incorporation assays (Figure 2a and 2b). The results demonstrated that BART5-3p significantly promoted cell proliferation in both the cell lines. Importantly, according to the in vitro migration assays, BART5-3p levels markedly improved the migration of both Ramos-BART5-3p and CA46-BART5-3p compared to the respective negative controls (Figure 2c). Additionally, using flow cytometry, we examined the cell cycle phase in cells with high levels of BART5-3p to determine whether the increased cell growth induced by this substance was associated with cell cycle regulation. Cells overexpressing BART5-3p displayed a longer S phase or G2/M phase in the cell cycle distribution, along with a shorter G0/G1 phase, than the negative control cells (Figure 2d). Remarkably, western blot analysis demonstrated significantly higher protein levels of the cell cycle-related genes cyclinD1, in cells with exogenous BART5-3p expression (Figures 2e). Collectively, these findings imply that BL cell replication is stimulated by BART5-3p, potentially contributing to cancer growth by regulating the cell cycle. Figure 2 | Highly expressed BART5-3p dysregulates the cell cycle enhanced the proliferation of BL cells. (a-c) The CCK8 cell proliferation assay, EdU incorporation assays and transwell migration assay were used to detect cell proliferation. (d-e) Ramos and CA46 cells were transfected with BART5 plasmid for 48 h, the cell cycle distribution was detected by a flow cytometer and the protein levels of cell cycle related genes cyclinD1 by western blots, original blots are presented in Supplementary Figure 2e. Three independent experiments were performed, and data are shown as the means. *, P <0.05; **, P <0.01; ***, P <0.001 compared with the control group. EBV-miR-BART5-3p directly downregulates human TP53 gene P53 is a widely recognized gene that acts as a tumor suppressor. It has garnered significant interest for its involvement in the progression of different types of cancers, including Burkitt's lymphoma. To investigate the relationship between EBV infection and P53 expression in Burkitt lymphoma, we conducted a comparative analysis of P53 expression levels in normal and Burkitt lymphoma tissue samples using the GEO database (GSE65674 and GSE4464). Based on our analysis, we discovered that, in contrast to normal tissues, P53 level were decreased in BL tissues (Figure 3a). Moreover, we used the CCLE database to examine how EBV infection affected P53 expression in Burkitt lymphoma cell lines and confirmed that P53 expression was substantially lower in EBV-infected Raji cells than in EBV-uninfected CA46 cells (Figure 3b). To further validate the findings from the microarray data, we performed RT-qPCR to ascertain P53 expression levels in BL cells, eventually yielding outcomes that are in line with the front (Figure 3c). These findings imply that the downregulation of P53 expression in BL cells may be caused by EBV infection. The predicted gene targets of EBV-miR-BART5-3p were first obtained from TargetScan and RNAhybrid to further establish the connection between EBV-miR-BART5-3p with P53. According to biological informatics analysis, the EBV-miR-BART5-3p seed sequence and the 3'-UTR of P53 matched quite well (Figure 3d). Additionally, we used luciferase reporter assays and observed that the BART5-3p mimic markedly lowered the luciferase activity of the 3'-UTR when compared to the control mimic. This finding is consistent with a study conducted by Zheng et al. (Reference: Zheng et al.[35]), which raises the possibility that the BART5-3p mimic specifically targets target sites in the 3' UTR of TP53 mRNA. Next, we examined how BART5-3p affects the levels of P53 mRNA, as well as protein in BL cell lines. As demonstrated by RT-qPCR and western blot analysis, higher levels of BART5-3p drastically decreased the level of P53 (Figure 3e, 3f). Based on the aforementioned information, it is highly likely that BART5-3p targets TP53, implying that BART5-3p may be involved in the reduction of P53 levels in Burkitt's lymphoma. Figure 3 | EBV-miR-BART5-3p directly downregulates human TP53 gene. (a)P53 expression in normal tissues and Burkitt lymphoma tissues from the GEO database. (b) P53 expression in different cell lines in the CCLE database. (c) The expression level of P53 in Burkitt lymphoma cells was determined by RT-qPCR. (d) Bioinformatics predictions of one binding sites by BART5-3p in the P53 3’-UTR region. (e)The dual-luciferase reporter assay determined that EBV-miR-BART 5-3p could be targeted to degrade TP53. (f-g) The expression levels of TP53mRNA and protein in BL cells following EBV-miR-BART5-3p were determined by RT-qPCR and Western Blot, * P <0.05, * * * P <0.001, original blots are presented in Supplementary Figure 3g. Inhibiting TP53 promotes BL proliferation To ascertain whether P53 downregulation produces a phenotype similar to that attained by BART5-3p, we used P53 inhibitors to downregulate P53 expression in Ramos and CA46 cells. After 48 h, we assessed P53 expression levels using RT-qPCR (Figure 4a). As expected, downregulation of P53 by miR-BART5-3p led to increased cell growth. This led us to hypothesize that maybe P53 controls how cell function. To verify this assumption, we explored the role of P53's activity in modulating the cell cycle and proliferation. Cell growth and migration were measured using CCK-8 proliferation and transwell assays, and the findings indicated that the proportion of proliferating and migrating cells increased in Ramos and CA46 cells treated with the P53 inhibitor compared to those treated with the control inhibitor (Figure 4b, 4c). In addition, in comparison to the control inhibitor, downregulating P53 increased the proportion of cells that participated in the S and G2/M phases and lowered the proportion of cells in the G0/G1 phase (Figure 4d). The results presented indicate that in Burkitt's lymphoma cells, BART5-3p negatively regulates P53 thus encouraging cell proliferation, migration, and alterations in cell cycle distribution. Figure 4 | Inhibiting TP53 promotes BL proliferation. (a) Expression levels of P53 after application of P53 inhibitor in Ramos and CA46 cells. (b-c) The CCK8 cell proliferation assay and transwell migration assay were used to detect cell proliferation and migration. (d) Cell cycle distribution of Ramos and CA46 cells after the use of P53 inhibitors was determined using flow cytometry. Three independent experiments were performed, and data are shown as the means. *, P <0.05; **, P <0.01; ***, P <0.001 compared with the control group. Inhibit the proliferation of BL after knockdown of BART5-3p in Raji cells To understand how innately expressed BART5-3p influences P53 expression in Burkitt's lymphoma cells infected with EBV, we conducted experiments using BART5-3p and P53 inhibitors in Raji cells. First, we assessed the effect of a P53 inhibitor on P53 mRNA levels. After treating Raji cells with the P53 inhibitor, we found that P53 mRNA levels decreased by 5% compared with the control (Figure 5a). We subsequently examined the effect of the BART5-3p inhibitor on P53. In comparison to the control group following inhibitor transfection, we noted a 7-fold higher level of P53 protein levels after transfecting Raji cells using the BART5-3p inhibitor (Figure 5b, 5c). The results of these studies indicate that BART5-3p, which is naturally expressed, effectively reduces P53 expression in Raji cells, elevations of P53 mRNA and P53 protein are caused by BART5-3p inhibition. Overall, these findings further support the notion that BART5-3p has a major impact on controlling the activity of P53 in EBV-infected Burkitt's lymphoma cells. To validate the regulatory significance of BART5-3p on P53 activity in BL growth, as well as to research fundamental biological processes, we conducted experiments using the Raji cell line. First, we performed CCK-8 proliferation and Transwell migration assays to assess the influence of BART5-3p on cell migration. The results indicated that upregulation of P53 by the miR-BART5-3p inhibitor reduced cell migration, whereas knockdown of P53 alone increased cell migration. Interestingly, co-transfection with the P53 inhibitor eliminated the inhibition of Raji cell growth caused by the BART5-3p inhibitor compared to the control inhibitor (Figure 5d-5f). Flow cytometry was used for additional analysis of cell cycle distribution. Compared with the control group, transfection with the BART5-3p inhibitor increased the percentage of cells in the G0/G1 phase and weakened the ratios of cells in the G2/M and S phases. In contrast, when the P53 inhibitor was used alone, it had a contrary impact on cell cycle distribution. Notably, when the miR-BART5-3p inhibitor and P53 inhibitor were co-transfected, the inhibitory effect of BART5-3p on the cell cycle was diminished (Figure 5g-h). Additionally, Similar outcomes were observed when cell cycle-related proteins were analyzed using western blotting, and the expression levels of these proteins were in line with the modifications observed in the distribution of the cell cycle. (Figure 5i). Taken together, these results imply that BART5-3p stimulates cell growth and cycle change through the direct downregulation of P53 in Raji cells. The upregulation of P53 by the miR-BART5-3p inhibitor leads to the suppression of cell growth and alterations in cell cycle distribution, while knockdown of P53 reverses these effects. Figure 5| Inhibit the proliferation of BL after knockdown of BART5-3p in Raji cells. (a-b) The expression levels of BART5-3p andP53 after application of P53 inhibitor and BART5-3p inhibitor in Raji cells. (c) Relative protein expression levels of P53 after transfection with BART5-3p inhibitor. (d-f) The CCK8 cell proliferation assay and transwell migration assaywere used to detect Raji cell proliferation and migration,（g-h）Cell cycle distribution of Raji cell was determined using flow cytometry. (i) Relative cell cycle protein expression levels after using with BART5-3p inhibitor and P53 inhibitor in Raji cell, original blots are presented in Supplementary Figure 5c and 5i. Three independent experiments were performed, and data are shown as the means. *, P <0.05; **, P <0.01; ***, P <0.001.

The miR-BARTs encoded by EBV play a crucial role not only in the capacity of the EBV virus to cause cancer but also in its control over the cell cycle. The BART gene produces 44 full-length miRNAs that are significantly expressed in EBV-associated lymphoma from 22 precursor miRNAs (miR-BART1-22)[36]. In our study, we showed that EBV-positive Raji cells have elevated levels of BART5-3p. To further explore how EBV-encoded genes promote the development of Burkitt lymphoma, we altered BART5-3p levels in BL cells that were linked to EBV. Our findings indicate that BART5-3p considerably enhances proliferation in vitro. Importantly, previous studies have indicated that cells in the S phase are more numerous and those in the G0/G1 phase are fewer when BART5-3p is present[35]. These findings were consistent with our results. Additionally, we determined that TP53 was the target of BART5-3p. leading to upregulation of cyclinD1 proteins. This involvement in regulating the cell cycle of BL cells contributes to cell growth and tumorigenesis. The p53 gene is one of the most well-known tumor suppressors[37]. Zheng et al. reported that BART5-3p has been identified in EBV-associated nasopharyngeal carcinoma along with gastric cancer cells compared to normal cells, promoting tumor growth. Furthermore, p53 can be degraded by BART5-3p overexpression[35]. Similarly, other studies have highlighted that downregulating BART5-3p can increase P53 expression by targeting it, thus enhancing the radiosensitivity[38] of human EBV-positive nasopharyngeal carcinoma cells [38]. In line with these findings, our research team performed bioinformatics analysis and dual-luciferase reporter assays, confirming that BART5-3p may bind to P53's 3'-UTR regions and cause the protein to be reduced. These findings provide further evidence for the close association between the P53 gene and the oncogenic mechanism of EBV. To understand the fundamental processes of BART5-3p in BL proliferation, we employed an integrated strategy that involved process enrichment analysis, literature retrieval, computational biology prediction, and verification of gene regulation in cells. Using this approach, we identified 285 differentially expressed genes between EBV-negative and EBV-positive samples, along with several significant pathways. Interestingly, we found that the cell cycle is a major pathway involved in the oncogenic mechanisms of EBV infection in BL. This suggests that EBV-miR-BART5-3p regulates the cell cycle to promote BL proliferation. Further investigation into the specific genes and molecular events involved in this pathway could provide valuable insights into the mechanisms of EBV-associated BL. P53 plays a crucial role in the regulation of the cell cycle and interacts with other cell cycle regulators to maintain the normal progression of the cell cycle[39]. P53 and cyclin-CDK complex: P53 can inhibit the activity of the cyclin-CDK complex, thereby preventing cells from entering the S phase[40, 41]. Consistent with these findings, when we added P53 inhibitors to EBV-infected Raji cells, we noticed significantly elevated levels of cyclinD1 protein genes. In order to further verify the effect of BART5-3p on the cell cycle of Burkitt lymphoma through P53, we added BART5-3p inhibitor to Raji cells before and found that the proportion of cells entering the S phase was much lower than that of the control group, and when BART5-3p inhibitor and P53 inhibitor were used together, It was found that P53 inhibitors could partially reverse the cell cycle induced by BART5-3p into S phase. This suggests that BART5-3p may induce the proliferation of Burkitt lymphoma cells by down-regulating P53. In summary, our findings indicate that EBV promotes cell growth in Burkitt lymphoma (BL) cells through miR-BART5-3p-mediated targeting of P53. Our data also demonstrated that BART5-3p plays a significant role in the cancer cell cycle by regulating P53. These results offer new perspectives on the carcinogenic mechanism of EBV in Burkitt lymphoma.

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