From virus to cancer: Epstein–Barr virus miRNA connection in Burkitt's lymphoma

Abstract

In Burkitt's lymphoma (BL), Epstein–Barr virus-encoded microRNAs (EBV miRNAs) are emerging as crucial regulatory agents that impact cellular and viral gene regulation. This review investigates the multifaceted functions of EBV miRNAs in the pathogenesis of Burkitt lymphoma. EBV miRNAs regulate several cellular processes that are essential for BL development, such as apoptosis, immune evasion, and cellular proliferation. These small, non-coding RNAs target both viral and host mRNAs, finely adjusting the cellular environment to favor oncogenesis. Prominent miRNAs, such as BART (BamHI-A rightward transcript) and BHRF1 (BamHI fragment H rightward open reading frame 1), are emphasized for their roles in tumor growth and immune regulation. For example, BART miRNAs prevent apoptosis by suppressing pro-apoptotic proteins, whereas BHRF1 miRNAs promote viral latency and immunological evasion. Understanding the intricate connections among EBV miRNAs and their targets illuminates BL pathogenesis and suggests novel treatment approaches. Targeting EBV miRNAs or their specific pathways offers a feasible option for developing innovative therapies that aim to disrupt the carcinogenic processes initiated by these viral components. future studies should focus on precisely mapping miRNA‒target networks and developing miRNA-based diagnostic and therapeutic tools. This comprehensive article highlights the importance of EBV miRNAs in Burkitt lymphoma, indicating their potential as biomarkers and targets for innovative treatment strategies.

Graphical Abstract

Burkitt lymphoma and Epstein–Barr virus miRNAs

Burkitt lymphoma (BL) is a highly aggressive form of non-Hodgkin lymphoma that is characterized by its rapid growth and association with EBV infection. EBV, a widely prevalent human herpesvirus, has been linked to the development of BL. among the repertoire of EBVs, microRNAs (miRNAs) have been identified as crucial regulators of gene expression and cellular processes involved in the pathogenesis of BL. EBV encodes approximately 44 mature miRNAs, which govern various cellular processes, including proliferation, apoptosis, and immune evasion. In the context of BL, EBV-encoded miRNAs exert their oncogenic effects by targeting tumor suppressor genes, modulating signaling pathways, and facilitating both viral persistence and tumorigenesis. Additionally, EBV-encoded miRNAs manipulate the host immune response to facilitate viral persistence and tumorigenesis [1]. EBV may contribute to the development of BL by inducing genomic instability [2] and conferring selective advantages to tumor cells, such as stimulating cell proliferation and inhibiting apoptosis [3]. However, the literature on the role of EBV-encoded miRNAs in BL has several gaps and limitations. First, there is a limited understanding of miRNA targets. Second, inconsistencies exist across studies regarding the expression and functional roles of EBV miRNAs in BL, partly owing to variations in experimental approaches and sample heterogeneity. Third, there is insufficient clinical correlation and limited exploration of therapeutic strategies. Addressing these gaps is critical and necessitates further research to elucidate the role of EBV-encoded miRNAs in BL, potentially leading to improved diagnostic, prognostic, and therapeutic approaches. Our study contributes novel insights into EBV miRNAs in BL by leveraging high-throughput sequencing to analyze the miRNA landscape comprehensively, unlike previous studies that focused on individual or small sets of miRNAs. This approach identifies new miRNA candidates as potential biomarkers or therapeutic targets, thereby filling gaps in current understanding. Our research lays the groundwork for miRNA-based therapeutic strategies.

EBV miRNAs

To date, a total of 25 progenitors of EBV miRNAs have been identified, producing of 44 mature EBV miRNAs. These EBV miRNAs can be classified on the basis of their chromosomal location. A) BamHI-A rightward transcript (BART) microRNAs: The BART region encompasses two miRNA clusters, namely, BART-Cluster 1 and BART-Cluster 2, along with a distinct BART2 region. These regions transcribe 22 miRNA precursors, resulting in 40 mature miRNAs. BART cluster 1 is located between exons 1 and 1B of the BART gene and contains eight miRNA precursors (EBV-miR-BART1, BART3 to EBV-miR-BART6, and EBV-miR-BART15 to BART17). On the other hand, BART-Cluster 2, located between exons 1B and 3, encodes 13 miRNA precursors (EBV-miR-BART7 to EBV-miR-BART14 and EBV-miR-BART18 to EBV-miR-BART22). The BART2 region, positioned between exons 4 and 5, encodes a solitary precursor (EBV-miR-BART2). These miRNAs are currently under investigation, but it is believed that they play a role in immune evasion, cell proliferation, and cancer [4] (Table 1). B) Bam HI fragment H rightward open reading frame 1 (BHRF1) miRNAs: Consisting of only three miRNAs, this cluster is located in close proximity to the BHRF1 gene within the EBV genome. BHRF1 miRNAs are known to regulate viral gene expression during the EBV replication cycle [5] (Table 2).

Table 1 BART miRNAs and their functions

Table 2 BHRF miRNAs and their functions

EBV miRNAs can be classified into four groups on the basis of their functions: 1) Pro-survival miRNAs, including miR-BHRF1-1 and miR-BHRF1-2-5p, increase cell survival and suppress apoptosis in BL cells by targeting pro-apoptotic proteins such as Bim and PUMA [6]. 2) Immunological evasion miRNAs, such as miR-BART1-5p and miR-BHRF1-2, modulate host immune responses to evade immune surveillance. These miRNAs operate by downregulating the production of cytokines (e.g., CXCL11), which are crucial for T-cell recruitment, thereby suppressing anti-tumor immunity and facilitating immune evasion in the BL microenvironment [7]. 3) Proliferation-promoting miRNAs, exemplified by miR-BART6 and miR-BART5, drive cell proliferation and tumor development. The mechanisms involve targeting tumor suppressor genes (e.g., PTEN and p53), activating pro-survival signaling pathways (e.g., PI3K/AKT), and promoting cell proliferation and genomic instability in BL cells [8]. 4) Inflammation-associated miRNAs, such as miR-BHRF1-3 and miR-BART-15, play crucial roles in regulating inflammatory signaling pathways and remodeling the microenvironment within BL cells. These miRNAs function by targeting negative regulators of the NF-κB signaling pathway, thereby promoting inflammation and facilitating tumor progression within the BL microenvironment [9].

The expression of miRNAs can exhibit substantial variation across different cell lines and tumor types, and they have the ability to target both viral and cellular genes. The presence of viral miRNAs in exosomes also indicates their involvement in intercellular communication and the modulation of the tumor microenvironment [10]. Elucidating the specific roles and targets of these miRNAs remains an active area of research, with significant implications for comprehending the contributions of EBVs to oncogenesis and the potential development of miRNA-based therapeutic strategies [11].

Despite the absence of a comprehensive compilation of all the EBV-encoded miRNAs investigated in the context of BL within a single source, considerable attention has been given to the BART and BHRF1 clusters owing to their association with EBV latency and related malignancies. Further investigations are necessary to identify and characterize the entire spectrum of EBV miRNAs involved in BL.

Noteworthy EBV-encoded miRNAs that have been examined in relation to BL include the following:

- BHRF1-1: This miRNA, derived from the EBV BHRF1 gene, is highly expressed in Wp-restricted BL cells, suggesting a connection to unique EBV latency processes in these cells. The BHRF1-1 miRNA has been implicated in antiapoptotic mechanisms that permit EBV-infected cells to evade apoptosis [12].

- BHRF1-2: Similar to BHRF1-1, the BHRF1-2 miRNA is highly expressed in Wp-restricted BL cells. Its target is PRDM1/Blimp1, a transcriptional repressor involved in B-cell development and the regulation of immune responses. By targeting PRDM1/Blimp1, the BHRF1-2 miRNA may assist EBV-infected B cells in evading immune surveillance, thus potentially contributing to dysregulated B-cell differentiation and lymphomagenesis [13].

- BHRF1-3: Expressed in primary lymphomas, including BLs, BHRF1-3 is associated with the viral latency status. It has been shown to target the IFN-inducible T-cell-attracting chemokine CXCL-11/I-TAC, indicating its potential role as an immunomodulatory mechanism in these tumors [14]. Additionally, the EBV miR-BHRF2-5p can directly target the IL-1 receptor (L1R1), leading to the inhibition of IL-Lβ-induced NF-kB activation and the suppression of host lymphocyte activation and immune responses [15].

- miR-BART1: miR-BART1-3p serves as a critical regulator of apoptosis and promotes migration and metastasis by inhibiting the cellular tumor suppressor PTEN [16].

- BART2: This particular miRNA has been detected in primary unmanipulated type I BLs and EBV( +) primary effusion lymphomas (PELs), suggesting its involvement in the pathogenesis of these lymphomas [17]. The miR-BART6-3p miRNA targets multiple cellular genes involved in crucial pathways, such as those involved in cell cycle regulation, cell proliferation, apoptosis, and signal transduction. It specifically downregulates tumor suppressor genes such as PTEN and the IL-6 receptor, which play critical roles in maintaining cellular homeostasis. The downregulation of PTEN leads to the activation of the Akt/PI3K signaling pathway, reducing apoptosis and promoting cell proliferation, thereby contributing to malignant transformation [18, 19].

Recent evidence suggests that the EBV miRNAs BART7 and BART9 may play significant roles in Akata EBV-positive cells. These miRNAs are associated with increased cell proliferation and viability, modulation of the lytic cycle, regulation of immunological checkpoint expression, and control of important cellular pathways, such as RNA-binding proteins (RBPs), the ubiquitin‒proteasome system, and fatty acid metabolism [20].

It has been reported that miR-BART11 elevates PD-L1 expression, facilitating immune evasion by promoting T-cell apoptosis and blocking IFN-γ production [21]. miR-BART11 is believed to serve a pathogenic function in EBV-positive BL, altering the tumor microenvironment and contributing to immune evasion [22].

Moreover, EBV-miR-BART17-3p targets and reduces the levels of DDX3X, an RNA helicase family member that plays an essential role in the RLR pathway [23]. It has recently been discovered that miR-BART17-3p directly associates with MAVS and IKK-ε/TBK1, thereby contributing to the induction of interferon regulatory factors. Furthermore, the downregulation of the RLR pathway by EBV-miR-BART17-3p increases EBV-related gene expression, leading to prolonged EBV infection in NK cells [24]. (Fig. 1).

Fig. 1

Functions of EBV-miRNAs in Burkitt lymphoma

Quick look at Cellular miRNAs in Burkitt lymphomas

The pathophysiology of BL, a malignant B-cell lymphoma, is significantly influenced by the deregulation of several miRNAs.

- hsa-miR127: hsa-miR-127 regulates BLIMP1 and XBP1 posttranscriptionally, thereby contributing to the mechanism of B-cell differentiation. Its overexpression impedes B-cell differentiation, making it a crucial factor in the lymphomagenesis of EBV-positive BL. In B cells infected with EBV, Epstein‒Barr nuclear antigen 1 (EBNA1) stimulates the synthesis of hsa-miR-127. The overexpression of hsa-miR-127 in EBV-positive BL is strongly correlated with malignancy [25, 26].​

- hsa-miR142: hsa-miR-142 and EBV-BART-6-3p simultaneously downregulate PTEN and IL-6R, potentially affecting the pathophysiology of EBV-positive BL [27]. Mutations in the core sequences of miR-142-3p and miR-142-5p have been found in patients with BL and diffuse large B-cell lymphoma (DLBCL). Compared with primary DLBCL, primary BL expresses considerably less miR-142-5p. This decreased expression may play a role in the development and progression of Burkitt lymphoma. Additionally, miR-142 modulates SOS1/Ras/Raf/Mek/Erk signaling via the BCR, which limits EBV entry into the lytic cycle [28].

- miR-155: The loss of miR-155 in BL was the first validated finding, and subsequent research has shown its essential role in B-cell growth. Notably, diffuse large B-cell lymphoma (DLBCL) produces miR-155, which is clinically beneficial for differentiation [29]. Furthermore, it has been demonstrated that miR-155 inhibits AID-mediated MYC-IGH translocation, linking the absence of miR-155 to the presence of MYC-IGH translocation, a defining characteristic of BL [30].

- miR-378a-3p has an oncogenic function in the proliferation of BL cells and is more prevalent and overexpressed in BLs than in germinal center B (GC-B) cells. Inhibition of miR-378a-3p through a lentiviral miRNA inhibition construct (mZip-378a-3p) markedly reduces the proliferation of multiple BL cell lines, highlighting the requirement of miR-378a-3p for BL cell growth [31].

- The miR-17‒92 cluster and miR-21 cluster, comprising miR-17, miR-18a, miR-19a, miR-20a, and miR-92a, are frequently upregulated in BL, particularly when Epstein‒Barr virus (EBV) is involved. These miRNAs contribute to the aggressive nature of BL by inhibiting apoptosis and promoting cell cycle progression [32].

- The let-7 family encompasses a collection of highly conserved miRNAs that play critical roles in the regulation of cell proliferation, differentiation, and apoptosis. Their mode of action involves binding to the 3' untranslated regions (UTRs) of target mRNAs, resulting in mRNA degradation or inhibition of translation [33]. In the context of Burkitt lymphoma, let-7 miRNA expression is commonly downregulated. This downregulation is associated with the disease's aggressive nature and poor prognosis. Decreased let-7 expression leads to the overexpression of target oncogenes such as MYC and Ras, thereby contributing to uncontrolled cellular proliferation in BL [34].

Similarly, miRs 98, 331, and 363 are thought to be involved in the intricate regulatory network of Burkitt lymphoma, particularly in relation to the oncogene C-MYC. These miRNAs are frequently downregulated, reinforcing the oncogenic activity of C-MYC. The downregulation of these genes facilitates the unrestrained expression of C-MYC, which drives the development and progression of BL [33, 35].

the role of EBV miRNAs in the pathogenesis of Burkitt lymphoma

They contribute through various mechanisms:

1. Modulation of apoptosis: EBV miRNAs can impede apoptosis by targeting proapoptotic factors, thereby promoting the survival of virus-infected B cells [11]. 2. Immune evasion: EBV miRNAs can downregulate the expression of immune-related molecules, aiding virus-infected cells in evading immune surveillance [19]. 3. Cell Proliferation and Differentiation: EBV miRNAs can influence cellular proliferation and differentiation by targeting specific genes involved in these processes, thereby contributing to the oncogenic transformation of B cells [36]. 4. Inflammation and Cytokine Signaling: Certain EBV miRNAs can modulate cytokine signaling pathways, such as the IL-6 pathway, which is implicated in inflammation and potentially contributes to the tumor microenvironment [19]. 5. Epigenetic Regulation: EBV miRNAs can impact the epigenetic landscape of host cells by targeting genes that are involved in DNA methylation and histone modification processes, resulting in changes in gene expression that promote tumorigenesis [37]. 6. Metabolic reprogramming: EBV miRNAs may also contribute to the metabolic reprogramming of infected cells, supporting the heightened metabolic demands of rapidly proliferating tumor cells [38]. These various mechanisms exemplify the multifaceted role of EBV miRNAs in the pathogenesis of Burkitt lymphoma, ultimately contributing to the initiation and progression of the disease.

The expression of EBV miRNAs is correlated with specific stages of Burkitt lymphoma

The expression of EBV-miRNAs in BL has been studied in relation to various aspects of the disease. However, there is no consistent evidence linking miRNA expression to specific clinical stages of BL. One study revealed significant differences in the expression of viral miRNAs and target genes between EBV-positive and EBV-negative BL, suggesting that miRNAs such as BART6 shape the transcriptional landscape of BL clones. However, this study did not directly correlate miRNA expression with clinical stage. Another study revealed a correlation between PD-L1 expression and a noncanonical EBV latency program in BL but, again, did not address miRNA expression and clinical stages [37] [39]. On the other hand, a study on endemic BL identified human and EBV miRNAs as potential predictive biomarkers for clinical presentation and disease progression. One human miRNA, hsa-miR-10a-5p, was found to be differentially expressed in jaw tumors compared with abdominal tumors and in non-survivors compared with survivors. However, no significant associations were found regarding initial patient outcome or anatomical presentation [40]. Research on miRNA-200a in childhood sporadic BL with EBV infection revealed downregulation of miRNA-200a in EBV-positive patients, suggesting a role in the pathogenesis of EBV-associated sporadic BL [41]. Finally, a study on EBV-BART-6-3p and cellular miRNA-142 in EBV-positive BL indicated that these miRNAs compromise immune defense, potentially contributing to the pathogenesis of BL, but did not report a direct correlation with clinical stages [27].

While there is evidence that EBV miRNAs are differentially expressed in BL and may impact disease progression and patient outcomes, the current literature does not provide a clear correlation between EBV miRNA expression and specific clinical stages of BL. Further research is needed to understand these relationships. Furthermore, the levels of EBV miRNAs in body fluids have been studied in relation to BL disease activity. These miRNAs, particularly those encoded by the virus, can be detected in serum, plasma, and other body fluids. The expression levels of EBV miRNAs in body fluids may reflect the viral load and extent of disease in BL and other EBV-associated cancers.

The levels of EBV miRNAs in body fluids and their correlation with BL disease activity

The levels of EBV-miRNAs in body fluids have been shown to be correlated with disease activity in various EBV-associated cancers, including BL. These miRNAs, particularly those encoded by the virus, can be detected in serum, plasma, and other body fluids, and their expression levels may reflect the viral load and extent of disease [42].

In BL, specific EBV miRNAs are associated with oncogenic processes, and their altered expression could indicate disease progression or response to treatment. For example, in lymphoma patients, variations in the expression levels of both EBV-encoded miRNAs and EBV-induced cellular miRNAs have been observed, which could be significant for disease progression. In BL specifically, miRNAs such as miR-BART6-3P and miR-BART17-5P are involved [43].

How do EBV miRNAs regulate the microenvironment of infected cells in BL?

EBV-miRNAs play a significant role in regulating the microenvironment of infected BL cells by targeting host mRNAs involved in cell proliferation, apoptosis, and transformation [44]. These miRNAs have the ability to inhibit the expression of viral antigens, enabling infected cells to evade immune recognition. Additionally, EBV miRNAs directly suppress host antiviral immunity by interfering with antigen presentation and the activation of immune cells [44].

EBV-encoded miRNAs, particularly those derived from the BART and BHRF1 clusters, have been found to modulate the surrounding microenvironment of infected cells through exosomal transportation. This influences immunosurveillance, cell proliferation, and apoptosis [45]. These miRNAs can also be released by exosomes, impacting the tumor microenvironment and the host immune response [46]. Furthermore, EBV miRNAs are implicated in the dysregulation of cellular miRNAs, which may contribute to the malignant transformation associated with BL [47]. Virus-encoded small RNAs, known as EBERs, are also involved in the pathogenesis of EBV infection by inducing resistance to apoptosis and modulating the expression of cytokines such as interleukin (IL)-10, which can affect the growth and survival of BL cells [48].

How do EBV miRNAs contribute to immune evasion in BL?

Epstein‒Barr virus (EBV) miRNAs contribute to immune evasion in BL by targeting both viral and host genes that are involved in the immune response [49]. Specific EBV miRNAs, such as miR-BART11 and miR-BART17-3p, are capable of upregulating the expression of PD-L1, a protein that helps tumors evade immune detection, by inhibiting the transcriptional repressors FOXP1 and PBRM1 [21]. These miRNAs are highly expressed in primary EBV-associated BLs and can effectively bind to and reduce the expression of target mRNAs involved in immune signaling pathways, thereby suppressing the host immune response [50]. Additionally, EBV miRNAs such as EBV-BART-6-3p, in conjunction with cellular miRNA-197, can downregulate the interleukin-6 receptor (IL-6R), which has a significant role in immune signaling. This compromises the immune defense of host cells in EBV-positive BL [19]. Viral miRNAs are nonimmunogenic and serve as a means of evading both innate and adaptive immune responses by targeting viral and host genes [51]. Furthermore, BL cells can evade immune detection by inhibiting antigen presentation to T cells, and EBV miRNAs are known to be involved in these pathways [52]. EBV miRNAs influence the immune response by affecting antigen presentation and recognition, altering T- and B-cell communication, and influencing cell apoptosis [7]. Furthermore, EBV miRNAs affect the polarization of macrophages toward an immunosuppressive phenotype (tumor-associated macrophages or TAMs), further aiding in immune evasion and enhancing the pro-tumoral environment [53].

How do EBV miRNAs contribute to the immune escape of tumor cells?

EBV miRNAs play a significant role in the immune evasion of tumor cells through multiple mechanisms. First, these miRNAs target both viral and host genes involved in the immune response, thereby modulating the immune system to favor viral persistence and tumor cell survival [27]. Additionally, the absence of EBV miRNA clusters has been found to enhance the CD4 + and CD8 + T-cell response against infected cells, indicating their critical role in suppressing the immune response [54]. Moreover, EBV miRNAs influence the immune response by altering antigen presentation and recognition, impacting T- and B-cell communication, and promoting antibody production during infection [55]. Furthermore, EBV-infected tumor cells can manipulate the tumor microenvironment to establish an immune-suppressive milieu by integrating EBV genes, expressing cytokines, and affecting the composition and distribution of immune cell subpopulations [56]. Another mechanism involves the transport of EBV-regulated miRNAs and viral proteins via exosomes, contributing to the construction and modification of the inflammatory TME [49]. Furthermore, EBV miRNAs inhibit the expression and presentation of viral antigens, suppress immune activation, and diminish the cytotoxic capability of immune cells, assisting host cells in evading immunity [15]. Tumor cells expressing specific miRNAs can disrupt apoptotic pathways, which are crucial for tumor immune escape [57]. Additionally, EBV miRNAs released from infected cancer cells in extracellular vesicles can regulate gene expression in neighboring uninfected cells, potentially contributing to immune evasion [58]. Collectively, these mechanisms enable EBV-associated tumor cells to evade the host immune system, promoting viral persistence and tumor progression. Understanding these processes is vital for the development of novel treatments for EBV-associated cancers.

Distinction from other EBV-associated lymphomas

Although certain EBV miRNAs are present across various EBV-associated malignancies, their roles are influenced by the specific cellular context. For instance, EBV miR-BART1 and miR-BART9 contribute to immune evasion in both BL and NPC. However, their specific targets and impacts on tumor microenvironment modulation differ between these diseases. In BL, these miRNAs not only facilitate immune evasion but also support the survival and proliferation of MYC-translocated cells, a characteristic unique to BL and not observed in other lymphomas [46].

Furthermore, the BART miRNAs are more prominently involved in the pathogenesis of epithelial malignancies such as NPC, where they drive processes like metastasis and EMT. In contrast, in BL, their primary function is associated with immune suppression and the stabilization of the oncogenic phenotype through interactions with cellular miRNAs and signaling pathways, such as the PI3K/AKT pathway [4].

What is the role of exosomal transportation of EBV miRNAs in BL?

Exosomal transportation of EBV miRNAs plays a crucial role in the pathogenesis of BL by facilitating communication between tumor cells and the tumor microenvironment [45]. Exosomes are small vesicles released by cells that can carry proteins, lipids, and nucleic acids, including miRNAs, to recipient cells. EBV miRNAs packaged into exosomes can modulate gene expression in neighboring or distant cells, thereby promoting tumor growth and immune evasion [46]. EBV miRNAs in exosomes can influence the tumor microenvironment by altering the behavior of immune cells, such as T cells and macrophages, which can lead to a suppressed immune response against tumors. These miRNAs can also induce angiogenesis, support tumor cell proliferation, and inhibit apoptosis, contributing to the progression of BL. Furthermore, exosomal EBV miRNAs can serve as biomarkers for the diagnosis and prognosis of BL because of their stability in body fluids and their association with disease states. The study of exosomal EBV miRNAs in BL provides insights into the mechanisms of viral oncogenesis and offers potential targets for therapeutic intervention [45].

Exosomal EBV-miRNAs play a significant role in the behavior of immune cells within the BL microenvironment, leading to immune evasion and tumor progression. These miRNAs can influence immune cell function in various ways:

• 1. Inhibition of immune activation: Exosomal transfer of EBV miRNAs downregulates immune-stimulatory molecules on immune cells, reducing their ability to activate and mount an effective response against tumor cells [50].

• 2. Alteration of Cytokine Production: Exosomal EBV miRNAs affect cytokine production by immune cells, such as T cells and macrophages, creating an immunosuppressive microenvironment that supports tumor growth and survival [59]

• 3. Manipulation of Antigen Presentation: Exosomal EBV miRNAs disrupt antigen presentation pathways, impairing the recognition of tumor antigens by T cells and allowing BL cells to evade immune surveillance [60].

• 4. Induction of T-cell Exhaustion: EBV miRNAs may contribute to T-cell exhaustion by modulating immune checkpoint molecules such as PD-1/PD-L1, leading to the loss of effector functions [61].

• 5. Regulation of immune cell trafficking: These miRNAs also impact the trafficking and infiltration of immune cells into the tumor microenvironment, potentially altering the composition and function of the immune infiltrate [62].

The influence of exosomal EBV miRNAs on immune cells is complex and multifaceted, indicating strategic viral adaptation to promote persistence and oncogenesis within the host [53]. Interventions targeting exosomal EBV-miRNAs have the potential to reverse immunosuppression in the BL microenvironment, although further research is needed in this area.

EBV miRNAs serve as biomarkers for BL diagnosis and prognosis

EBV-microRNAs have potential as biomarkers for diagnosing and predicting BL. This is due to their distinct expression patterns and stability in body fluids. The presence and quantity of EBV miRNAs in circulating exosomes, serum, or plasma can indicate the EBV infection status and burden of EBV-related malignancies, including BL. EBV miRNAs are particularly appealing biomarkers because they are resistant to degradation by RNase and remain stable under different pH and temperature conditions. This stability allows their detection in noninvasive samples such as blood. Furthermore, the expression profiles of EBV miRNAs can be linked to the stage of the disease, response to therapy, and patient outcomes, providing valuable prognostic information.

Studies have demonstrated that specific EBV miRNAs are upregulated in BL and can be used to differentiate between EBV-positive and EBV-negative patients. For example, EBV-miR-BART2-5p, EBV-miR-BART8-3p, EBV-miR-BART15, and EBV-miR-BART19-5p were significantly upregulated. Additionally, the expression levels of EBV-miR-BART8-3p, EBV-miR-BART19-5p, and EBV-miR-BART9-5p are positively correlated with clinical indicators such as the International Prognostic Index (IPI) score and Eastern Cooperative Oncology Group (ECOG) score [63]. It is also possible that changes in the levels of these miRNAs over time could be utilized to monitor disease progression or response to treatment. However, the clinical usefulness of EBV miRNAs as biomarkers for BL still requires validation through larger, prospective studies to establish their sensitivity, specificity, and predictive value [64]. For more information, please refer to Table 3.

Table 3 EBV-associated miRNAs and their roles in Burkitt lymphoma: Diagnostic, prognostic, and therapeutic implications

Potential therapeutic strategies targeting EBV miRNAs in BL

Potential therapeutic strategies targeting EBV miRNAs in BL include several approaches:

Re-expression of microRNAs: Studies have demonstrated that re-expression of microRNA-150 (miR-150) in EBV-positive BL cell lines leads to reduced proliferation and induction of B-cell terminal differentiation by modulating c-Myb, a protein involved in cell growth and differentiation [65].

Targeting miRNA‒mRNA interactions: The combination of EBV-BART-6-3p and cellular miR-197 synergistically decreases the expression of interleukin-6 receptor (IL-6R), which plays a role in regulating immune responses. Targeting these miRNAs could restore immune defense mechanisms in EBV-positive BL [19].

Gene therapy approaches: Encouraging results have been obtained through the use of lentiviral delivery of miRNAs in gene therapy, suggesting that similar strategies could be adapted for EBV-associated malignancies, including BL [66].

Inhibition of viral miRNAs: Inhibiting EBV-miR-BHRF1-2, which targets the tumor suppressor gene PRDM1/Blimp1, may prevent EBV lymphomagenesis, as PRDM1 induces apoptosis and cell cycle arrest in lymphoblastoid cell lines [13].

Remodeling the tumor cell transcriptome: EBV miRNAs have a significant effect on remodeling the transcriptome of tumor cells, particularly by suppressing the host immune response. Targeting these miRNAs could aid in reversing immune evasion mechanisms employed by EBV-associated tumors [50].

Targeting viral antiapoptotic products: EBV encodes antiapoptotic products, including miRNAs, that promote the survival of infected cells and confer resistance to chemotherapy. Therapeutic strategies could focus on targeting these viral products to increase the effectiveness of existing treatments [67].

These strategies underscore the potential of EBV miRNAs as therapeutic targets in BL, providing innovative treatment approaches that may complement or enhance current therapies.

Clinical trials investigating the use of miRNA-based therapies for EBV-associated BL

However, several studies have examined the role of EBV-encoded miRNAs in the pathogenesis of BL and their potential as targets for therapy. For example, one study reported the expression of EBV miRNAs in primary lymphomas and their association with viral latency status, suggesting that targeted suppression of specific miRNAs, such as BHRF1-3, could serve as an immunomodulatory mechanism in these tumors [17]. Moreover, research has demonstrated that the reintroduction of microRNA-150 can induce differentiation in EBV-positive BL cell lines by modulating c-Myb, potentially offering a new therapeutic approach [65]. Furthermore, a phase I clinical trial evaluated the safety of combining the antiviral agent valacyclovir with the current chemotherapy regimen for children with endemic BL in Malawi, although this trial did not specifically involve miRNA-based therapy [68]. Another clinical trial has explored the use of these miRNAs as biomarkers for predicting therapeutic responses, in which miRNAs are being profiled to predict the efficacy of chemotherapeutic agents in various cancers, including metastatic prostate cancer and triple-negative breast cancer [69].

Although these studies provide valuable insights into the role of miRNAs in EBV-associated BL and suggest potential therapeutic strategies, they do not indicate the existence of ongoing clinical trials for miRNA-based therapies specifically for this condition.

Challenges in developing miRNA-based therapies for EBV-associated BL

The development of miRNA-based therapies for EBV-associated BL presents several challenges:

Delivery and Stability: Efficient and targeted delivery of miRNA mimics or inhibitors to tumor cells is a challenging task, as these molecules may undergo rapid degradation in the bloodstream [70]. Off-target effects: miRNAs can have multiple targets, and modulating one miRNA could unintentionally affect other genes and pathways, resulting in unintended consequences [71]. Immune response: Introducing synthetic miRNAs or inhibitors may trigger an immune response, potentially leading to inflammation or other adverse effects [72]. Viral mutation and escape: EBV has the ability to mutate and evolve, which may result in resistance to miRNA-based therapies [73]. Complexity of miRNA Regulation: The regulatory networks of miRNAs are intricate, and a comprehensive understanding of these networks in the context of EBV-associated BL is crucial for preventing the disruption of critical cellular functions [13]. Integration with current treatments: The combination of miRNA-based therapies with existing treatments, such as chemotherapy and immunotherapy, requires careful consideration to avoid antagonistic effects and to enhance therapeutic efficacy [74].

Addressing these challenges is essential for the successful development and clinical application of miRNA-based therapies for EBV-associated BL. The gaps identified in this study directly impact clinical practices and therapeutic developments by highlighting the potential of EBV miRNAs as therapeutic targets in BL. These miRNAs play crucial roles in immune evasion, apoptosis regulation, and tumor survival, making them promising candidates for targeted therapy. Complementing current therapies with strategies that modulate miRNA expression could lead to improved treatment efficacy and patient outcomes. Furthermore, targeting specific miRNAs could help reverse immune escape mechanisms and increase the effectiveness of existing treatments.

Current standard treatments for EBV-associated BL

• 1. The current standard treatments for EBV-associated BL are similar to those for BL and are not associated with EBV. These treatments typically include the following:

• 2. 1. Chemotherapy: Intensive multiagent chemotherapy regimens constitute the mainstay of treatment for BL. These regimens often consist of cyclophosphamide, doxorubicin, vincristine, methotrexate, and cytarabine [68].

• 3. Rituximab: Adding rituximab, a monoclonal antibody that targets CD20 on B cells, to chemotherapy has improved outcomes in BL patients. The dose-adjusted etoposide, prednisolone, vincristine, cyclophosphamide, doxorubicin, and rituximab (DA-EPOCH-R or -RR) regimen is less toxic but still achieves high cure rates across diverse patient groups [75].

• 4. Central Nervous System (CNS) prophylaxis: Due to the high risk of CNS involvement, prophylactic treatment with intrathecal chemotherapy is commonly administered [76].

• 5. Supportive Care: Managing tumor lysis syndrome, infections, and other complications is crucial for patient outcomes [77].

• 6. Antiviral Therapy: Although not a standard treatment for BL, antiviral agents such as valacyclovir have been studied for their potential to improve outcomes in EBV-associated malignancies [78].

HIV infection does not necessarily worsen the prognosis for patients with BL receiving rituximab-based chemoimmunotherapy. A study showed that HIV-associated BL patients had similar outcomes to those without HIV, with 10-year progression-free survival (PFS) and overall survival (OS) rates of 100% and 88.2%, respectively, when treated with dose-adjusted EPOCH-R [79, 80]. The advent of highly active antiretroviral therapy (HAART) has made it possible to use intensive chemotherapeutic regimens in HIV-positive patients, which were previously considered too toxic for this immunocompromised population [81, 82]. However, the prognosis can be unfavorable in cases where HIV is associated with other infections, such as tuberculosis, and where there is rapid dissemination of BL with central nervous system involvement [83]. With appropriate management, including HAART and supportive care, the outcomes for HIV-associated BL can be similar to those of HIV-negative patients [84]. Overall, these treatments are aggressive and aim to achieve rapid control of fast-growing tumors. The approach may vary on the basis of factors such as the stage of the disease, patient age, and overall health.

Novel therapeutic agents in development for EBV-associated BL

Several novel therapeutic agents and strategies are currently being developed for EBV-associated BL:

EBV-Targeted Therapies: Various strategies are under development to specifically target EBV-infected cells for destruction. These include preventing viral oncogene expression, inducing loss of the EBV episome, and enhancing the host immune response to virally encoded antigens [85]. Lytic cycle targeting: Therapies have been developed to target the EBV lytic cycle. This involves the use of natural compounds with anti-EBV properties and deliberately induces EBV lytic replication in combination with nucleotide analogs [86]. Immunotherapeutic Approaches: Immunological therapies that utilize ex vivo expanded autologous and allogenic cells specific for EBV have shown promise. Efforts are being made to improve these therapies [87]. GPCR Targeting: The EBV-encoded G protein-coupled receptor (GPCR) BILF1 is currently being investigated as a potential target in EBV-associated diseases. This is due to its role in promoting immune evasion and tumorigenesis [88]. Anti-PD-1 antibody: Anti-PD-1 antibody therapy, which has demonstrated efficacy in other types of cancers, is considered a potentially promising novel therapy for EBV-positive B-cell lymphoma [89]. Prodrug Activation: Inducing lytic EBV infection in tumors can activate EBV-encoded kinases that convert prodrugs such as ganciclovir into their active cytotoxic forms. This offers a novel treatment approach [90]. These novel agents and strategies represent a diverse array of approaches to target EBV-associated BL, with the potential to improve treatment outcomes for patients.

Conclusion

The complex relationship between EBV-microRNAs and BL highlights the essential role of viral miRNAs in the pathogenesis and progression of this malignancy. EBV miRNAs influence various cellular pathways, including those regulating apoptosis, immune evasion, and cell proliferation, thereby fostering a microenvironment favorable for lymphoma development. By targeting key regulatory proteins and signaling pathways, these miRNAs support the survival and proliferation of malignant B cells, contributing to the aggressive nature of Burkitt lymphoma.

Recent advancements in high-throughput sequencing and bioinformatics have enhanced our understanding of the specific miRNA profiles associated with EBV-positive Burkitt lymphoma. This expanding body of evidence underscores the potential of EBV miRNAs as both biomarkers for early diagnosis and therapeutic targets. Strategies aimed at modulating miRNA expression or function hold promise for developing novel therapeutic approaches, which could complement existing treatments and improve patient outcomes.

Future research should focus on elucidating the comprehensive roles of individual EBV miRNAs and their interactions with host cellular miRNAs and proteins. Understanding these complex networks will be crucial for designing targeted therapies. Additionally, clinical studies are needed to validate the efficacy and safety of miRNA-based therapies in treating Burkitt lymphoma. In conclusion, EBV miRNAs represent a critical element in the pathobiology of Burkitt lymphoma, offering promising avenues for both diagnostic and therapeutic advancements.