Mechanism of Action of circRNA/miRNA Network in DLBCL

Abstract

Circular RNAs (circRNAs) make up approximately 10% of the human transcriptome. CircRNAs belong to the broad group of non-coding RNAs and characteristically are formed by backsplicing into a stable circular loop. Their main role is to regulate transcription through the inhibition of miRNAs' expression, termed miRNA sponging. CircRNAs promote tumorigenesis/lymphomagenesis by competitively binding to miRNAs at miRNA binding sites. In diffuse large B-cell lymphoma (DLBCL), several circRNAs have been identified and their expression is related to both progression and response to therapy. DLBCL is the most prevalent and aggressive subtype of B-cell lymphomas and accounts for about 25% to 30% of all non-Hodgkin lymphomas. DLBCL displays great heterogeneity concerning histopathology, biology, and genetics. Patients who have relapsed or have refractory disease after first-line therapy have a very poor prognosis, demonstrating an important unmet need for new treatment options. As more circRNAs are identified in the future, we will better understand their biological roles and potential use in treating cancer, including DLBCL. For example, circAmotl1 promotes nuclear translocation of MYC and upregulation of translational targets of MYC, thus enhancing lymphomagenesis. Another example is circAPC, which is significantly downregulated in DLBCL and correlates with disease aggressiveness and poor prognosis. CircAPC increases expression of the host gene adenomatous polyposis coli (APC), and in doing so inactivates the canonical Wnt/β-catenin signaling and restrains DLBCL growth. MiRNAs belong to the non-coding regulatory molecules that significantly contribute to lymphomagenesis through their target mRNAs. In DLBCL, among the highly expressed miRNAs, are miR-155-5p and miR-21-5p, which regulate NF-ĸB and PI3K/AKT signaling pathways. The aim of this review is to describe the function and mechanism of regulation of circRNAs on miRNAs' expression in DLBCL. This will help us to better understand the regulatory network of circRNA/miRNA/mRNA, and to propose novel therapeutic targets to treat DLBCL.

Keywords: B-cells; circRNA; gene expression; lymphoma; miRNA.

Figure 1

Scheme of classification of aggressive B-cell lymphomas in WHO-HAEM5 in the light of MYC, BCL2, and BCL6 rearrangement. R means rearrangement and G means germline configuration. The classification based on morphology should be confirmed by genetics to obtain correct diagnosis (modified and adapted from [17]).

Figure 2

Molecular classification systems of diffuse large B-cell lymphoma according to gene expression profiling and the potential relationship between the molecular entities and the cell of origin groups. From left to right, first classification is based on the cell of origin and distinguishes: ABC, unclassifiable and GCB subtypes of DLBCL. Second, based on the genetic analysis called “LymphGen”, there are seven distinguished subtypes of DLBCL: MCD, N1, A53, BN2, ST2, EZB MYC⁺, and EZB MYC^-. Third classification, also based on genetic analysis, distinguishes only four subtypes of DLBCL: MCD, BN2, N1, and EZB. The last, and current classification, C1–C5 represents clusters with different genetic aberrations and outcomes (adapted, modified from “Reproduced with permission from (Vodicka Prokop), (Onco Targets Ther); published by (Taylor & Francis), (2022).” [22]).

Figure 3

Management, scheme, and cure rates of patients with newly diagnosed DLBCL. Diagnosis of new DLBCL is based on histopathology, FISH method, and cell of origin. Staging of DLBCL is determined by laboratory analysis, FDG PET/CT scan, bone marrow biopsy, and prognostic factors. Front line treatment represents R-CHOP. Abbreviations: CNS = Central Nervous system; GCB = Germinal Center B-cell like; HBV = Hepatitis B virus; HCV = Hepatitis C virus; HIV = Human Immunodeficiency virus; IPI = International Prognostic Index; LDH = Lactate dehydrogenase; NCCN-IPI = National Comprehensive International Prognostic Index; R-IPI = Revised International Prognostic Index; R-CHOP = Rituximab (R) in combination with cyclophosphamide, doxorubicin, vincristine, and prednisone (adapted, modified from “Reproduced with permission from (Vodicka Prokop), (Onco Targets Ther); published by (Taylor & Francis), (2022).” [22]).

Figure 4

Therapeutic approaches in patients with newly diagnosed diffuse large B-cell lymphoma. There are two main therapeutic approaches: (1) Non molecular biology-based, includes Antibody-drug conjugates, Radioimmunotherapy, Monoclonal antibodies, CAR T-cell, Bispecific antibodies. (2) Tailored therapy—by targeting BCR/NF-kB pathway, BCL-2, XPO-1, EZH2, and Proteasome inhibitors. Abbreviation: CAR = Chimeric Antigen Receptor, BTK = Bruton tyrosine kinase (adapted, modified from “Reproduced with permission from (Vodicka Prokop), (Onco Targets Ther); published by (Taylor & Francis), (2022).” [22]).

Figure 5

Scheme of miRNA biogenesis and mechanism of action. The first step is the formation of the primary transcript—pri-miRNA from DNA. After cleavage by the microprocessor complex, Drosha enzyme and DiGeorge Syndrome Critical Region 8 (DGCR8), the precursor pre-miRNA is formed. Then, the pre-miRNA is transported from the nucleus into the cytoplasm by Exportin 5 (by RanGTP-dependent manner). The mature miRNA duplex is processed (cleaved by Dicer) and 5p or 3p strands of the mature miRNA duplex are further loaded into the Argonaute (AGO) family of proteins to form a miRNA-induced silencing complex (miRISC). Finally, based on the complementarity with target mRNA, miRISC could degrade mRNA or result in translational repression, in case of partial complementarity (modified, adapted from [32]).

Figure 6

Linear splicing and backsplicing, biogenesis of circRNAs—Schematic of linear splicing (top) and backsplicing (bottom). Linear splicing is common for mRNAs of coding genes. CircRNAs are generated via backsplicing; from exons of coding genes, concretely the upstream 3′ splice site of exon is joined to a downstream 5′ splice site that results in the junction of the 3′ end of an exon with the 5′ end of the same or upstream exon(s). Here, exon 2 gives rise to circRNA. Intron pairings and some RBPs (RNA-binding proteins) can enhance back splicing. By contrast, in A-I editing (adenosine-to-inosine), some helicases and other RBPs can inhibit backsplicing and hence inhibit circRNA production (modified, adapted from [56]).

Figure 7

Molecular general functions of circRNAs. (1) circRNAs can act as competing endogenous RNAs (ceRNAs) by sequestering and/or stabilizing miRNAs. This mechanism of acting is also called miRNA sponge—inhibition of miRNA function by occupying binding sites (by competition) of miRNAs by circRNAs. (2) circRNAs can function as scaffolds or decoys for RBPs; the examples of circFOXO3 and circANRIL are shown here. CircFOXO3 inhibits cell cycle progression by binding to or blocking CDK2. Similarly, circANRIL blocks protein PES1 that suppresses ribosome biogenesis. (3) circRNAs can also regulate the transcription of their parental (linear counterpart) gene, as occurs in the case of elciRNA, which controls the expression of its mRNA counterpart through the binding of small nuclear ribonucleoproteins (snRNPs) and the modulation of RNA polymerase II activity. (4) Some circRNAs can act as a template for cap-independent translation (modified, adapted from [52]).

Figure 8

Schematic diagram of circRNA functioning as miRNA/RBP sponge. CircRNA contains several conserved miRNA target sites that can bind miRNAs, facilitate a specific miR-AGO interaction, and thus suppress concrete miRNA activity in many diseases (leukemia, lymphoma, hepatocellular carcinoma, colorectal cancer, diabetes, and others). AGO = Argonaute protein; RBP = RNA-binding protein.

Figure 9

Summary scheme of circRNA and miRNA interaction in homeostasis vs. in malignant cells. In the case of homeostasis, cells express a low level of circRNAs with a high level of miRNAs that results in the translation repression of targeted mRNAs. In the case of malignancy, levels of circRNA increase and inhibit expression of miRNAs by sponging, which results in increased expression of oncogenes.