Role of microRNA deregulation in the pathogenesis of diffuse large B-cell lymphoma (DLBCL)

Abstract

MicroRNAs (miRNAs) are small endogenous RNA molecules that regulate gene expression at the post-transcriptional level through its sequence complementation with target mRNAs. An individual miRNA species can simultaneously influence the expression of multiple genes and conversely, several miRNAs can synchronously control expression of specific gene product mRNA levels. Thus, miRNAs expression in cells has to be precisely regulated and alterations in miRNA levels may cause an aberrant expression of genes involved in oncogenic pathways and consequently result in cancer development. Indeed, miRNA expression is often deregulated in many cancers, including B-cell lymphomas. Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous group of B-cell lymphomas with different genetic backgrounds, morphologic features, and responses to therapy. Over the past decade, miRNAs emerged as a new tool for understanding DLBCL biology, and promising candidate molecular markers in DLBCL classification and treatment. In this review, we will focus on miRNAs aberrantly expressed in DLBCL and discuss the putative mechanisms of this deregulation. Additionally, we will summarize miRNAs’ involvement in the identification of DLBCL subgroups, and their potential role as diagnostic/prognostic biomarkers as well as specific therapeutic targets for DLBCL.

1. Introduction

1.1. MicroRNA - biogenesis and function

MicroRNAs (miRNAs) are endogenous short (~22-nucleotides) non-protein-coding regulatory RNA molecules. They are synthesized in the nucleus from long primary miRNA (pri-miRNA) transcripts and then cleaved by ribonucleases, Drosha and DGCR8/Pasha, to precursor (pre-miRNA) transcripts [1]. Next, Exportin 5 exports the pre-miRNA to the cytoplasm, where pre-miRNA is further cleaved by ribonuclease Dicer in complex with TRBP and PACT proteins to 22-nucleotide long miRNAs duplex [2,3]. Subsequently, one strand of the duplex is loaded onto Argonaute (Ago) proteins to generate the ribonucleoprotein inhibitory complex (miRISC) and serve as a mature functional miRNA. The other strand of the duplex was previously believed to be a carrier strand that degrades [4–6] however; increased numbers of recent reports provide evidence for its expression and functional activity in gene regulation [7–11]. The miRISC assembly associates with specific messenger RNAs (mRNAs) based on its sequence complementation (“seed region”) in mRNA’s 3′untranslated regions (3′UTR), coding sequences or 5′untranslated regions (5′UTR). The association of miRNA with mRNA regulates gene expression at the post-transcriptional level by either suppressing stability, translation of the mRNA or both of these processes [12,13].

1.2. MicroRNA implication in cancer

Since the discovery of the first miRNA, lin-4, in nematode worms in 1993 [14,15], miRNAs have been assessed to regulate nearly 60% of protein-coding mammalian genes [16,17], and this number will most certainly increase with the future research. Single miRNA can usually simultaneously control different target mRNAs and consequently influence production of multiple proteins [18–20]. Conversely, multiple miRNAs can often synchronously target the same mRNA and jointly control expression of the particular gene product [21–23]. Recent data from miRNA-based high-throughput functional screens revealed that miRNAs have the potential to regulate multiple functionally related genes involved in a specific biological pathway [24], reviewed in [25]. The cooperative miRNA interactions with the target mRNAs can influence a variety of critical biological programs such as cell division, apoptosis, differentiation, development, senescence, metabolism, control of hematopoiesis and tumorigenesis [26,27]. In fact, the regulation of whole biological pathways is the result of not only direct miRNAs-mRNAs interactions but also indirect effects through miRNAs-mediated alteration of components of transcription, translation or RNAi systems [20]. This extensive deregulation of miRNA expression may result in the activation of several oncogenic pathways and subsequently lead to cancer development. Indeed, gene expression profiling studies and bioinformatic analysis have discovered that cancer cells retain specific miRNA signatures different from those of normal cells [28–31]. Interestingly, the majority of human miRNAs genes are located at the genomic loci frequently implicated in cancers, called cancer-associated genomic regions (CAGRs) [32,33]. Many reports have described miRNAs that function exclusively as either oncogenes or tumor suppressors. However, a number of miRNAs have also been shown to play both oncogenic or tumor-suppressor function in distinct types of cancer [27,34], reviewed in [35,36]. Recognition of miRNA participation in cancer pathogenesis has prompted investigators in the development of preclinical studies targeting specific miRNAs. Consequently, numerous miRNAs have become candidates for diagnostic and prognostic biomarkers and targets for cancer therapeutic intervention [37–40], including hematologic malignancies [29,30,41–47]. Recent investigations have established miRNAs as predictive of chemo- and radiosensitivity in cancer as well as miRNAs’ efficacy in resensitizing resistant cancer cells [48–51].

1.3. Diffuse Large B-cell Lymphoma (DLBCL)

Diffuse Large B-cell Lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma (NHL) in the adult population, representing approximately 30–40% of all NHLs, and accounting for more than 80% cases of aggressive lymphomas in the world [52]. DLBCL is highly heterogeneous group of B-cell lymphomas with different genetic abnormalities, molecular grounds, clinical characteristics, therapy responses, and prognosis [53]. Based on gene expression profiling and immunohistochemical studies, DLBCL has been classified into three major molecular subtypes: germinal center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL and primary mediastinal large B-cell lymphoma (PMLBCL) [53–57]. GCB-DLBCL has frequent BCL2 translocations, REL amplifications, and somatic hypermutation of the immunoglobulin genes [55]. Both ABC-DLBCL and PMBCL are characterized by constitutively activated NF-κB signaling with the more aggressive ABC-DLBCL having a poorer clinical prognosis [54,55]. The Rituxin-CHOP (Cyclophosphamide, Doxorubicin, Vincristine, Prednisolone) immuno-chemotherapy regimen is the standard therapy used for the treatment of DLBCL. Unfortunately a large number of DLBCL patients ultimately develop refractory disease [58–60] and the precise mechanism underlying DLBCL drug resistance has not yet been determined. Although recent advances in combined therapy improved overall survival rates [58,61–64], the response to treatment remains variable and a heterogenic nature of DLBCL adds additional complexity in the treatment of the disease. As a consequence, a substantial proportion of DLBCL patients remain uncured and the identification of appropriate second-line therapies for them is challenging. Hence, better understanding of lymphoma biology, identification of additional biologic markers that can accurately predict survival and novel molecular targets is crucial for the improvement of the current diagnostic and treatment tools, and clinical outcome of patients with DLBCL (reviewed in [53]).

In the last few years miRNAs have provided a new platform for the understanding of DLBCL biology and emerging candidates for both predictive and prognostic biomarkers in DLBCL. miRNA signatures may provide information for specific tumor drug resistance or sensitivity and clinical outcome in chemo-immunotherapy-treated DLBCL patients [50,51]. The definitive proof for direct miRNA contribution to initiation and progression of B-cell lymphoma has been established by the generation of animal models in which modulation of miRNA levels directly impacts B-cell lymphoma development. In this review we will summarize the literature on miRNAs that have been implicated in B-cell lymphoma, with a special focus on DLBCL. We will discuss the deregulation of miRNAs in DLBCL, its implication in the identification of DLBCL subgroups and potential as diagnostic and prognostic biomarkers as well as specific therapeutic targets for DLBCL.

2. miRNA signature in DLBCL diagnosis and classification

In light of miRNAs’ potential as diagnostic markers for cancer prognostication there is increasing interest in the possible role for miRNAs as markers for both B-cell differentiation stage and malignant transformation. It has been shown that miRNA expression patterns can characterize stages of human B-cell differentiation [29,30,65,66], reviewed in [67]. To date, a large number of microRNA signatures characterizing lymphomas were identified, and the role of miRNAs in the development, classification and in the regulation of target genes is under intensive investigation [30,42,68]. Here, we discuss in detail four of the miRNAs deregulated in DLBCL (miR-17-92, miR-155, miR-21 and miR-34a) which are among the best studied owing to the availability of both gain and loss of function in mouse models.

2.1. miR-17-92 cluster

The first study to discover that miRNAs could affect lymphomagenesis appeared in 2004 when a novel gene, C13orf25, was identified to be upregulated in B-cell lymphoma cell lines and DLBCL patients with 13q31-q32 amplifications [69]; subsequent investigation discovered that C13orf25 contains the miR-17-92 polycistron encoding six, processed from the same precursor, miRNAs (miR-17-5p, miR-18, miR-19a, miR-19b, miR-20a and miR-92-1) [34,69]. The aberrant overexpression of the miR-17-92 polycistron in B-cell lymphomas suggested its oncogenic potential. Functional evidence for mir-17-92 tumorigenic activity came from a mouse B-cell lymphoma model, in which virus-mediated constitutive overexpression of mir-17-19b (a truncated mir-17-92 lacking mir-92) in coordination with the c-myc oncogene, accelerated B-cell lymphomagenesis and progression [34]. Remarkably, mice overexpressing miR-17-92 in wild type lymphocytes also developed lymphoproliferative disease, autoimmunity and died prematurely but did not developed lymphoma [70]. Lymphocytes from these mice exhibited reduced expression of miR-17-92 targets, the pro-apoptotic PTEN and Bim. These and other data suggest that Myc and the miR-17-92 cluster synergistically contribute to aggressive cancer development mainly by repressing tumor suppressor genes [71]. Ventura and colleagues by demonstrating that germline deletion of miR-17~92 cluster inhibits the pro-B to pre-B transition confirmed that miR-17~92 is a major participant in B-cell differentiation [72]. An additional report showed that deletion of the complete miR-17-92 cluster slows Myc-induced oncogenesis [73]. Furthermore, modulation of levels of individual member-miRNAs from miR-17-92 polycistron in a Eμ-myc B-cell lymphoma mice model revealed that miR-19 is a key component for the oncogenic activities of the whole miR-17-92 cluster [73,74]. Lenz and coworkers found that frequent amplification of the oncogenic mir-17-92 cluster was connected to deletion of the tumor suppressor PTEN in GCB type DLBCL but not in in ABC like DLBCL [75]. Moreover, miR-17-92 microRNA cluster members were found to be upregulated in GC-DLBCL in comparison with high-grade follicular lymphoma (FL) [76]. Gene expression analysis of lymphoma cell lines transfected with miR-17-92 suggests the miR-17-92 polycistron regulates distinct targets in different B-cell lymphoma subtypes [77]. Although further studies are needed to investigate the role of miR-17-92 in DLBCL, existing reports show that the miR-17-92 cluster plays a crucial role in B cell development and proliferation, and may be considered as a diagnostic tool to differentiate large B-cell lymphoid neoplasms.

2.2. miR-155

Encoded by BIC, miR-155 has been demonstrated to function in hematopoiesis and the immune response [78–80], acting as an oncogenic miRNA (oncomiR) in many malignancies, especially B-cell lymphoproliferative disorders. On the physiological level, miR-155 was shown to play a critical role in B-cell maturation and lymphocyte activation (reviewed in [80]). miR-155 has been found overexpressed in several lymphomas, including DLBCL [44,68,81–83]. Upregulation of miR-155 was shown to overexpress SH2-domain containing inositol-5-phosphatase-1 (SHIP-1), which subsequently activates the AKT pathway, inducing B-cell proliferation [84,85]. Further studies exploring the regulatory functions of miR-155 in DLBCLs demonstrated that miR-155 represses SHIP1 levels in TNF-α-dependent manner [86,87]. miR-155 was also found to regulate expression of activation-induced cytidine deaminase (AID), a major DNA mutator in GCB cells [88,89]. Recent studies demonstrated that BCL6 transcriptionally modulates miR-155 and consequently influences expression of AID and other genes relevant for the GC phenotype [90]. Furthermore, DLBCL cell lines and primary samples exhibit positive correlation between miR-155 upregulation and NF-κB activation [91,92]. Importantly, higher levels of mir-155 were discovered in ABC [30,42–44,81–83,91] and PMBCL [81] phenotypes when compare to GCB type of DLBCL. Differential miR-155 expression in subtypes of DLBCL can therefore serve as a valuable marker for DLBCL diagnosis and prognostication. In vivo research demonstrated that ectopic expression of miR-155 in a transgenic mouse model results in B-cell malignancy [84,93]. Recent results obtained from a mouse xenograft model for anti–miR-155–mediated inhibition of miR-155 in low-grade B-cell lymphomas emphasize the potential of therapeutic targeting of miR-155 in lymphoma malignancies [94]. In summary, despite a large body of evidence supporting the therapeutic potential of targeting miR-155, the exact mechanism by which it promotes transformation is not yet fully understood and appears to involve several different targets and pathways. Therefore, targeted miR-155 inhibition in the treatment of DLBCL and other B-cell malignancies is an area of active investigation.

2.3. miR-21

Another miRNA, which has been validated to play a major role in lymphomagenesis is miR-21. An oncogenic function for miR-21 has been supported by elevated miR-21 expression in most tumor types including B-cell malignancies [42,44,95,96]. Medina and colleagues established strong evidence for the role of miR-21 in cancer by employing an in vivo model of miRNA-21-induced pre-B-cell lymphoma; utilizing Cre and Tet-off technologies the authors generated a mouse model of conditional and inducible miR-21 expression. In this model, elevation of miR-21 levels caused pre-B malignant lymphoid-like phenotype, and miR-21 inactivation completely regressed tumors via apoptosis and cell arrest [96]. These data not only reinforced a strong oncogenic role of miR-21 but also demonstrated the first case of tumor addiction to an oncomiR. Microarray analysis identified elevated levels of miR-21 to be associated with ABC-type compared to GCB-type DLBCL cell lines [30,42–44]. A recent study demonstrated that miR-21 knockdown could significantly increase DLBCL cell sensitivity to the CHOP chemotherapy regimen, supporting the importance of this miRNA for DLBCL drug resistance [60]. Although further studies are necessary for validation, the previous work provides support for miR-21 as a potential DLBCL therapeutic target.

2.4. miR-34

In contrast to the miRNAs presented above, miR-34a has been shown to be repressed in diffuse large B-cell lymphoma (DLBCL) cells and has emerged as a potent tumor suppressor [97,98]. miR-34a is a member of the miR-34 family in which alteration has been described in many type of cancers (reviewed in [99]). A number of reports revealed that the gene encoding miR-34a is transcriptionally regulated by the tumor suppressor p53 [100–102]. Rao and colleagues found that constitutive overexpression of miR-34a causes a partial inhibition in B cell development, while it’s silencing increased the numbers of mature B cells [103]. These findings suggest that, similar to the miR-17-92 cluster, miR-34a is important for B cell proliferation and development. Therapeutic benefit of miR-34a modulation was demonstrated in vivo, in a xenograft mouse model of DLBCL, in which the systemic administration of miR-34a suppressed growth of DLBCL via its pro-apoptotic properties [98]. Further investigation of the mechanistic basis underlying miR-34a tumor-suppressor activity showed, that miR-34a suppresses growth of DLBCL, primarily through targeted repression of transcription factor FoxP1 [97,98,103], a hematopoietic oncoprotein and prognostic marker in DLBCL patients [104]. Details of the underlying mechanisms behind miR-34a pro-apoptotic effects are actively being investigated.

2.5. Other miRNAs aberrantly expressed in DLBCL

In addition to the miRNAs described above, which have been experimentally verified in pre-clinical animal models, a number of miRNA profiling assays performed in lymphoma patient samples have identified other candidate microRNAs putatively involved in DLBCL pathogenesis (Table 1). Because of the heterogeneous nature of DLBCL, in recent years studies have attempted to distinguish DLBCL from other lymphoma types while also finding specific miRNAs differentially expressed between DLBCL subtypes (Table 2). Comparison of miRNA profiles in reactive lymph nodes, DLBCL and follicular lymphoma (FL) revealed four miRNAs (miRs-210, -155, -106a and -17-5p) significantly highly expressed, and 11 repressed (miRs-150, -145, -328, -139, -99a, -10a, -95, -149, -320, -151, -let 7e) in DLBCL when compared to normal tissue [68]. The same analysis revealed, eight miRNAs (miRs-21, -127, -34a, -195, -let 7g) correlating with survival in DLBCL and four miRNAs (miRs-330, -17-5p, -106a, -210) separating DLBCL and FL cases. By using a microarray approach, Lawrie and coworkers obtained distinct miRNA expression patterns between DLBCL and FL. These specific microRNA signatures that were able correctly predict lymphoma type in 95% of cases [44]. Other microarray analysis in B-cell lymphoma cell lines identified discriminatory microRNA signatures between DLBCL vs. FL and lymphoma against normal CD19+ PB B-cells [105]. Recent studies by Di Lisio and colleagues have identified a signature of 128 specific miRNAs, which enable the characterization of different B-cell lymphoma types [106]. In this work, comparative microarray analysis data were validated by paraffin-embedded samples 19 miRNAs discriminating BL and DLBCL (miRs -155, -29b, -146a, -17-3p, -365, -30b, -595, -663, -573, -26b, -374, -520d, -92, -let7f, -516-3p, -9, -629, -9*, -34b). Lenze and coworkers, by microarray profiling of patient samples, identified 38 MYC regulated and NF-κB pathway-associated miRNAs, which were differentially expressed between DLBCL and Burkitt lymphoma (BL) [107]. Five of the miRNAs (miRs-155, -221, -222, -146a and -146b) found in this study to be upregulated in DLBCL compare to BL, have also been reported among other discovered unique miRNAs for the two main DLBCL classes. These five miRNAs distinguished the GCB–DLBCL type from ABC–DLBCL by being upregulated in ABC type [30,42,44,51,91]. Montes-Moreno and coworkers also found miR-144 and miR-451 to be elevated in ABC type and conversely miR-331, miR-151, miR-28, and miR-454-3p were upregulated in the GC-type DLBCL [51]. Culpin and colleagues, using microRNA profiling technology and a number of B-cell lymphoma cell lines, identified an additional nine miRNAs that separated ABC- and GC-type DLBCL [105]. Together, these reports confirm that differences in the expression of specific miRNAs can be considered as a potential indicator to define DLBCL subtypes.

Table 1.

MicroRNAs deregulated in DLBCL

miRNA name	Expression in DLBCL	Comparison cell type	References
miR-9	high	normal lymphocytes	[43,44]
		BL	[106]
miR-17-5p	high	non-neoplastic LN, FL	[68]
		normal B-cells	[34,42]
		normal lymphocytes	[43]
miR-18a	high	normal tonsillar lymphocyte	[30]
		normal lymphocytes	[43]
miR-18b	high	non-transforming FL	[44]
		normal B-cells	[42]
		normal lymphocytes	[43]
miR-19a	high	normal B-cells	[34]
miR-19b	high	non-transforming FL	[44]
		normal B-cells	[34,42]
miR-20	high	normal B-cells	[34,42]
miR-20a	high	non-transforming FL	[44]
		normal tonsillar lymphocytes	[30]
		normal lymphocytes	[43]
miR-21	high	normal lymphocytes	[44]
		serum from healthy patients	[126]
miR-93	high	non-transforming FL	[44]
		normal tonsillar lymphocyte	[30]
		normal B-cells	[42]
miR-100	high	normal lymphocytes, non- transforming FL	[44]
miR-106a	high	non-neoplastic LN	[68]
		non-transforming FL	[44]
		normal tonsillar lymphocytes	[30]
		normal B-cells	[34.42]
miR-106b	high	normal tonsillar lymphocyte	[30]
		normal B-cells	[42]
miR-143	high	normal lymphocytes	[44]
miR-146a	high	BL	[106,107]
		reactive lymphoid nodes	[83]
miR-155	high	non-neoplastic LN	[68]
		normal lymphocytes	[44,81]
		BL	[106,107]
		serum from healthy patients	[126,127]
		reactive lymphoid nodes	[83]
miR-182	high	BL	[106]
		normal lymphocytes	[43]
miR-210	high	non-neoplastic LN	[68]
		serum from healthy patients	[126]
miR-451	high	normal lymphocytes, non- transforming FL	[44]
		FL	[68]
		BL	[106]
miR-15a, miR-16-1	high	serum from healthy patients	[127]
miR-7, miR-33, miR-105a, miR-183	high	normal lymphocytes	[43]
miR-34b, miR-146b, miR-221, miR-365	high	BL	[106,107]
miR-15b, miR-16, miR-17, miR-18, miR-146	high	normal B-cells	[42]
miR-30a, miR-30d, miR-103, miR-107, miR-142-5p, miR-222, miR-342-3p	high	BL	[107]
miR-17, miR-25, miR-92a, miR-128a, miR-130b, miR-181b, miR-425	high	normal tonsillar lymphocyte	[30]
miR-30b, miR-98, miR-126*, miR-191, miR-196b, miR-199a*, miR-374, miR-582, miR-660	high	BL	[106]
miR-10b, miR-22, miR-24, miR-199a, miR-199b, miR-200c, miR-206, miR-362, miR-518a, miR-636, miR-638	high	non-transforming FL	[44]
let-7e	low	non-neoplastic LN	[68]
		normal B-cells	[34]
let-7i	low	normal CD19+ B-cells	[105]
miR-34a	low	-	[94]
		serum from healthy patients	[127]
miR-150	low	non-neoplastic LN, FL	[68]
		normal lymphocytes, non- transforming FL	[44]
		normal tonsillar lymphocyte	[30]
		normal lymphocytes	[43]
miR-320	low	non-neoplastic LN	[68]
		normal tonsillar lymphocyte	[30]
miR-328	low	non-neoplastic LN	[68]
		BL	[106,107]
miR-485-3p	low	BL	[106,107]
let-7d, let-7g	low	normal CD19+ B-cells	[105]
		normal B-cells	[34]
miR-24, miR-101	low	normal tonsillar lymphocyte	[30]
miR-487b, miR-92b	low	FL	[105]
let-7a, let-7b, let-7c	low	normal B-cells	[34]
miR-10a, miR-139, miR-149, miR-151	low	non-neoplastic LN	[68]
miR-154, miR-342, miR-405, miR-708-3p	low	normal lymphocytes	[43]
miR-213, miR-126, miR-135a, miR-330, miR-338	low	FL	[68]
miR-181a, miR-189, miR-217, miR-361, miR-363, miR-584, miR-625, miR-634, miR-768-5p	low	normal lymphocytes	[43,44]
miR-197, miR-453, miR-483, miR-516-3p, miR-520c, miR-520d, miR-520f, miR-560, miR-573, miR-574, miR-595, miR-609, miR-615, miR-629, miR-663	low	BL	[106]
miR-26a-1*, miR-26b*, miR-93*, miR- 105*, miR-124, miR-185, miR-192, miR-193a-5p, miR-202*, miR-326, miR-339-5p, miR-340, miR-371-5p, miR-429, miR-448, miR-483-3p, miR-497	low	BL	[107]
let-7f	high	BL	[106]
	low	normal B-cells	[34]
miR-9*	high	BL	[106]
	low	BL	[107]
miR-17-3p	high	normal B-cells	[34]
	low	BL	[106]
miR-20b	high	normal tonsillar lymphocytes	[30]
		normal lymphocytes	[43]
	low	FL	[105]
miR-26a	high	BL	[107]
	low	FL, normal CD19+ B-cells	[105]
		normal lymphocytes	[43]
miR-26b	high	BL	[106,107]
	low	non-transforming FL	[44]
miR-29b	high	BL	[106,107]
	low	normal tonsillar lymphocyte	[30]
miR-29c	high	serum from healthy patients	[127]
	low	normal tonsillar lymphocyte	[30]
miR-92	high	FL	[68]
		non-transforming FL	[44]
		normal B-cells	[34]
	low	BL	[106]
miR-95	high	normal lymphocytes	[43]
	low	non-neoplastic LN	[68]
miR-99a	high	non-transforming FL	[44]
	low	non-neoplastic LN	[68]
miR-125b	high	normal lymphocytes	[44]
		BL	[106]
	low	FL	[68]
miR-145	high	normal lymphocytes	[44]
	low	non-neoplastic LN	[68]
miR-223	high	BL	[106]
	low	normal lymphocytes	[43,44]
miR-301	high	normal lymphocytes	[44]
	low	FL	[68]
miR-324-5p	high	normal lymphocytes	[43]
	low	BL	[107]
miR-23a, miR-23b	high	BL	[107]
	low	normal tonsillar lymphocyte	[30]
miR-27a, miR-27b	high	non-transforming FL	[44]
	low	normal tonsillar lymphocyte	[30]

Abbreviations: DLBCL, diffuse large B-cell lymphoma; BL, Burkitt’s lymphoma; FL, follicular lymphoma; LN, lymph nodes

Table 2.

MicroRNA signatures that differentiate GCB from ABC subtype of DLBCL

Expression in GCB vs ABC	miRNA name	References
High	miR-421	[43,44]
	miR-181a, miR-324, miR-590	[43]
	miR-28, miR-151, miR-331, miR-454-3p	[51]
	miR-129, miR-138, miR-199b, miR-520h, miR-569, miR-616, miR-620, miR-653	[44]
Low	mir-21	[30,42–44]
	miR-155	[30,42–44,81–83,91]
	miR-221	[42–44]
	miR-222	[30,42–44,51]
	miR-363	[30,43,44]
	miR-146a	[83]
	miR-518a	[43,44]
	miR-194, miR-246	[42]
	miR-144, miR-221, miR-451	[51]
	miR-146a, miR-146b-5p, miR-574-5p, miR-574-3p	[30]
	miR-17, miR-19b, miR-20a, miR-29a, miR-92a, miR-106a, miR-720, miR-1260, miR-1260	[105]
	miR-30d, miR-132, miR-146b, miR-190, miR-213, miR-302, miR-340, miR-422b, miR-660	[44]

Abbreviations: GCB, germinal center B-cell; ABC, activated B-cell

3. Mechanisms involved in miRNAs deregulation in DLBCL

Since an individual miRNA species can influence the expression of multiple genes, miRNA levels in cells have to be precisely regulated. As we described above, an alteration in miRNA levels causes an aberrant expression of miRNA target genes and consequent disruption of the gene expression profile, which can result in cancer development. Studies have identified multiple mechanisms that contribute to the modulations of miRNA expression levels. These alterations can originate from several reasons; the most thoroughly studied are genomic mutation of miRNA loci, epigenetic changes and deregulation of transcription factors (reviewed in [108]).

Deletions at 13q14.3 are present in both chronic lymphocytic leukemia (CLL) and DLBCL cases [109]. Located at 13q14, miR-15a and miR-16a, are often deleted or repressed in B-cell chronic lymphocytic leukemia [110]. However, no reduced miR-15a and miR-16a expression was found in DLBCL cases bearing the del (13q14.3) [111,112]. Translocation at t(3;7)(q27;q32) in DLBCL was shown to fuse BCL6 to a noncoding region of FRA7H near miR-29 and down-regulate the levels of miR-29 [113]. Genome-wide miRNA profiling revealed a mutation in the seed sequence of miR-142 in approximately 20% of DLBCL patients [114]. Additional studies, suggest that loss of tumor-suppressive function of miR-142 may contribute to cell growth stimulation and induction of DLBCL [115].

Another mechanism that has recently emerged in the regulation of miRNAs expression is epigenetics. Specifically, DNA hypermethylation of microRNA gene promoters has been found to be responsible for miRNAs repression in cancers. In this topic, tumor suppressor miRNAs such as miR-203, miR-124-1, miR-34a and miR-129-2 are frequently silenced by hypermethylation in many cases of hematologic malignancies including NHLs and were suggested to corroborate in lymphomagenesis [116–119].

Like other genes, miRNAs expression can also be regulated by conventional transcription factors. The most extensively investigated is c-Myc, a transcription factor that is frequently disrupted in different types of lymphoma. O’Donnell and colleagues demonstrated that c-Myc specifically targets and upregulates the miR-17-92 cluster [120]. Extensive miRNA microarray analysis has identified large set of c-Myc-regulated miRNAs in aggressive B-cell malignancies including miR-34a and miR-15a/miR-16-1 [97,121,122].

Beside the widely investigated mechanisms described above, the other causes may add further complexity for understanding the loss or gain of miRNAs in cancers. In particular, miRNAs deregulation in B-cell lymphoma may be additionally complicated by Epstein-Barr virus (EBV) infections, which are found in around 15% of DLBCL cases. EBV encoded miRNAs were found to be active players in the lymphoma pathogenesis by employing the cellular miRNAs to modulate cellular miRNAs levels and subsequently the expression of target genes in infected DLBCL [114,123]. Overall, a growing body of evidence suggests that aberrant expression of a single miRNA is the result of a variety of deregulating mechanisms, which altogether account for changes in miRNA levels (reviewed in [124]).

4. miRNAs as biomarkers in prognosis and treatment of DLBCL

Discovery of extracellular miRNAs in body fluids has raised the possibility of utilizing levels and composition of circulating miRNAs as novel non-invasive biomarkers for identification of malignant and non-malignant diseases [125]. Lawrie and colleagues were the first to investigate circulating miRNAs in DLBCL [126]. They found increased levels of DLBCL-associated miRNAs-155, miRNA-210 and miRNA-21 in serum DLBCL when compared with healthy controls patients. They also found that high levels of miR-21 in the serum of DLBCL patients were associated with an increased relapse-free survival. Recently published by Fang and coworkers, examination of miRNAs in serum samples from patients with DLBCL and healthy controls found significantly elevated miR-15a, miR-16-1, miR-29c and miR-155, while lowered miR-34a in DLBCL when compared with normal controls, suggesting circulating miRNAs as potentially useful tools for diagnosis of DLBCL [127]. So far, circulating miRNAs is an intense area of investigation and a growing numbers of studies continue to discover the importance of circulating miRNAs in cancer and other diseases.

An increasing body of evidence links miRNAs levels with chemotherapy drug resistance in tumor cells. Recent data have shown that expression of miRNAs may indicate sensitivity or resistance to specific drugs in B-cell NHL lymphoma treatment and correlate with clinical outcome of uniformly treated DLBCL patients. For example, studies by Montes-Moreno and colleagues identified a set of miRNAs associated with prognosis in immuno-chemotherapy treated DLBCL patients [51]. These and several other investigations found a relationship between miR-222, miR-181a, miR-18a expression and clinical outcome of R-CHOP treated DLBCL [30,50,51]. Recent work demonstrated, that low expression of miR-129-5p in DLBCL patients correlates with a significantly worse prognosis then DLBCL patients with high miR-129-5p levels [128]. Although further clinical studies are necessary to corroborate the prognostic impact of individual miRNAs in DLBCL, it is becoming quite apparent that miRNA profiling will help in identification of a subgroup of candidate patients for individualized therapeutic regimens.

5. Concluding Remarks

In the last few years, multiple publications have addressed the deregulation of miRNAs in many types of cancer, including DLBCL. It has become evident that an aberrant expression of these small RNAs is one of the crucial steps in the pathogenesis of lymphomas. Recently, an increasing number of miRNAs have been incorporated into models for distinguishing different types of DLBCL while predicting therapeutic response or survival outcome. However, the precise mechanisms governing miRNAs expression and their impact on targeted genes/gene pathways are still being elucidated. Furthermore, much more work is needed in order to understand the mechanisms and role of these small RNAs in lymphomagenesis as well as to determine their potential as biomarkers and/or therapeutic targets. The recently described serum circulating miRNAs hold potential as a noninvasive biomarker for the diagnosis of DLBCL. The question of whether miRNA-based therapy in B-cell malignancies is ultimately clinically feasible is both a promising and challenging area of intense research.