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Keywords:

  • multiple myeloma;
  • miRNA;
  • epigenetics;
  • expression

Abstract

  1. Top of page
  2. Abstract
  3. MicroRNAs
  4. MiRNA in the development of normal and malignant plasma cells
  5. miRNA expression in MM
  6. miR-21
  7. The miR-17-92 cluster
  8. miR-15a/miR-16-1 cluster
  9. The miR-29 family
  10. miR-34
  11. Circulating miRNAs in MM
  12. Future implications
  13. Acknowledgements
  14. References

Multiple myeloma (MM) is a devastating disease with a complex biology, and in spite of improved survivability by novel treatment strategies over the last decade, MM is still incurable by current therapy. MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate gene expression at a post-transcriptional level. More than half of all protein coding genes are estimated to be controlled by miRNAs, and their expression is frequently deregulated in many diseases, including cancer. Recent studies have reported aberrant miRNA expression patterns in MM, and the function of individual miRNAs in MM has been investigated in detail in cell culture and animal models. Here, we review the current knowledge on the role of miRNAs in MM pathogenesis and discuss their potential as prognostic biomarkers and targets for treatment.

Multiple myeloma (MM) is a malignant disorder characterized by neoplastic growth of plasma cells in the bone marrow [1]. It is suggested to arise by clonal expansion of plasma cells leading to a condition known as monoclonal gammopathy of undetermined significance (MGUS), which may evolve to smoldering myeloma and finally to symptomatic myeloma [2]. Features of symptomatic myeloma are, apart from the production of a clonal protein detected in the serum or urine in most of the cases, the presence of end-organ damage, appearing as anemia, hypercalcemia, renal impairment, and bone disease [1]. The interplay between malignant plasma cells and bone marrow stromal cells is important for the development of the disease, as the bone marrow microenvironment stimulates growth of plasma cells, promotes angiogenesis and bone disease, and thus is critical in the pathogenesis of MM [3]. The introduction of high-dose chemotherapy with autologous stem cell support, and more recently, novel biologically targeted agents for the treatment of MM, such as bortezomib or immunomodulatory drugs (lenalidomide and thalidomide), has improved the overall survival of patients, but the disease is still considered incurable with current approaches [4].

Although morphologically similar, MM is clinically heterogeneous, due to a complex pathobiology. The use of fluorescent in situ hybridization (FISH) has revealed specific cytogenetic abnormalities, some of which are associated with prognosis, and FISH is now used as a standard tool for a biological and prognostic classification of MM [5]. Hyperdiploidy is seen in almost 50% of cases and is characterized by trisomy of odd-numbered chromosomes (3, 5, 7, 9, 11, 15, 17, 19, and 21) as well as gains of 1q and del(13q), while cases with non-hyperdiploidy are characterized by translocations involving the IGH gene located at chromosome 14q32 region or chromosomal deletions [3, 6]. Recently, gene expression profiling (GEP) was used to further classify MM in seven distinct subgroups with unique clinical and prognostic features [7]. Similar studies have established prognostic models for MM based on gene expression [8, 9]. It has been suggested that novel deep sequencing technologies may further elucidate the pathogenesis of MM and may reveal targets for new therapeutic strategies [10]. Recent studies have shown epigenetic mechanisms involved in the pathobiology of MM [11], as well as deregulation of microRNAs (miRNAs), which not only gives information about disease development and prognosis, but can also provide potential therapeutic targets [12-14].

MicroRNAs

  1. Top of page
  2. Abstract
  3. MicroRNAs
  4. MiRNA in the development of normal and malignant plasma cells
  5. miRNA expression in MM
  6. miR-21
  7. The miR-17-92 cluster
  8. miR-15a/miR-16-1 cluster
  9. The miR-29 family
  10. miR-34
  11. Circulating miRNAs in MM
  12. Future implications
  13. Acknowledgements
  14. References

miRNAs were discovered in 1993 in Caenorhabditis elegans by Lee et al., who described the production of small RNA molecules (22nt and 61nt) from the transcript of the lin-4 gene, which had antisense complementarity to the 3-UTR (3′ untranslated region) of the lin-14 mRNA transcript [15]. Further studies have elucidated the biogenesis and function of these molecules. miRNAs are endogenous, non-coding small RNAs (18–22 nt in length) that regulate gene expression at a post-transcriptional level [16]. They are either located in non-coding, independent DNA loci acting as ‘miRNA genes’ (intergenic miRNAs) or in the intronic regions of protein-coding genes (intragenic or intronic miRNAs) [17]. Approximately 50% of miRNAs are located next to each other, forming clusters with a common promoter, and thus transcribed from a single polycistronic transcription unit [18]. The transcription of intergenic miRNAs from non-coding loci (canonical pathway) is mediated by RNA polymerase II, producing primary transcripts (pri-miRNA), which are further processed by the RNase III type enzyme Drosha, to form ~70nt precursor molecules (pre-miRNAs), with a characteristic hairpin structure (Fig. 1). Intronic miRNAs are transcribed by RNA polymerase II together with the mRNA molecule of their host gene, and pre-miRNAs are produced either by Drosha processing of the spliced introns (canonical intronic miRNAs), or directly from the splicing process, thus bypassing Drosha (non-canonical intronic miRNAs or miRtrons) [18]. The pre-miRNAs are then exported to the cytoplasm by the protein Exportin-5, where the enzyme Dicer cleaves the loop of the hairpin structure of pre-miRNAs, resulting in the formation of a ~20nt mature miRNA-3p/miRNA-5p duplex (previously called miRNA/miRNA*). One strand of the duplex is degraded and the remaining strand is then loaded on an Argonaut (AGO) protein, to form together with other proteins the miRNA-induced silencing complex (miRISC) [17].

image

Figure 1. Biogenesis of miRNAs. Intergenic miRNAs: Transcription of DNA to a primary miRNA molecule (pri-miRNA) is catalyzed by RNA polymerase II (Pol-II) (or for certain miRNAs Pol-III). The pri-miRNA is processed in the nucleus by the Drosha/Di George syndrome critical region gene 8 (DGCR8) complex, which has RNase III endonuclease activity, to a final ~70nt long pre-miRNA with a characteristic hairpin structure. The pre-miR is recognized by Exportin 5 and exported to the cytoplasm. Intronic miRNAs: These miRNAs are transcribed with their host genes. After the splicing process, some will follow the canonical pathway and undergo processing by Drosha/DGCR8, but others (miRtrons) will bypass this step and be directly processed to pre-miRNAs by a debranching enzyme. In the cytoplasm, pre-miRNAs are further processed by Dicer in complex with TAR RNA-binding protein (TRBP), and the loop of the hairpin structure is removed, yielding a ~20nt miRNA duplex. A helicase unfolds the miRNA duplex in the mature miRNA (guide strand) and the complementary passenger strand, the latter being destroyed by nucleases (not shown). The mature miRNA is then loaded onto the RNA-induced silencing complex (RISC), with the help of Dicer and Argonaut (Ago). The RISC protein complex deliver the miRNA to its target mRNA.

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miRNAs regulate gene expression at a post-transcriptional level through direct binding to the target mRNAs [16]. Interaction between miRNAs and their target mRNAs usually results in translational repression, but upregulation has also been noted by several groups [19, 20]. Base pairing takes place between the ‘seed’ region of the miRNA, which consists of nucleotides 2–8 from the 5′ end of miRNA, and the 3′-UTR region of the target mRNA, although binding to the 5′-UTR or exonic region has also been reported [19-21]. Perfect binding of the miRNA to its target mRNA results in degradation of the mRNA molecule, while imperfect pairing, which is far more common in animals, results in either inhibition of translation initiation or, if translation has already begun, ribosome removal and proteolysis of the synthesized peptides [21] (Fig. 2). As of August 2012, 1600 mature human miRNAs have been recognized in the recent version 19 of miRbase (http://mirbase.org). It is estimated that more than 50% of protein-encoding genes are under regulatory control of miRNAs [17]. Early studies have shown that miRNAs participate in the regulation of important cellular functions, such as cell proliferation, differentiation, and apoptosis in a cell type-dependent manner [16].

image

Figure 2. Post-translational repression by miRNAs. miRNAs loaded on RISC exhibit their action by binding with their seed region (nucleotides 2–8) to the 3′-UTR (3′ untranslated region) of the target mRNA. Perfect complementarity between the miRNA and its target mRNA leads to degradation of the mRNA transcript by endonucleases. Near-perfect base-pair complementarity results in translational repression by two main mechanisms: Inhibition of translation initiation or inhibition of ribosomal elongation with subsequent ribosomal release and degradation of the produced peptides by proteases.

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Deregulation of miRNA expression is associated with a variety of human diseases, including cancer [22]. miRNAs, which are upregulated in the cancer cells and contribute to carcinogenesis by inhibiting tumor suppressor genes, are considered oncogenic miRNAs (or oncomiRs), while downregulated miRNAs, that normally prevent cancer development by inhibiting the expression of proto-oncogenes, are known as tumor suppressor miRNAs [23]. Whether a miRNA acts as an onco- or a tumor suppressor miRNA is highly dependent on the cellular context [24].

MiRNA in the development of normal and malignant plasma cells

  1. Top of page
  2. Abstract
  3. MicroRNAs
  4. MiRNA in the development of normal and malignant plasma cells
  5. miRNA expression in MM
  6. miR-21
  7. The miR-17-92 cluster
  8. miR-15a/miR-16-1 cluster
  9. The miR-29 family
  10. miR-34
  11. Circulating miRNAs in MM
  12. Future implications
  13. Acknowledgements
  14. References

Hematopoiesis is a differentiation process that requires fine, cell-specific regulation of gene expression, and several studies have shown that miRNAs play an essential role in the production of mature hematopoietic cells from the bone marrow [25-27]. After their exit from the bone marrow, mature B-lymphocytes (naïve B-cells) undergo further maturation and differentiation in the lymph nodes upon antigenic stimulation, which results in the production of plasma cells. The differentiation process starts with the formation of a germinal center (GC) and subsequent proliferation of B cells, as well as somatic hypermutation (SHM), where mutations occur in the variable regions of the immunoglobulin genes that may result in a higher antigen affinity. During GC formation, activation of BCL6 is essential and results not only in proliferation, but also in protection against DNA damage-induced apoptosis caused by SHM, through repression of TP53 and other pro-apoptotic genes [28]. BCL6 is also responsible for downregulation of Blimp1 (called PRDM1 in humans), a protein that induces plasmacytic differentiation, and which is expressed upon the transition of GC B-cells to post-GC B-cells, driving the formation of plasma cells [29]. IL-21 is responsible for downregulation of BCL6 after SHM, as well as activation of STAT3 and IRF4, a combination that results in upregulation of Blimp1 [30].

Absence of the processing enzyme Dicer in an experimental mouse model resulted in compromised GC formation, suggesting a critical role of miRNAs in the process of B-cell maturation [31]. Indeed, differential expression of miRNAs is seen in naïve B-lymphocytes, GC B-cells, and post-GC cells (plasma cells and memory cells) [32, 33]. GC B-cells are shown to have high expression of miR-125b, which is found to downregulate IRF4 and Blimp1 [33, 34]. MiR-155-deficient mice have impaired immune response with reduced numbers of both GC B-cells and IgG1-producing plasma cells [35, 36]. MiR-155 is downregulated during BCL6 activation and GC formation, while its subsequent upregulation targets BCL6 to allow B cells to transit from the GC [37, 38]. Supporting these findings, patients with diffuse large B-cell lymphoma deriving from post-GC B-cells exhibit upregulation of miR-155, in contrast to those deriving from GC B-cells [39, 40].

Interestingly, aberrant miRNA expression has been found to be sufficient to drive malignant transformation. In two different mouse models, increased expression of the miR-17-92 cluster and miR-21 resulted in lymphomagenesis [41, 42]. Downregulation of miR-21 expression led to tumor regression and restoration of a normal phenotype, suggesting that single miRNAs can play a critical role in cancer development and thus have potential as therapeutic targets [42].

miRNA expression in MM

  1. Top of page
  2. Abstract
  3. MicroRNAs
  4. MiRNA in the development of normal and malignant plasma cells
  5. miRNA expression in MM
  6. miR-21
  7. The miR-17-92 cluster
  8. miR-15a/miR-16-1 cluster
  9. The miR-29 family
  10. miR-34
  11. Circulating miRNAs in MM
  12. Future implications
  13. Acknowledgements
  14. References

The association of miRNAs with cancer has led to extensive research in miRNA deregulation in different malignancies, including MM. There are two types of studies addressing this issue: Global miRNA expression studies and functional studies in in vitro models with human myeloma cell lines (HMCL) or in vivo mouse models. A summary of the studies performing global miRNA expression in HMCL or patient samples, and their results, is given in Table 1. These studies have highlighted the critical role of particular deregulated miRNAs in MM, which will be presented here. A summary of the up- and downregulated miRNAs in MM is given in Tables 2 and 3. The miRNAs discussed below are not included in the tables.

Table 1. Summary of studies on global miRNA expression in MM
StudySamplesMethodsmicroRNAsResultsComments
  1. GEP, gene expression profiling; FISH, fluorescent in situ hybridization; HMCL, human myeloma cell lines; HMCL, monoclonal gammopathy of undetermined significance; MM, Multiple myeloma.

Pichiorri et al. [12]

41 HMCL

10 MM, 5 MGUS, 4 controls

Microarrays + qRT-PCR345 human miRNAs

•HMCL + MM vs. controls:

[UPWARDS ARROW]60 miRNAs, [DOWNWARDS ARROW]36 miRNAs

•MM vs. controls: [UPWARDS ARROW]37 miRNAs, [DOWNWARDS ARROW]37 miRNAs

•MGUS vs. controls: [UPWARDS ARROW]41 miRNAs, [DOWNWARDS ARROW]7 miRNAs

Upregulation of miR-21, miR-106b-25 in both MGUS and MM, but miR-17-92 only in MM
Lionetti et al. [53]38 MM, 2 PCL, 3 controls (previously GEP defined)Microarrays723 human miRNAs[UPWARDS ARROW]74 miRNAs 26 miRNAs correlating with GEP subtypesAssociation with FISH subtypes
Unno et al. [45]7 HMCL, 2 MM, 2 controlsMicroarrays + qRT-PCR757 human miRNAs[UPWARDS ARROW]22 miRNAs, [DOWNWARDS ARROW] 6 miRNAsUpregulation of miR-193b-365
Gutiérrez et al. [58]60 MM, 10 controlsMicroarrays + qRT-PCR365 human miRNAs[DOWNWARDS ARROW]11 miRNAsAssociation with FISH subtypes
Zhou et al. [46]52 MM, 2 controlsMicroarrays + qRT-PCR464 human miRNAs[UPWARDS ARROW]39 miRNAs, [DOWNWARDS ARROW]1 miRNAAssociation of a signature with high-risk patients
Corthals et al. [44]44 MM, 4 controlsMicroarrays + qRT-PCR365 human miRNAsFour MM clusters with specific miRNA expression signaturesAssociation with survival
Chi et al. [13]33 MM, 5 MGUS, 4 HMCL, 9 controlsMicroarrays + qRT-PCR655 human miRNAs

MGUS vs. controls:

[UPWARDS ARROW]28 miRNAs, [DOWNWARDS ARROW]11 miRNAs

MM vs. controls: [UPWARDS ARROW]109 miRNAs, [DOWNWARDS ARROW]20 miRNAs

Association with FISH subtypes, isotype, survival
Table 2. Upregulated miRNAs in MM, reported in two or more studies
miRNAChromosomal locationReferences
  1. FISH, fluorescent in situ hybridization; MM, Multiple myeloma.

  2. a

    miR-1 was found upregulated only in the samples depicting t(14;16) by FISH in the study of Gutiérrez et al. [58]

miR-120q13.33/18q11.2([13], [53], [58])a
miR-79q21.32/15q26.1/19p13.3([13], [45])
miR-15b3q26.1([12], [45], [46])
miR-27a19p13.2([12], [13])
miR-27b9q22.32([12], [13])
miR-30a-5p6q13([12], [46])
miR-30d8q24.22([12], [46])
miR-329q31.3([12], [13])
miR-34b11q33.1([12], [13])
miR-99a19q13.33([13], [46], [53])
miR-10011q24.1([12], [13], [44])
miR-125b11q24.1/21q21([12], [13], [46], [53])
miR-130a11q12.1([12], [13], [44])
miR-130b22q11.21([12], [13])
miR-133a6p12.2([12], [13], [53])
miR-133b6p12.2([12], [13], [53])
miR-1383p21.32/16q13([12], [13], [53])
miR-142-5p17q22([12], [13])
miR-146a5q34([12], [13])
miR-15019q13.33([13], [46])
miR-181a1q32.1/9q33.3([12], [13], [46], [53])
miR-181b1q32.1/9q33.3([12], [46])
miR-1837q32.2([13], [45])
miR-188Xp11.23([13], [46])
miR-1913p21.31([12], [46])
miR-19517p13.1([12], [46])
miR-200a12p.13.31([13], [44])
miR-2066p12.2([13], [44])
miR-21217p13.3([13], [46])
miR-221-3pXp11.3([12], [13], [46])
miR-221-5pXp11.3([46], [52])
miR-222-3pXp11.3([13], [46], [53])
miR-3208p21.3([13], [44])
miR-3357q32.2([13], [44])
miR-34214q32.2([13], [44])
miR-365a16p13.12([13], [46], [53])
miR-520 g19q13.42([13], [44])
miR-54915q25.1([13], [44])
miR-5744p14([13], [46])
let-7c21q21.1([46], [52])
let-7e19q13.41([46], [52])
Table 3. Downregulated miRNAs in multiple myeloma (MM), reported in two or more studies
miRNAChromosomal locationReferences
  1. a

    Downregulated miRNAs in samples with del(13q) in the study of Gutiérrez et al. [58]

  2. b

    Downregulated miRNAs in samples with t(4;14) in the study of Gutiérrez et al. [58]

miR-140-5p16q22.1([12], [58])a
miR-1867p15.2([12], [58])a
miR-193a-3p17q11.2([12], [58])a,b
miR-196b7p15.1([12], [58])b
miR-223Xq12([12], [13], [45], [58])a
miR-373-5p19q13.42([12], [13])

miR-21

  1. Top of page
  2. Abstract
  3. MicroRNAs
  4. MiRNA in the development of normal and malignant plasma cells
  5. miRNA expression in MM
  6. miR-21
  7. The miR-17-92 cluster
  8. miR-15a/miR-16-1 cluster
  9. The miR-29 family
  10. miR-34
  11. Circulating miRNAs in MM
  12. Future implications
  13. Acknowledgements
  14. References

The first study to address the role of miRNAs in MM was performed in 2007 in two HMCL. In this study, miR-21 was found to be upregulated by the IL-6/STAT3 pathway, and its overexpression resulted in inhibition of apoptosis, even in the absence of IL-6 [43] (Fig. 3). Pichiorri et al. [12], performed global miRNA expression arrays in 41 HMCL and 15 patient samples (10 with MM and five with MGUS) and showed that miR-21 was upregulated in both MGUS and patients with MM. Several genome-wide miRNA profiling studies have since then confirmed miR-21 as an oncomiR in MM [13, 44-46]. Munker et al. [47] showed that upregulation of miR-21 in HMCL was related to resistance to melphalan. Wang et al. [48] found low expression of miR-21 in HMCL cultured alone, but high expression of miR-21 in HMCL cultured together with bone marrow stromal cells, suggesting that upregulation of miR-21 in myeloma cells is mediated by their interaction with the bone marrow microenvironment (Fig. 3). In the same study, bortezomib was found to reduce expression of miR-21, while downregulation of miR-21 expression with an antagomiR resulted in increased dexamethasone and doxorubicin sensitivity, suggesting a role of miR-21 expression in drug resistance [48]. Other studies have addressed the potential of miR-21 as a target for treatment, as downregulation of miR-21 has lead to apoptosis of myeloma cells in vitro as well as in vivo [49-51].

image

Figure 3. miRNA network and Multiple myeloma (MM) pathogenesis. Bone marrow stromal cells (BMSC) downregulate miR-15a/16-1 and upregulate miR-21 in plasma cells through the IL-6/STAT3 pathway, leading to downregulation of the pro-apoptotic protein PIAS3. miR-15a/16-1 downregulation stimulates angiogenesis through upregulation of VEGF and increases cell survival via activation of NF-κB. miR-106b-25 upregulation leads to p53 and, in turn, miR-34a downregulation, resulting in increased cell growth, as miR-34a targets CDK4, c-MET, and c-Myc. In a complex circuit, c-Myc stimulates miR-17-92 expression and thus BIM downregulation. These combined mechanisms lead to increased myeloma cell growth.

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The miR-17-92 cluster

  1. Top of page
  2. Abstract
  3. MicroRNAs
  4. MiRNA in the development of normal and malignant plasma cells
  5. miRNA expression in MM
  6. miR-21
  7. The miR-17-92 cluster
  8. miR-15a/miR-16-1 cluster
  9. The miR-29 family
  10. miR-34
  11. Circulating miRNAs in MM
  12. Future implications
  13. Acknowledgements
  14. References

The miR-17-92 cluster is an example of polycistronic miRNAs. In humans, the miR-17-92 cluster is located on chromosome 13 and encodes six miRNAs: miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and miR-92a-1. It has two paralogs, thought to have arisen by gene duplications: The miR-106b-25 cluster located on chromosome 7, coding for three miRNAs (miR-106b, miR-93, and miR-25), and the miR-106a-363 located on the X chromosome, coding for six miRNAs (miR-106a, miR-18b, miR-20b, miR-19b-2, miR-92a-2, and miR-363) [52].

Upregulation of the miR-106b-25 cluster was reported in both MGUS and patients with MM, but the miR-17-92 cluster was only upregulated in patients with MM [12]. In this study, potential targets for these miRNA clusters were also defined: miR-106b-25 was found to downregulate p300-CBP-associated factor (PCAF), a histone acetyltransferase involved in the transcriptional control of various genes, including TP53, while the miR-17-92 cluster downregulated the pro-apoptotic genes BIM and SOCS-1, a negative regulator of IL-6/STAT3 pathway, both yielding an anti-apoptotic effect [12] (Fig. 3). Another study also reported upregulation of the miR-106b-25 cluster in 16 HMCL, and found a correlation between the expression of this intronic miRNA cluster and the host gene, MCM7 [53].

The oncogene c-MYC, which is overexpressed in almost half the cases of MM [3], directly targets and upregulates the miR-17-92 cluster [46, 54]. Silencing of MYC in HMCL resulted in downregulation of miR-17-92, and upregulation of BIM, leading to inhibition of cell growth [12, 54]. This effect could be induced pharmaceutically using the histone deacetylase (HDAC) inhibitor, ITF2357. ITF2357 downregulated the expression of MIRHG1 (also called C13orf25), which hosts the miR-17-92 cluster; however, it was not investigated whether the downregulation of the MIRHG1 transcript was an effect of MYC downregulation, or a direct effect of the drug [55]. Bortezomib has also been found to downregulate miR-17-92 in vitro [56].

The oncogenic role of the miR-17-92 cluster in MM has been further supported by the demonstration of miR-17-92 upregulation in patient samples [13, 44-46]. Upregulation of miR-17-92 was associated with a shorter progression-free survival in two studies, but the median follow-up was relatively short (under 2 years) for both studies [54, 57].

Interestingly, even though the miR-17-92 cluster is located on chromosome 13q31.3, monoallelic deletion of chromosome 13 detected by FISH in MM samples was not associated with normal or reduced expression of the miR-17-92 cluster [46, 54]; however, in other studies, monoallelic del(13q) was associated with downregulation of members of the miR-17-92 cluster [13, 58].

miR-15a/miR-16-1 cluster

  1. Top of page
  2. Abstract
  3. MicroRNAs
  4. MiRNA in the development of normal and malignant plasma cells
  5. miRNA expression in MM
  6. miR-21
  7. The miR-17-92 cluster
  8. miR-15a/miR-16-1 cluster
  9. The miR-29 family
  10. miR-34
  11. Circulating miRNAs in MM
  12. Future implications
  13. Acknowledgements
  14. References

The miR-15a/miR-16-1 cluster on 13q show tumor suppressor properties as they target oncogenes such as BCL2 and CCDN1 [59]. Functional studies in HMCL have shown that miR-15a/16-1 target VEGF, so that their downregulation may contribute to angiogenesis during MM development, and it also correlates with advanced disease [60, 61]. IL-6 produced by bone marrow stromal cells downregulates the expression of miR-15/16-1 in HMCL, an effect that is inhibited by bortezomib and melphalan, which restores expression of these miRNAs and lead to apoptosis [62].

In addition to their role to angiogenesis, miR-15a/16-1, along with miR-192, miR-194, and miR-215, affect the homing and migration ability of MM cells in in vivo models [60, 63].

Approximately 50% of the patients with MM exhibit del(13q) by FISH [6]. The effect of this chromosomal abnormality on the expression of miR-15a/16-1 is unclear. Even though Roccaro et al. [60] reported total absence of expression in cases with del(13q), other studies find no effect of monoallelic del(13q) on the expression of these miRNAs, which is either normal or even increased [57, 58, 64].

The miR-29 family

  1. Top of page
  2. Abstract
  3. MicroRNAs
  4. MiRNA in the development of normal and malignant plasma cells
  5. miRNA expression in MM
  6. miR-21
  7. The miR-17-92 cluster
  8. miR-15a/miR-16-1 cluster
  9. The miR-29 family
  10. miR-34
  11. Circulating miRNAs in MM
  12. Future implications
  13. Acknowledgements
  14. References

The miR-29 family consists of three miRNAs, miR-29a, miR-29b, and miR-29c. MiR-29b is encoded from two different chromosomal regions (miR-29b-1 on chromosome 7q32 and miR-29b-2 on chromosome 1q32) [65]. Aberrant expression of the miR-29 family members is reported in different forms of cancer, where they mostly function as tumor suppressor miRNAs [65]. Global miRNA expression array studies of both HMCL and MM patient samples show diverging results. In three studies, no difference in expression of the members of the miR-29 family was observed between MM patient samples and/or HMCL and normal plasma cells [13, 44, 58]. In one study, upregulation of the miR-29 family was observed in both MGUS and MM samples compared with normal plasma cells [12], while in two different studies, miR-29s were downregulated only in MM patient samples [45, 46].

However, functional studies suggest that the miR-29 family, and especially miR-29b, plays a tumor suppressor role in MM. MiR-29b was shown to directly target the anti-apoptotic gene MCL-1, as overexpression of miR-29b resulted in MCL-1 downregulation and apoptosis through caspase 3 activation [66, 67]. In addition, CDK6 was shown to be targeted by miR-29b, demonstrating that miR29b may also inhibit proliferation [67]. Treatment with bortezomib restored miR-29b expression and apoptosis through downregulation of Sp1, while treatment with antago-miR-29b conferred resistance to bortezomib [67]. MiR-29b is also found to directly target DNA methyltransferase 3B (DNMT3B), and transfection of miR-29b mimics reduced global DNA methylation and caused cell cycle arrest and apoptosis in in vitro and in vivo models [68]. In line with this, a high frequency of promoter methylation of miRNA genes is observed in both HMCL and MM patient samples [63, 69, 70].

Apart from their role in cancer, the miR-29s are also found to regulate bone remodeling. All three miR-29 family members are found to be upregulated during the differentiation of osteoblasts [71]. A recent study revealed that the expression of miR-29b follows the opposite direction in the maturation process of osteoclasts and declines toward the late stages of osteoclast differentiation [72]. High expression of miR-29b in osteoclasts resulted in reduced bone resorption in vitro, even after stimulation by myeloma cells, suggesting a that miR-29b mimics may potentially be used in the treatment of myeloma-related bone disease [72].

miR-34

  1. Top of page
  2. Abstract
  3. MicroRNAs
  4. MiRNA in the development of normal and malignant plasma cells
  5. miRNA expression in MM
  6. miR-21
  7. The miR-17-92 cluster
  8. miR-15a/miR-16-1 cluster
  9. The miR-29 family
  10. miR-34
  11. Circulating miRNAs in MM
  12. Future implications
  13. Acknowledgements
  14. References

The miR-34 family consists of three miRNAs, miR-34a, miR-34b, and miR-34c, the latter two often referred to as miR-34b/c, as they are transcribed from a common promoter. Being directly upregulated by the tumor suppressor protein p53, members of the miR-34 family are suggested to play essential roles in cell cycle and apoptosis control by targeting c-MYC, CDK6, and c-MET [73, 74] (Fig. 3). A functional study of six HMCLs confirmed miR-34a as well as the miR-194-2-192 cluster as direct targets of p53, and transfection with these miRNAs resulted in cell growth arrest [12]. Wong et al. [69] performed methylation-specific PCR in eight HMCL and 123 MM samples (95 primary, 28 relapsed) and reported methylation of the promoter of the MIR-34b/c gene in the majority of the HMCL and relapsed MM samples, suggesting that DNA methylation of miRNA genes can be an acquired event in MM, contributing to relapse. Treatment of the cells with the demethylating agent 5-azacytidine leads to upregulation of miR-34b and apoptosis [69]. Di Martino et al. [14] showed the potential of miR-34a as a treatment in MM, as overexpression of miR-34a in MM cells, resulted in downregulation of BCL2 and NOTCH1 and induced apoptosis both in vitro and in a xenograft mouse model. Nevertheless, global miRNA expression studies fail to confirm miR-34 downregulation in MM, either reporting normal or even increased expression of the miR-34 family in MM [12, 13, 44-46, 58].

Circulating miRNAs in MM

  1. Top of page
  2. Abstract
  3. MicroRNAs
  4. MiRNA in the development of normal and malignant plasma cells
  5. miRNA expression in MM
  6. miR-21
  7. The miR-17-92 cluster
  8. miR-15a/miR-16-1 cluster
  9. The miR-29 family
  10. miR-34
  11. Circulating miRNAs in MM
  12. Future implications
  13. Acknowledgements
  14. References

Recently, it was shown that miRNAs can also circulate in a stable form in body fluids such as blood or urine. The source of circulating miRNAs is still unclear, but they are thought to derive by either passive leakage from destructed cells or by active secretion, suggesting a role as intercellular messengers [75]. Since their discovery, circulating miRNAs have been intensively investigated, and there is now created a comprehensive database of human extracellular circulating miRNAs [76].

Tumor-derived circulating miRNAs can be measured in plasma and can be used as biomarkers for diagnosing and monitoring cancer [77]. It seems although that the collection process is crucial for the quantity of miRNAs, as for example, heparinized plasma cannot be used for this purpose, as heparin may act as a polymerase inhibitor [78]. The first study of circulating miRNAs in MM revealed downregulated plasma miR-92a levels in patients with MM, but not in patients with smoldering MM or MGUS [79]. Moreover, the downregulation of miR-92a in plasma correlated with response to treatment, as patients in complete response or very good partial response, showed normal levels of plasma miR-92a, together with upregulation of miR-92a in T-lymphocytes [79]. Six upregulated circulating miRNAs were identified in the plasma of 28 myeloma patients in another study, and two of them were associated with relapse-free survival [80]. In another study, three serum miRNAs could be used as biomarkers for distinguishing MM or MGUS from healthy individuals [81]. Interestingly, the deregulated circulating miRNAs are different from miRNAs detected in the myeloma cells, especially in the case of miR-92a, which is downregulated in plasma, but is part of the upregulated miR-17-92 cluster in MM cells.

Future implications

  1. Top of page
  2. Abstract
  3. MicroRNAs
  4. MiRNA in the development of normal and malignant plasma cells
  5. miRNA expression in MM
  6. miR-21
  7. The miR-17-92 cluster
  8. miR-15a/miR-16-1 cluster
  9. The miR-29 family
  10. miR-34
  11. Circulating miRNAs in MM
  12. Future implications
  13. Acknowledgements
  14. References

The data on miRNA and the understanding of their role in cancer development are steadily growing; however, facts about miRNA deregulation in MM are limited, and the results are confounding. This is probably because the data originates from relatively small patient samples, differently treated patient populations, and different analytical platforms. Accordingly, there is a need of global miRNA expression studies in comprehensive, uniformly treated patient cohorts, to establish the role of deregulated miRNAs in MM. In addition, miRNAs may be used as biomarkers for early detection of MM, and in the case of MGUS, as early predictors for the development of MM. However, as miRNAs may be potential targets for therapy, it is essential to perform functional studies to rule out the role miRNA in MM pathogenesis and to identify ‘driver’ miRNAs with a direct involvement in MM development.

Establishment of miRNA regulatory networks may contribute to the development of a targeted treatment, as there are quite a few studies showing that deregulation of miRNAs can affect the response to a specific treatment or confer resistance [47, 48, 56, 82]. Moreover, several studies in HMCL have shown that MM-related drugs such as bortezomib, but also other drug categories unrelated to MM, exhibit their action through restoring the expression of deregulated miRNAs in the malignant plasma cells or bone marrow microenvironment [48, 50, 55, 62, 83, 84]. This could possibly mean that, in the future, miRNA signatures from plasma cells can be used as indicators for a specific, more targeted treatment, but also as biomarkers of drug resistance.

What is even more interesting is that miRNAs, as well as other small non-coding RNAs, may be used as novel targets for treatment, and this possibility is currently being explored in a panel of diseases [85]. Synthetic oligonucleotides can either mimic or antagonize the endogenous miRNAs, and their potential has been widely examined [86]. Regarding MM, administration of synthetic oligonucleotides in xenograft mice models has been reported, with either intratumoral or intravenous administration with promising results [14, 51, 61, 68]. Recently, it was shown that administration of a synthetic locked nucleic acid (LNA) oligonucleotide antagonizing miR-122 is effective for treating hepatitis C in a primate in vivo model [87], and this LNA-oligonucleotide is now the first miRNA-based treatment being tested in humans (http://ClinicalTrials.gov, NCT01646489). There are still no cancer-related miRNA therapies developed for humans, but the research in this field is rapidly growing. There are several challenges to the implementation of miRNA-based therapy. Firstly, the oligonucleotides must be effectively delivered into target tissue, and secondly, systemically administered oligonucleotides may be removed by endonucleases or by phagocytic cells. Several attempts are being carried out to overcome these essential issues, including conjunction of oligonucleotides with liposome nanoparticles or application of a viral system with transfection and reintroduction to target cells [86].

Circulating miRNAs, detected in patient serum or plasma, are easily accessible, non-invasive biomarkers that may be useful in diagnostics, as predictive and prognostic tools, in response evaluation, and as indicators for relapse. There are very few data on circulating miRNAs and their clinical use in MM, and the results are neither similar nor conclusive. However, it is likely that miRNAs may have great potential both in the diagnosis and in the treatment of MM, and more studies are needed to reveal the full potential of these small molecules.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MicroRNAs
  4. MiRNA in the development of normal and malignant plasma cells
  5. miRNA expression in MM
  6. miR-21
  7. The miR-17-92 cluster
  8. miR-15a/miR-16-1 cluster
  9. The miR-29 family
  10. miR-34
  11. Circulating miRNAs in MM
  12. Future implications
  13. Acknowledgements
  14. References

KG is supported by Rigshospitalets Research Foundation, The Danish Cancer Society, and The Novo Nordisk Foundation. We thank Lasse Sommer Kristensen for critical reading of the manuscript.

References

  1. Top of page
  2. Abstract
  3. MicroRNAs
  4. MiRNA in the development of normal and malignant plasma cells
  5. miRNA expression in MM
  6. miR-21
  7. The miR-17-92 cluster
  8. miR-15a/miR-16-1 cluster
  9. The miR-29 family
  10. miR-34
  11. Circulating miRNAs in MM
  12. Future implications
  13. Acknowledgements
  14. References