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Keywords:

  • microRNAs;
  • multiple myeloma;
  • target genes;
  • regulatory networks;
  • cancer

Abstract

  1. Top of page
  2. Abstract
  3. MicroRNAs and Cancer
  4. MicroRNAs in Hematopoiesis: General Considerations
  5. MicroRNAs in MM
  6. Conclusions
  7. Search Strategy
  8. FUNDING SOURCES
  9. REFERENCES

MicroRNAs are short noncoding RNAS involved in gene expression regulation under physiological and pathological situations. They bind to mRNA of target genes and are potential regulators of gene expression at a post-transcription level through the RNA interference pathway. They are estimated to represent 1% to 2% of the known eukaryotic genome, and it has been demonstrated that they are involved in the pathogenesis of neurodegenerative diseases, cancer, metabolism disorders, and heart disease. MicroRNAs are known to act as tumor suppressors or oncogenes in cancer biology. The authors describe the current knowledge on microRNA involvement in regulatory pathways that characterize multiple myeloma pathogenesis gained from in vitro and in vivo studies. These small molecules interact with important factors such as p53, SOCS1, IGF-1, IGF-1R, vascular endothelial growth factor, NF-κB, and others. As such, microRNAs represent an attractive therapeutic target in the context of multiple myeloma interfering with the myeloma regulatory networks. Further studies are needed to better understand their role in myelomagenesis and their therapeutic potential. Cancer 2012;. © 2011 American Cancer Society.

MicroRNAs (miRNAs) are short noncoding RNAs of ∼22 nucleotides that play important roles in gene regulation during physiological or disease-associated processes. They are capable of binding to the mRNA of target genes, resulting in translation suppression, and they are potential regulators of gene expression at a post-transcription level through the RNA interference pathway.1 MiRNAs are transcribed by RNA polymerase II or III in primary miRNAs characterized by a stem loop structure; they are then processed in the nucleus by a complex of the RNase enzyme Drosha and a double-stranded RNA-binding domain protein named DGCR8/Pasha, resulting in pre-miRNAs. The pre-miRNAs in turn are exported into the cytoplasm by the protein Exportin-5, and under the action of the enzyme Dicer and in collaboration with the TRBP/PACT proteins become double-stranded miRNAs.2 Next in the maturation process, the 2 miRNA strands are separated, and 1 of the strands connects with the Argonaut 2 (AGO2) protein within the RNA-induced silencing complex, where it acts as a guide to repress target mRNAs.2 miRNA genes are distributed in all human chromosomes except for the Y chromosome. Half of them are found in clusters, and they are transcribed as polycistronic primary transcripts. However, miR-18a represents a particular miRNA located in a cluster that is regulated independently from the other miRNAs in the same cluster, indicating that individual miRNAs can be post-transcriptionally regulated by factors that confer specificity and function at various points during the biogenesis pathway.2, 3

Some miRNAs are generated from noncoding transcription units, whereas others are encoded in protein-coding transcription units.4 Almost 40% of miRNA loci are located in the intronic region of noncoding transcripts, whereas 10% are located in the exonic regions of noncoding transcripts. miRNAs are also found in intronic regions of protein-coding transcription units, which account for the 40% of all miRNA loci, including imprinted genes.3-7 One of the largest miRNA clusters, including miR-127, miR-134, miR-342, and many others, is located in the DLK1/MEG3 imprinted region, which contains about 40% to 50% of the currently known miRNAs.7 They are expressed exclusively by the maternally inherited allele MEG3, which encodes a noncoding transcript, with a tissue-specific expression pattern. It has been suggested that they might be able to simultaneously reduce the expression of numerous genes in distinct cell types targeting more than 100 genes contained in the human genome.8, 9 Interestingly, both MEG3-imprinted and SNRPN-imprinted genes presented an aberrant CpG methylation profile in various hematological malignancies and were associated with disease stage and overall survival.10, 11

miRNAs play an important role in determination and/or maintenance of lineage during development, and most of them are not expressed in embryonic stem cells but are induced during development in a tissue-specific pattern.1 They are associated with phenotypic variation in closely related species during animal evolution, and alterations in the regulation of miRNA target genes can be expected to contribute in phenotypic variation such as disease susceptibility in humans.12 miRNAs act on a broad spectrum of genes including numerous transcription factors already present in the metazoan ancestor, and it is very probable that miRNAs contributed to the emergence of animal phenotypy.13

miRNA expression falls under multiple regulatory mechanisms, including post-transcriptional events such as inefficient processing of miRNAs through Drosha or Dicer. The genomic location of miRNAs also represents an important regulatory factor, as many deregulated miRNAs are located in cancer-associated genomic regions.14 However, it seems that the most important regulatory mechanism in miRNA biogenesis is the transcriptional control of miRNA genes, which is driven by promoter hypermethylation or global hypomethylation.14 In addition to the control of miRNA expression by epigenetic mechanisms, miRNAs themselves can regulate the expression of the components of the epigenetic machinery, especially in cancer, as miRNAs such as miR-29 directly regulate the levels of DNMT3a and DNMT3b, with consequent silencing of tumor suppressor genes.14

miRNAs have important functions that include regulation of development, differentiation, apoptosis, stress response, and gene expression regulation.15 To date, almost 9539 mature miRNAs have been described in various species varying from viruses to animals, and are estimated to represent 1% to 2% of the known eukaryotic genome16; it has been demonstrated that they are involved in the pathogenesis of neurodegenerative diseases, cancer, metabolism disorders, and heart disease.5 Another significant functional characteristic is that a single miRNA is capable of regulation of >200 mRNAs, and a single mRNA may be regulated by multiple miRNAs.16 Prediction of miRNA targets with the use of computational programs including PicTar, miRanda, and TargetScan is based on sequence alignment. However, these methods have limitations in identifying targets, especially in animals. Other methods used to identify miRNA targets are described elsewhere.16

MicroRNAs and Cancer

  1. Top of page
  2. Abstract
  3. MicroRNAs and Cancer
  4. MicroRNAs in Hematopoiesis: General Considerations
  5. MicroRNAs in MM
  6. Conclusions
  7. Search Strategy
  8. FUNDING SOURCES
  9. REFERENCES

miRNA alterations have been observed in various types of cancer, including chronic lymphocytic leukemia (CLL), acute promyelocytic leukemia (APL), acute myeloid leukemia (AML), multiple myeloma (MM), monoclonal gammopathy of undetermined significance (MGUS), non-Hodgkin lymphoma (NHL), breast cancer, esophageal cancer, gastric cancer, clear-cell kidney cancer, cervical cancer, and others.17 miRNAs have multiple roles in tumor formation, as they are capable of modulating oncogenic, tumor suppressor, and metastatic pathways including c-MYC, p53, RAS, BCR/ABL, and TWIST1-miR10b-HOXD10 pathway, whereas the expression of miRNAs themselves can be regulated by oncogenes or tumor suppressors.17, 18 The aberrant miRNA levels in neoplastic disease are caused by genetic and epigenetic (separately or in combinations), transcriptional, and post-transcriptional modifications resulting in the altered cellular homeostasis and in a less differentiated cellular state, which is a hallmark of cancer.19, 20

Selected groups of distinct miRNAs were commonly and concurrently up-regulated or down-regulated in distinct types of human cancers and were often associated with distinct cytogenetic abnormalities; their deregulation seems not to be a random event in cancer.21 Specific miRNAs, acting upstream or downstream of p53, or by repressing members of the BCL family, can result in successful apoptosis in normal cells. In constrast, miRNA epigenetic silencing could result in altered apoptosis rates, a finding characteristic of cancer, and miRNAs can indirectly promote the synthesis of vascular endothelial growth factor (VEGF) by targeting HIF-1.22 miRNAs can act as oncogenes, affecting all hallmarks of cancer including neoplastic cell independence from external growth factors signals, uncontrolled proliferation, and survival, and can result in insensitivity of tumor cells to antigrowth stimuli. Single nucleotide polymorphisms, and somatic and germline mutations in miRNAs have been observed in patients and experimental models of breast cancer and thyroid cancer, and in CLL patients conferring oncogenic properties of several miRNAs.22, 23

The potential use of miRNAs as prognostic markers in clinical practice has already been demonstrated, as expression levels of miRNAs could predict the time to first treatment in CLL patients, were associated with mutations of established molecular prognostic factors,24 and were also associated with overall survival in patients with hepatocellular carcinoma, pancreatic cancer, colon adenocarcinoma, lung cancer, esophageal cancer, and melanoma.25 Moreover, miRNAs have been evaluated in the context of chemosensitivity to assess the individual chemoresponse in both in vitro and in vivo models.26

The measurement of miRNAs levels in plasma or serum has rendered them useful in the diagnosis of solid malignancies such colorectal cancer, lung, prostate, and kidney cancer.21, 25 However, it has not been elucidated whether miRNA circulating levels are tumor produced or represent a systemic response, and it is not clear yet which is the best specimen among serum, plasma, and peripheral blood mononuclear cells to be used for miRNA signature detection.27

The important functions of miRNAs in cancer make them an attractive therapeutic target. Therapeutic modulation of miRNAs could be used to achieve clinical benefit against cancer, as has been demonstrated by Kota et al in their elegant work.28

MicroRNAs in Hematopoiesis: General Considerations

  1. Top of page
  2. Abstract
  3. MicroRNAs and Cancer
  4. MicroRNAs in Hematopoiesis: General Considerations
  5. MicroRNAs in MM
  6. Conclusions
  7. Search Strategy
  8. FUNDING SOURCES
  9. REFERENCES

In 2004 Chen and colleagues29 first established the connection between miRNAs and hematopoiesis regulation, determining that individual hematopoietic cell types differentially expressed miRNAs, given that the same miRNAs were not always expressed in all lineages. Their findings suggested that the specific miRNAs are induced during lineage differentiation and could influence hematopoietic lineage differentiation in mice, although differences in the expression pattern of the same miRNAs in humans have been reported.29, 30

Moreover, 31 mature miRNAs were found to regulate hematopoietic differentiation-associated mRNA on CD34+ cells, and especially miR-155 represented an inhibitor of hematopoietic stem progenitor differentiation.31 It has also been reported that members of the miR-30 family by targeting the transcription factor PRDM1 regulate the differentiation of lymphocytes to plasma cells.32 More than the deregulation of miRNAs, hematopoietic deletion of AGO2 or of Dicer resulted in disruption of erythropoiesis, with severe anemia, splenomegaly, and maturation arrest of erythroid precursors.33 Aberrant miRNA expression profiles have been reported in almost all types of hematological malignancies, including various types of NHL, Hodgkin lymphoma, CLL, AML, APL, acute lymphoblastic leukemia, and MM. The miRNAs currently known to be involved in normal and malignant hematopoiesis are described extensively in excellent recent reviews.30, 33-41

MicroRNAs in MM

  1. Top of page
  2. Abstract
  3. MicroRNAs and Cancer
  4. MicroRNAs in Hematopoiesis: General Considerations
  5. MicroRNAs in MM
  6. Conclusions
  7. Search Strategy
  8. FUNDING SOURCES
  9. REFERENCES

MM is a chronic, still incurable, disease characterized by a clonal proliferation of malignant plasma cells. Numeric chromosomal abnormalities, translocations, gene mutations, epigenetic alterations, and direct interactions between malignant plasma cells and stromal cells in the bone marrow microenvironment are all involved in the pathogenesis of MM.42 The pathogenesis of MM is a multistep process, with the primary early chromosomal translocations involving chromosomes 14q32.33 and 4p16.3 resulting in the deregulation of the MMSET and FGFR3 genes. These early chromosomal alterations are observed in both MM and MGUS as well. As the disease progresses, secondary chromosomal translocations and gene mutations including activation of KRAS and NRAS, complex karyotype abnormalities in the MYC gene, mutations in the TP53 and FGFR3 genes, and inactivation of CDKN2A and CDKN2C. These genetic abnormalities result in altered expression of adhesion molecules on malignant plasma cells and consequent abnormal interactions between plasma cells and bone marrow stroma.43 An extensive description of MM biology, clinical presentations, and treatment are beyond the scope of this report. The potential involvement of miRNAs in the MM biology has become a promising field of research in an effort to elucidate their role in the pathogenesis of the disease.

The first evidence of miRNA involvement was reported in a meeting abstract by Al Masri et al,44 who assessed miRNA expression profiles in both human myeloma cell lines and MM patient samples. They noted that malignant plasma cells in both cell lines and patient samples presented significant variation in miRNA expression levels (including miR-125b, miR-133a, miR-1, and miR-124a) compared with those of healthy subjects.

Bakkus et al,45 also in a meeting abstract, reported the expression of 10 miRNAs using quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR), and observed higher expression levels of let-7a, miR-16, miR-17-5p, and miR-19b in both MM patients and cell lines in comparison with healthy controls, whereas for miR-15a and miR-21 there were no significant variations in expression among cell lines, patients, and control subjects. Down-regulation of miR-372, miR-143, and miR-155 was also described. Importantly, mir-16 presented higher expression in plasma cells of MM compared with those of MGUS, indicating an association not only with disease pathogenesis but also with disease progression.

In contrast to the previous results, miR-21 has been identified as an oncogenic miRNA with antiapoptotic function. Chromatin immunoprecipitation studies revealed that STAT3 is recruited to the miR-21 upstream region in human myeloma cells in response to IL-6. miR-21 transcription seemed to be controlled by IL-6 and mediated by STAT3 activation in MM cell lines, promoting survival of malignant plasma cells. Ectopic expression of miR-21 in the absence of IL-6 resulted in significantly higher levels of nonapoptotic cells, providing evidence of miR-21 contribution to the antiapoptotic function of STAT3. These data suggest a cardinal role of miR-21 in the oncogenic activity of STAT3 and therefore support its involvement in myelomagenesis.46

The differential expression of miRNAs located within transcription units in human MM cell lines and MM patients has also been investigated.47 The overexpression of 3 intronic miRNAs—miR-561, miR-335, and miR-342-3p—whose modulated expression was not associated with genomic alterations, correlated with the expression of their corresponding host genes (GULP1, MEST, and EVL, respectively) and were also sense oriented with respect to their genes. The miRNAs and their host genes were found involved in cell cycle control, apoptosis, proliferation, angiogenesis, bone marrow microenvironment interactions, and migration, processes all altered in MM. The authors concluded that miRNAs/host genes are regulated depending on each other, and their altered expression may contribute to the disease pathogenesis.47

Picchiorri et al48 studied the expression of miRNAs in plasma cells of patients with MM and MGUS, and in MM cell lines, providing new data about miRNA function in MM. They observed that 41 miRNAs were up-regulated and 7 miRNAs were down-regulated in MGUS, with the most up-regulated miRNAs being miR-181, miR-21, miR-106a, miR-106b, miR-25, and miR-93, thus describing an miRNA signature that distinguishes MGUS from healthy plasma cells. Their microarray analysis, further validated with Q-RT-PCR, revealed up-regulation of 60 miRNAs and down-regulation of 36 miRNAs in both cell lines and MM patients, whereas 37 up-regulated and 37 down-regulated miRNAs were found only in MM subjects in comparison with healthy controls. Up-regulated miRNAs included miR-25, miR-32, miR-20a, miR-93, miR-106b, miR-106a, miR-181a, miR-21, miR-19b, miR-181b, and miR-92a. Interestingly, miR-32 and the miR-17-92 cluster were up-regulated only in the case of MM and not in healthy cases or MGUS cases, suggesting a potential role in the disease progression from MGUS to MM, and probably representing MM-specific genetic changes. The up-regulated miR-32, miR-181a, and miR-181b, and the miR106b-25 cluster were found to target and negatively regulate histone acetyltransferase P300/CBP-associated factor (PCAF), which is involved in the acetylation of p53, and thus indirectly controls p53 activity in MM. miR-19a and miR-19b down-regulated the negative regulator of the cytokine IL-6 pathway, SOCS1, resulting in constitutive activation of the Jak/STAT3 signaling pathway, whereas overexpression of the miR-17-92 cluster negatively modulated the expression of the proapoptotic protein Bim, contributing in myelomagenesis. The oncogenic role of miRNAs in MM was also confirmed in a nude mice model in which tumor regression of transplanted tumors after treatment with miR-19a/b and miR-181a/b antagonists was achieved, also denoting a potential therapeutic use of antagomiRNAs.48 In their most recent report, the same authors identified a subset of down-regulated miRNAs in MM that are transcriptionally activated by p53 and modulate expression of the p53-negative regulator murine double minute 2 (MDM2).49 In MM cell lines treated with the MDM2 inhibitor nutlin-3a, the authors observed that miR-34 and miR-194 were up-regulated, confirming the dependence of both miRNAs on wild-type p53. Conversely, miR-192 and miR-215 were down-regulated in MM patients' plasma cells and up-regulated after p53 re-expression with nutlin-3a. miR-34a, miR-194, miR-192, and miR-215 expression were directly correlated with p53 protein up-regulation after nutlin-3a treatment and p21 activation, whereas miR-15, miR-29a, and miR-29b expression appeared more related to down-regulation of their repressor c-MYC than to p53 activation. Expression of miR-194, miR-192, and miR-215 in MM patients' plasma cells and MGUS plasma cells was consistently deregulated in MM samples compared with MGUS samples. This difference in expression probably reflects the involvement of the specific miRNAs in disease progression. The p53-dependent miRNAs induced a consistent G0/G1 arrest in cell lines with high expression of MDM2 mRNA. In contrast, in cells lines expressing lower levels of MDM2 mRNA were detected increases of sub-G1 fractions indicative of cell death, in cells transfected with miR-192, miR-215, and miR-194. Ectopic expression of the miRNAs studied in wild-type TP53 cells inhibited cell growth and enhanced apoptosis, effects that could be related to MDM2 regulation in MM cells. The authors have hypothesized that miR-192, miR-215, and miR-194 could target MDM2 expression. Overexpression of miR-192, miR-194, and miR-215 significantly increased the level of p53 and p21 after nutlin-3a treatment, and expression of MDM2 protein was dramatically decreased in both cell lines. Moreover, these 3 miRNAs, by targeting MDM2, indirectly modulated the expression of IGF-1R and its ligand IGF-1, influencing plasma cell migration in the bone marrow. The assays performed demonstrated that IGF-1R and IGF-1 were also directly targeted by miR-192, and miR-215 and the protein levels were decreased. Ectopic expression of miR-215 and miR-192 in IGF-1–treated cell lines resulted in decreased cell adhesion, migration, and tissue invasion. The results were verified in a NOD/SCID mouse model in which proliferated tumors were significantly suppressed. The nonuniform re-expression of the 3 miRNAs in all samples analyzed prompted authors to seek other mechanisms unrelated to the TP53 gene. In the cell lines studied, the miR-194-2-192 cluster presented promoter hypermethylation with consequent silencing of the cluster, supporting the hypothesis that the transition from MGUS to MM was favored by clonal selection of cells with aberrant promoter methylation of the specific cluster.

Lionetti et al50 sought to identify in vitro, performing global miRNAs profiling, modulated miRNAs resulting from deregulated gene expression profiles or altered gene copy number. They observed that the most common copy number alteration was gains (losses were rarer), with a minimum gain frequency of 12.5%. mir-548d-1 presented the highest frequency in copy number gains (94%), whereas mir-130b, mir-185, mir-648, and mir-649, interestingly all mapped on chromosome 22q11.21, presented gene losses, with maximum frequency of 38%. Interestingly, it was shown that 18 miRNAs were significantly more expressed in cells with a gain of the corresponding genomic regions than in cells without a gain. On the contrary, 15 miRNAs were underexpressed in cells with genomic losses/biallelic deletions. Moreover, it was demonstrated that the expression of a significant percentage (18%) of intronic miRNAs in MM are modulated in accordance with the expression of their host genes, suggesting that such association might contribute to MM pathogenesis. In another study, the same group51 identified 26 different miRNAs that were overexpressed in MM and plasma cell leukemia patients, and their expression varied according to the translocation/cyclin classification subgroups. Their study allowed the definition of distinct patterns of miRNA deregulation, the potential regulatory effects on their targets, and the prediction of the miRNA/mRNA regulatory networks in molecular subsets of MM. Specific patterns of miRNA expression could differentiate MM with distinct and well-defined genetic alterations. This latter finding was verified by another group,52 which identified 11, 8, 7, 37, and 18 miRNAs differentially expressed in MM patients with t(4;14), t(14;16), t(11;14), RB deletion as the only abnormality, and cytogenetically normal MM samples, respectively. A significant inverse correlation between deregulated miRNA expression levels and expression levels of the deregulated target genes was also observed. Predicted miRNA target gene overexpression correlated with miRNA underexpressed levels, and as target genes were implicated in cellular growth and proliferation, it can be deduced that down-regulation of these specific miRNAs can assign malignant plasma cells a growth advantage. There was no association between expression of the selected deregulated intronic miRNAs and their host transcripts, in contrast with previously published results.47

An important recent study evaluated the expression of various miRNAs in relapsed/refractory MM patients and in vitro.53 They observed that miR-15a and miR-16 expression was decreased, whereas miR-221, miR-222, miR-382, miR-181a, and miR-181b expression was increased compared with healthy subjects. Predicted target genes of overexpressed miRNAs included proapoptotic factors, tumor suppressors, cytokine signaling suppressors, NF-κB suppressors, and tyrosine phosphatases. Conversely, predicted target genes of underexpressed miRNAs included AKT3 and its downstream target ribosomal protein S6, angiogenic cytokines like VEGF, the NF-κB activator MAP3KIP3 (TAB3), Jag1, JUN, NEDD9, and Snai2, with the MAPK pathway being most affected, whereas β-globin and δ-globin were among the most up-regulated genes. miR-15a and miR-16 could regulate cell cycle by inhibiting cyclinD1, cyclinD2, CDC25A, and the level of phosphorylated Rb, resulting in G1 arrest, and also could induce reduced expression of BCL-2. Moreover, increased phosphorylation of the inhibitory protein IkB in the cytoplasm of pre-miRNA-15a–transfected and pre-miRNA-16-1–transfected cells was observed. These findings suggested a putative role of miR-15a and miR-16 in the regulation of both canonical and noncanonical NF-κB pathways. VEGF secretion was significantly decreased in pre-miRNA-15a–transfected and pre-miRNA-16-1–transfected MM.1S cells, compared with untransfected cells, suggesting an antiangiogenic role for their mature counterparts. Pre-miRNA-15a–transfected and pre-miRNA-16-1–transfected cells inhibited migration in response to SDF-1, whereas significant inhibition of adhesion of MM to primary bone marrow stromal cells was observed. The effect of miR-15a and miR-16 on migration was also evaluated in mouse models, confirming the in vitro results. The importance of miR-15a/16-1 cluster inhibition in promoting tumor progression by enhancing tumor cell survival, metastasis, and angiogenic properties of MM cells by up-regulating genes like NEDD9, Snai2, MALAT1, and VEGF was confirmed. Moreover, absent expression of miR-15a and overexpression of miR-181a and miR-181ab correlated with poor prognosis in patients with International Scoring System grade II/III.53 However, the findings of the study mentioned above on miR-15a and miR-16 expression were contradicted by others,54 who reported that miR-15a and miR-16 were displayed at a range of expression levels in MM patients that were higher than in normal plasma cells, and that the expression of these miRNAs varied independently of chromosome 13 status.

The group of Roccaro55 recently reported, in abstract form, miRNAs expression patterns in MM cell lines cocultured with primary bone marrow stromal cells and observed that miR-450, miR-432*, miR-299-5p, miR-409-3p, miR-29b, miR-542-5p, miR-184, miR-517*, miR-218, miR-128b, miR-142-5p, and miR-211 presented significantly increased expression in the cocultures compared with MM cells alone, and their predicted targets were represented by negative regulators of NF-κB, PI3K/Akt/mTOR, and MAPK/ERK signaling pathways, such as PTEN, KSR2, TWEAK, DUSP, as well as various tumor suppressors genes, proapoptotic factors, and cyclin-dependent kinase inhibitors.55

The members of the miR-125a and miR-125b clusters act by inhibiting the differentiation of centroblasts in germinal centers, with miR-125b demonstrating a dominant role because of its higher expression levels.56 miR-125b overexpression by down-regulating its target genes, BLIMP1 and IRF4, facilitated BCL-6 maintenance of the germinal center, thus negatively modulating the B-cell differentiation into plasma cells, antibody secretion, and myeloma cell survival in MM cell lines. Hence, impaired expression of the particular miRNA could contribute to MM pathogenesis.

miR-34a, a transcriptional target of p53 that mediates cellular apoptosis, has been found epigenetically silenced in MM samples.57 In particular, miR-34a was hypermethylated in 5.5% of the MM patients studied (no change in the methylation status of miR-34a was observed during disease progression), and was also hypermethylated in 37% of the MM cell lines, whereas no aberrant methylation was detected in healthy blood cells. These results supported the tumor suppressive function of the specific miRNA.57

Zhou and colleagues,58 in an analysis of both miRNA expression and protein coding gene expression profiles, observed that the total expression levels of 95 miRNAs evaluated was higher in the 52 newly diagnosed MM patient samples compared with those of control subjects. Nine miRNAs, all located on chromosome 13, were overexpressed in MM samples compared with normal samples, consistent with previous studies,48 and were also positively associated with risk score and proliferation index. Their results indicated that higher total expression levels of miRNAs might be associated with disease initiation, with an inferior clinical outcome, and with a high-grade undifferentiated stage of cancer that appears to be more aggressive than the low-grade stages. Notably, their findings suggested that although single miRNAs could not contribute to disease progression, they could act synergistically, contributing significantly to disease progression. miR-106a, miR-106b, miR-17-5p, and miR-20b were found to repress their target CDKN1A/p21Waf1/Cip1, deregulating its function in cell cycle progression in G1. Silencing of the enzymes AGO2 and Dicer resulted in decreased viability of malignant plasma cells, further illustrating the potential therapeutic option.58 In particular, AGO2 silencing led to cell cycle arrest in MM cell lines and enhanced expression of p21Waf1/Cip1, p27Kip1, CDK2, and CCND1, and finally induced increased rates of apoptosis through activation of caspase 3, caspase 8, and caspase 9. Similar results were also obtained when Dicer enzyme was experimentally knocked down.

The miR-193b-365 cluster, present only in vertebrates, has also recently been described as a novel overexpressed miRNA in MM patients and MM cell lines59; the deregulated expression of miR-17-92, miR-106b-25, and miR-106a-92 was reconfirmed. However, the role of this cluster in MM pathogenesis has to be elucidated.

Up-regulation and down-regulation of numerous miRNAs has also been observed in the drug-resistant myeloma cell lines RPMI8226 Dox6/LR5 and U266 Dox/LR7 (treated with doxorubicin-Dox6 or melphalan-LR5), suggesting that deregulated miRNA expression might be involved in the generation of drug resistance.60 The data obtained from that study argued for the involvement of multiple individual miRNA changes in drug-resistant cell lines, or for random gains and losses of genetic material during the induction of drug resistance.

Anastasiadou et al,61 to better understand the interaction between Epstein-Barr virus (EBV) and malignant plasma cells, using an LNA-based miRNA microarray, found that EBV-infected plasma cells could modulate the expression of the 36 miRNAs reported.

Genes such as HOX9, c-MYC, BCL-2, SHP1, and SHP2 represent targets of miR-146b, miR-140, miR-145, miR-125a, miR-151, miR-223, miR-155, and let-7f, and changes in expression of these miRNAs are thought to be involved in myelomagenesis and are also associated with overall prognosis.62

Neri et al,63 as they reported in their meeting abstract in 2009, were the first to evaluate miRNA expression patterns that are associated with response to bortezomib. They identified 22 deregulated miRNAs with overexpression of miR-155, miR-342-3p, miR-181a, miR-181b, miR-128, and miR-20b and down-regulation of miR-let-7b, miR-let-7i, miR-let-7d, miR-let-7c, miR-222, miR-221, miR-23a, miR-27a, and miR-29a in bortezomib-resistant versus bortezomib-sensitive cell lines. Predicted targets included genes involved in cell cycle regulation, cell growth, apoptosis, and ubiquitin conjugation pathways. To investigate the clinical importance of miRNAs, they correlated the miRNA expression profile of malignant plasma cells from bortezomib-sensitive and bortezomib-resistant MM patients with their response to therapy. Their data analysis revealed that the bortezomib-sensitive MM patients presented the same deregulated miRNAs as the sensitive cell line, whereas resistant patients segregated into the bortezomib-resistant cell line cluster. Moreover, they described a miRNA signature critical for MM pathogenesis and capable of prediction of response to bortezomib.

Most recently, miR-223 was reported to be down-regulated in extramedullary plasmacytomas compared with MM in patient samples, whereas miR-93, miR-181b, and miR-30a presented similar expression. The deregulated miR-223 expression and the CD19 positivity could distinguish MM from extramedullary plasmacytoma cases.64 miRNA expression profiling could be a useful tool for assessing medullary plasmacytomas and extramedullary plasmacytoma progression to overt MM, as has been determined by Mahindra and colleagues in their meeting report.65

The potential antimyeloma effect of various drugs by targeting and deregulating miRNA expression as a mechanism of action represent an interest and promising field of research. Two drugs, the cytostatic ribonuclease Onconase (known to reduce tumor growth in animal models with miRNAs being the intracellular targets of the drug), and the alkaloid cepharanthine (used to treat several acute and chronic diseases), as single agents or in combination, have been evaluated in the MM RPMI-8228 cell line.66 The cytotoxic effect was more pronounced when the 2 drugs were combined, probably by targeting the family of miRNAs that provide tumor resistance to cytotoxic drugs through mobilizing the cell defense mechanisms. Another possible explanation could be the modulation of expression of target miRNAs associated with the p53 network.66 The recently identified histone deacetylase inhibitor ITF2357 is capable of inducing apoptosis and inhibition of IL-6 and VEGF production in malignant plasma cells.67 It is also capable of down-regulating miR-19a and miR-19b, both members of the miR-17-92 cluster, with levels of inhibition ranging from 25% to 57% in various cell lines; it also down-regulated the expression of the C13orf25 transcript, which is the miR-17-92 cluster host. Hence, this particular drug affects MM cells biology by modulating transcription regulation genes, cell cycle and apoptosis regulator genes, genes involved in the tumor necrosis factor pathways, the production of proinflammatory cytokines, and the expression of the IL-6R. Interestingly, in this study a novel mechanism of action has been reported, delineating decreased expression of miR-19a and miR-19b by the specific drug and consequently reduced tumor cell growth.

The development of a screening assay capable of identifying miRNAs that negatively regulates p53 signaling through direct interaction with its coding gene TP53 has recently been developed, identifying miR-25 and miR-30d as putative regulators of p53 function.68 miR-25 and miR-30d were overexpressed in plasma cells from 31 MM patient samples compared with those from healthy donors, and miR-25 expression was inversely correlated with TP53 mRNA levels. TP53 mRNA was down-regulated about 60% or 85% with the introduction of miR-30d or miR-25. Introduction of miRNA inhibitors into an MM cell line with a wild-type p53 gene resulted in an increase of endogenous p53 levels, suggesting that dysregulation of miR-25 and miR-30d is pathologically important for p53 function in malignant plasma cells. The effect of miR-25 and miR-30d on cell apoptosis was also evaluated; Bax, p53, and p21 expression decreased with miRNA overexpression in the presence or absence of etoposide, and such effect was attributed to decreased expression of caspases. No significant alteration in PCAF or Bim expression was noted, suggesting that miR-25 did not indirectly contribute through modulation of PCAF or Bim in the down-regulation of p53.

Conclusions

  1. Top of page
  2. Abstract
  3. MicroRNAs and Cancer
  4. MicroRNAs in Hematopoiesis: General Considerations
  5. MicroRNAs in MM
  6. Conclusions
  7. Search Strategy
  8. FUNDING SOURCES
  9. REFERENCES

Since the first discovery of miRNAs in the nematode Caenorhabditis elegans almost 20 years ago, and 8 years since the first evidence of their involvement in human cancer,15 our knowledge has expanded enormously in understanding their importance and significance in cell biology and cancer pathogenesis. These small RNA molecules are present in several species, from viruses and plants to mammals, are tissue and developmental stage specific, and play pivotal roles in regulating the expression of genes involved in development, proliferation, differentiation, apoptosis, and stress response. Although the number of studies reporting miRNA involvement in cellular biology and in neoplastic diseases reaches some thousands, fewer reports have been published on the involvement of miRNAs in MM. The purpose of this paper was to summarize the current knowledge regarding the contribution of miRNAs to myelomagenesis, and prognosis and treatment of the particular disease. The location of miRNA genes in cancer-associated genomic regions, the presence of epigenetic mechanisms, and alterations in the miRNA processing machinery could explain the widespread differential expression of miRNA genes observed in cancer cells compared with normal cells69 and very probably in MM plasma cells compared with normal plasma cells. Moreover, several miRNAs have been found up-regulated or down-regulated in MM cell lines and patients, whereas for some miRNAs such as miR-15, miR-16, and miR-21 the results were conflicting. Discrepancies on miRNA differential expression among various studies have been reported, but that could be explained: 1) by the different platforms used for miRNA analysis; 2) by sampling methods, as in some studies sampling was performed according to patients' genetic abnormalities, whereas in other studies there was no such distinction; 3) by the different sample sizes; and 4) by the different statistical analyses regarding class comparisons and significance level (statistical analysis of microarray-SAM vs t test).

The studies described here established the deregulation of several miRNAs in MGUS and MM in association with well-known pathogenetic factors such as IL-6 and SOCS1 activating the STAT3 pathway, and apoptosis-related genes, thus contributing to malignant plasma cell survival. They are also capable of regulating the MDM2/p53 autoregulatory loop, and indirectly the p53 expression by targeting PCAF. miRNAs are also capable of targeting the IGF-1 axis controlling mobility and invasion of malignant plasma cells, as demonstrated by in vivo and in vitro experiments (Fig. 1). They also act synergistically, contributing to disease progression; their total expression levels are associated with clinical outcome, and correlate with the International Scoring System stage. miRNAs deregulate expression in MM cells, and the pattern of expression in MM seems associated with specific genetic abnormalities. Moreover, miRNAs observed in distinct genetic subsets characterized by chromosomal and molecular aberrations target several pathways contributing to cell cycle progression and cellular survival. Interestingly, different miRNAs that are deregulated in different cytogenetic subgroups putatively target the same genes.

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Figure 1. MicroRNAs established to interact with their target genes, already known to be involved in the pathogenesis of multiple myeloma, by either activating or blocking specific genes and pathways, are shown.

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Target prediction of miRNAs based on computational methods remains a challenge because of false-positive results, and better approaches to identify predicted targets are needed.16 This can be deduced by the observation that several miRNAs are identified to target mRNA by the currently known methods, whereas fewer miRNAs are found to deregulate experimentally reported gene expression.16 It is important, however, to identify target genes of miRNAs deregulated in MM, as miRNA/mRNA interactions could be useful in identifying relevant protein-coding genes involved in MM pathogenesis and could also represent future therapeutic targets.

As it seems that miRNAs play a crucial role in the pathogenesis of MM, development of drugs targeting miRNAs and modulating their expression represent a promising therapeutic approach.70 The development of drugs targeting these small RNA molecules is under continuous investigation in preclinical and clinical studies.69

In conclusion, the studies reported here aimed to place miRNAs into oncogenic and/or suppressive pathways to better understand how these pathways act in myelomagenesis.

Search Strategy

  1. Top of page
  2. Abstract
  3. MicroRNAs and Cancer
  4. MicroRNAs in Hematopoiesis: General Considerations
  5. MicroRNAs in MM
  6. Conclusions
  7. Search Strategy
  8. FUNDING SOURCES
  9. REFERENCES

A literature search of PubMed, using the keywords “multiple myeloma,” “microRNAs,” and “noncoding RNA” as single words or in combination, was performed. References of all articles pertinent to the topic were identified. We also separately browsed the following journals: New England Journal of Medicine, Science, The Lancet, Journal of Clinical Oncology, Blood, and Proceedings of the National Academy of Sciences of the United States of America, as well as the journals of Nature Publishing Group and Cell Press. Meeting abstracts of the American Society of Clinical Oncology and American Society for Hematology were also identified.

FUNDING SOURCES

  1. Top of page
  2. Abstract
  3. MicroRNAs and Cancer
  4. MicroRNAs in Hematopoiesis: General Considerations
  5. MicroRNAs in MM
  6. Conclusions
  7. Search Strategy
  8. FUNDING SOURCES
  9. REFERENCES

No specific funding was disclosed.

CONFLICT OF INTEREST DISCLOSURES

The authors made no disclosures.

REFERENCES

  1. Top of page
  2. Abstract
  3. MicroRNAs and Cancer
  4. MicroRNAs in Hematopoiesis: General Considerations
  5. MicroRNAs in MM
  6. Conclusions
  7. Search Strategy
  8. FUNDING SOURCES
  9. REFERENCES