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The small regulatory RNA microRNA-21 (miR-21) plays a crucial role in a plethora of biological functions and diseases including development, cancer, cardiovascular diseases and inflammation. The gene coding for pri-miR-21 (primary transcript containing miR-21) is located within the intronic region of the TMEM49 gene. Despite pri-miR-21 and TMEM49 are overlapping genes in the same direction of transcription, pri-miR-21 is independently transcribed by its own promoter regions and terminated with its own poly(A) tail. After transcription, primiR-21 is finally processed into mature miR-21. Expression of miR-21 has been found to be deregulated in almost all types of cancers and therefore was classified as an oncomiR. During recent years, additional roles of miR-21 in cardiovascular and pulmonary diseases, including cardiac and pulmonary fibrosis as well as myocardial infarction have been described. miR-21 additionally regulates various immunological and developmental processes. Due to the critical functions of its target proteins in various signaling pathways, miR-21 has become an attractive target for genetic and pharmacological modulation in various disease conditions.
miRNAs are a class of small non-coding RNAs whose mature products are ~22 nucleotides long. They negatively regulate gene expression by inducing translational inhibition or transcript degradation.1 Initially RNA polymerase III was described to mainly drive transcription of miRNAs. However, some primary-miRNA (pri-miRNA) transcripts are very long ranging up to several kilobases, and contain four or more uracils at a stretch, that would terminate polymerase III mediated transcription.2 MiRNA-21 was one of the first miRNAs to be identified as transcribed by RNA polymerase II, which subsequently has been identified as a major driver of miRNA transcription.
Annotation of miRNA genes has revealed that the majority of the miRNAs are intergenic. However, a significant number of miRNAs are located in sense or anti-sense strand in intronic regions.2 PrimiR-21 is one of the first human miRNA genes whose regulation was extensively studied and indeed, miR-21 is an example of an intronic miRNA. Despite that, primiR-21 has its own promoter region.3
miR-21 has been found to be upregulated in many pathological conditions including cancer and cardiovascular diseases.4 A non-transcriptional mechanism for miR-21 upregulation implying gene amplification, rather than promoter hyper-activation, has been proposed.5 However, most of the available data suggest that miR-21 expression is maintained by transcriptional and post-transcriptional regulation.3,6 Potential promoter regions of pri-miR-21 have been thoroughly studied. The actual size of pri-miR-21, the transcriptional start site (TSS) and minimal promoter region of pri-miR-21 are still subjects of debate.7
Due to its ubiquitous role in various biological processes, the interest in miRNA-21 has dramatically increased during recent years, especially in cancer and cardiovascular diseases. Several excellent reviews are already available highlighting the importance of miR-21 in cancer and cardiovascular diseases.4,7,8 Therefore, these entities are not focused in great detail in this review, but we put more emphasis on miR-21 regulation and its role in development, immunity and epithelial-to-mesenchymal transition.
Cullen's lab first described a miR-21 regulatory region mapping −3,403 to −2,395 upstream of the pri-miR-21.3 Three years later Loffler et al. described a very similar region mapping −3,565 to −2,415, which is inducible by IL6/Stat3.9 In a later study employing more stringent criteria, Fujita et al. described a new promoter mapping −3,770 to −3,337 upstream to the miR-21 hairpin. This has several conserved enhancer elements including binding sites for activation protein 1 (AP-1; composed of Fos and Jun family proteins), Ets/PU.1, C/EBPα, NFI, SRF, p53 and STAT3. Interestingly, the promoter region described by Fujita et al. has minimal overlap with the first described promoter in 20043 and therefore, this may suggest that these two promoter regions function independently as two separate promoters (Fig. 1). Ozsolak et al. compared transcriptional activity of different promoter regions of miR-21 and found that the promoter region described by Fujita et al. is stronger when compared to others and the first identified promoter3 is inducible in a cell line specific manner.11 More recently, Mudduluru et al. identified yet another regulatory region and two transcriptional start sites in miR-21 gene.12 In addition to positive regulators of miR-21 transcription, several transcriptional suppressors have also been reported. For example, miR-21 transcription was found to be repressed by NFI, C/EBPα.10 Dissociation of these factors from the promoter region after treatment with phorbol myristic acid (PMA) leads to enhanced promoter activity. In addition, Gfi1 13 and estrogen receptor14 were also shown to negatively regulate miR-21 promoter activity.
According to the standard nomenclature,15 the less abundant miRNA from each strand of a hairpin was designated with an asterisk mark. This asymmetry in the abundance is the result of asymmetric degradation of the opposing strand following Dicer processing. The strand with the less stable 5′ end has a better chance to remain undegraded.16,17 Analysis of 5′-end hairpin stability by the nearest-neighbor method for miR-21 revealed that 5′-end of miR-21* is slightly less stable than-miR-21.18 However, after Dicer mediated cleavage of 72-nt-long pre-miR-21 stem-loop to 22 nt miRNA-miRNA duplex, miR-21* is degraded and miR-21 becomes a mature miRNA.
In addition to transcriptional regulation, miR-21 expression is also regulated at the post-transcriptional level. Davis et al. showed that TGFβ and BMP4 (a member of TGFβ superfamily) upregulate pre-miR-21 expression by 4-fold within 30 min after treatment while the expression of pri-miR-21 is unaltered. They further showed that miR-21 upregulation is not affected by inhibition of RNA polymerase II by α-amanitin and a luciferase reporter construct containing the miR-21 gene promoter could not be activated by BMP4 or TGFβ treatment. In subsequent experiments, they showed that elevated miR-21 levels were due to an increase in the Drosha processing of the pri-miR-21 transcript, which is mediated by Smad proteins. After ligand stimulation, signal transducer SMADs (SMAD1/5 and SMAD2/3) were recruited to pri-miR-21 in a complex with the RNA helicase p68, a component of the Drosha microprocessor complex, which led to a fast processing of pri-miR-21 to pre-miR-21 and subsequent maturation. Interestingly, BMP6, which is also a member of the TGFβ superfamily has been shown to inhibit miR-21 expression. In breast cancer tissues an inverse correlation between BMP6 and miR-21 was observed.19 In luciferase reporter assays it has been shown that BMP6 inhibits miR-21 promoter activity through E2-box and AP1 binding sites. Similarly, BMPR1a signaling negatively regulated miR-21 expression in astrocytes, but in a post-transcriptional manner, as reduction in miR-21 levels were not accompanied by changes in pri-miR-21 levels.20 Results of these studies indicate that BMPs regulate miR-21 both positively and negatively through complex mechanisms.
After fertilization, embryos are dependent on maternally derived mRNAs for their transcriptional needs until their own transcription machinery is functional. When embryonic genome activation is initiated, maternal mRNA is degraded and miRNAs that are expressed during early developmental stages have thought to play an important role in this degradation.21 In studies employing vertebrate models of development such as zebrafish, miR-21 expression was detectable from very early stages of development. Chen et al.22 have shown that miR-21 could be detected from early developmental stages (12 h) and constituted up to 40% of all miRNAs in fibroblasts, irrespective of tissue of origin in developing zebrafish embryo. In another study involving rainbow trout (Oncorhynchus mykiss),23 it was shown that expression levels of miR-21 and Stat3, which is one of the transcription factors regulating miR-21,9 significantly increase during embryonic gene activation. They propose that miR-21 play an important role in degrading maternally inherited mRNAs by a yet unidentified mechanism.
The neuronal repressor REST (RE1-silencing transcription factor) is expressed at high levels in mouse embryonic stem (ES) cells.24 Heterozygous deletion of Rest and/or its short-interfering RNA-mediated knockdown in mouse ES cells cause a loss of self-renewal and lead to the expression of markers specific for multiple lineages.25 MiRNA expression has revealed that Rest represses this set of miRNAs including miR-21, that interfere with the expression of critical self-renewal regulators such as Oct4, Nanog and Sox2. In subsequent analyses it was found that overexpression of miR-21 by pre-miR-21, markedly decreased the self-renewing capacity of mouse ES cells by 60%. The effect of pre-miR-21 on self-renewal was specific as suppression of self-renewal by pre-miR-21 was rescued by anti-miR-21. Expression levels of the self-renewal markers Oct4, Nanog, Sox2 and c-myc were also found to be decreased in pre-miR-21-treated cells. These results suggest that miR-21, which is repressed by Rest, regulates, at least in part, the self-renewal of mouse ES cells. However, Jørgensen et al.26 and Buckley et al.27 showed that REST is not required for maintaining ESC pluripotency. Singh et al.22 reasoned that the discrepancies could be due to the differences in experimental procedures but future studies are needed to explore the exact role of miR-21 and its targets in the regulation of pluripotency.
MiR-21 has been shown to play an additional important role in branching morphogenesis28 which is a basic developmental process in the formation of many organs, such as exocrine glands, the lung or the kidney. The fetal murine submandibular salivary gland is a well analyzed model system for studying organogenesis, including branching morphogenesis. It has been shown that degradation of extracellular matrix by matrix metalloproteinases (MMPs) leads to enhancement of branching morphogenesis because extracellular matrix in the mesenchyme is remodelled during submandibular salivary gland development.29,30 Reck is a membrane-anchored inhibitor of MMPs. Therefore, inhibition of Reck results in degradation of ECM by MMPs, leading to enhancement of branching morphogenesis. At the transcriptional level, MMPs are regulated by transcriptional factors such as AP1.31,32 Incidentally PDCD4 inhibits AP1-mediated gene transactivation.33 Hayashi et al. have shown that miR-21 promotes branching morphogenesis possibly by modulating MMPs by targeting atleast RECK and PDCD4.28
The involvement of miRNAs in cancer emerged from several studies showing that expression of several miRNAs is deregulated in neoplastic tissues.34 The identification of several targets of miRNAs which are actually classical oncogenes or tumor suppressors has led to the widely accepted idea that miRNAs play pivotal roles in cancer initiation, progression and metastasization.35,36 miR-21 was first noted as an apoptotic suppressor in various cell lines.37 In a subsequent large scale study from 540 human samples, it was found that miR-21 is the only miRNA that is overexpressed in six solid cancers including that of lung, breast, stomach, prostate, colon and pancreas. In later studies miR-21 was established as an oncogenic miRNA and its overexpression was shown in most cancer types analysed so far.38–46 Additionally, miR-21 has been proposed as a biomarker of malignancy in circulation,45,47–49 sputum,50 cerebrospinal fluid51 and feces.52
Cancer is a multifactorial disease which evolves through a multistage process over a period of time driven by amassing mutations and epigenetic abnormalities in expression of multiple genes. However, despite severe disturbances in gene expression during cancer development, several studies reveal that the restoration of only one or a few of these abnormalities can profoundly inhibit the growth of cancer cells, and can lead to improvements in patient survival.53–55 This apparent dependency of some cancers on one or a few genes for the maintenance of the malignant phenotype is referred as “oncogene addiction.” Medina et al. have developed a mouse model using Tet-Off and Cre-recombinase technologies to achieve tissue-specific and doxycycline-controlled expression of miR-21 under Nestin (a protein marker for neural stem cells, also expressed in adult haematopoietic cells) promoter control. The resulting NesCre8, mir-21LSL-Tetoff mice had about ten-fold upregulation of miR-21 in the brain when animals were not treated with doxycycline compared with their siblings treated with doxycycline. Results indicate that overexpression of miR-21 has led to a pre-B malignant lymphoid-like phenotype, demonstrating that miR-21 is a genuine oncogene. When miR-21 was inactivated, the tumors regressed completely with in a few days. These results demonstrate that tumors can become addicted to oncomiRs and this study emphasizes the absolute dependence of at least some cancers on miR-21 for maintenance of the malignant phenotype. Oncogene addiction of some tumors has allowed the development of targeted therapeutic modalities that profoundly benefit cancer patients.54 Similarly addiction of some cancers on miR-21 can be exploited by developing pharmacological inhibitors of miRNA miR-21. In another recent elegant study employing K-ras G12D non-small-cell lung cancer (NSCLC) mouse model, Hatley et al. have showed that incidence of lung tumors significantly high in miR-21 overexpressing mice.57 Consistent with this, deletion of miR-21 has resulted in suppression of Ras-driven transformation in vitro and tumor development in vivo.
In view of the mechanism of action of miRNAs, it is not surprising that miRNAs govern myriad functions in immune cells. Indeed, several recent studies have identified the importance of miRNAs in immune cell development and function.58–62
During an immune response, antigen-specific naive T cells proliferate enormously and develop into effector T cells capable of executing effector functions such as cytotoxicity and cytokine secretion.63 After clearance of antigen, most of the effector T cells are eliminated by activation-induced cell death, but some antigen encountered cells differentiate into relatively quiescent T cells (memory cells), which persist over long periods of time and mount a rapid response upon re-challenge with the same antigen.64 Protein expression patterns differ enormously between effector and memory T cells.65 Wu et al. analyzed the miRNA expression profile in antigen-specific naive, effector and memory CD8 T cells and found that at least seven miRNAs are differentially expressed amongst these T-cell subsets among which six showed dramatically reduced frequencies in effector T cells compared to naive T cells, and the expression of these miRNAs tended to increase back in memory T cells. However miR-21 was an exception, showing reverse kinetics with highest frequency in effector T cells followed by memory T cells and lowest expression in naive T cells66 suggesting that miR-21 plays an important role in maintaining effector phase of the T cells.
In another study involving Sezary syndrome patients, it was observed that expression of miR-21 in neoplastic skin-homing CD4-memory T cells (Sezary cells) is higher when compared with CD4 T cells from healthy donors.67 Sezary Syndrome is characterized by constitutively activated signal transducer and activator of transcription 3 (STAT3). In this study, van der Fits et al. showed that miR-21 is a direct STAT3 target in Sezary cells. Further, stimulation of Sezary cells or healthy CD4 T cells with IL-21 results in a strong activation of STAT3, and subsequent upregulation of miR-21 expression suggesting a casual relationship between STAT3 and miR-21 67 and a more general role of miR-21 in STAT3 mediated immune functions.
Asthma is a chronic inflammatory disease characterized by inflammation of the airways, tissue remodeling and decreased respiratory function. MiRNAs may play an important role in asthma as it is characterized by marked changes in gene and protein expression in the lung.68,69 By using three different experimental animal models of asthma viz, OVA (ovalbumin), Aspergillus fumigatus, lung specific-IL-13 overexpression, Lu et al.70 have shown that IL-12p35 (a component of IL-12) expression is downregulated during asthma, which is correlated with increased expression of miR-21. Subsequent analyses have shown that miR-21 was primarily detected in the cytoplasm of mononuclear and multinucleated myeloid cells and its expression significantly increases in asthma. Target validation experiments show that IL-12p35 is a target of miR-21. IL-12 is a key cytokine derived from macrophages and dendritic cells and is involved in adaptive immune responses involving Th1 cell polarization, suggesting that in addition to asthma, miR-21 may play an important role in regulation of Th1 immune responses in general. However, this needs to be validated in further models of inflammation and infection.
miRNA expression analysis of monocytes from children with allergic rhinitis has indicated that miR-21 is one of the miRNAs that were most significantly downregulated in allergic rhinitis and this decrease in miR-21 expression is coupled with enhanced miR-21-target gene TGFβ receptor 2 (TGFBR2).71 However, it is not known whether targeting of TGFBR2 by miR-21 has any functional consequence in allergic rhinitis. None the less, the study reiterates that miR-21 may have a role in immune cell functions.
Lipopolysaccharide (LPS) is a membrane glycolipid of Gram-negative bacteria and recognition of LPS by the innate immune system can lead to uncontrollable cytokine production, which can result in cardiovascular collapse and hemodynamic instability, and can eventually cause fatal sepsis syndrome in humans. In humans, exposure to LPS increases severity of chronic obstructive pulmonary disease.72 Many negative regulatory mechanisms exist to counter the toxic effects of LPS. These include soluble decoy receptors, such as Toll-like receptor 4 (TLR4) and signal transduction proteins such as MyD88-S, IRAK-M and TAG.73 The inhibitor of transcription factor NFκB α-subunit (IκBα) is promptly resynthesized by NFκB in an autoregulatory way to block excessive transcription factor activity after treatment with LPS.74 PDCD4 is one of the transcriptional targets of NFκB, and it is expression is inducible by cytokine treatment.33 PDCD4 suppresses expression of IL-10 and IL-4 in an eLF4 dependent manner75 and PDCD4-deficient mice are resistant to models of inflammatory disease, such as experimental autoimmune encephalomyelitis and streptozotocin-induced type II diabetes. Therefore, by virtue of its ability to suppress IL-10 translation, PDCD4 is believed to function as a pro-inflammatory protein.76 Consistent with this, Sheedy et al. have shown that PDCD4 deficient mice were protected from LPS-induced death. In subsequent experiments they found that treatment of human peripheral blood mononuclear cells with LPS resulted in lower PDCD4 expression which was associated with enhanced miR-21 expression. Further they showed that expression of miR-21 was mediated directly by MyD88 and NFκB. These results are particularly interesting as they offer an opportunity to modulate TLR4 activity therapeutically by regulating miR-21 expression, which may have implications in the use of TLR4 in vaccine adjutancy77 or in inflammatory diseases such as sepsis, rheumatoid arthritis and allergic asthma.
Adult fibroblasts are considered to be derived directly from embryonic mesenchymal cells78 and increase in number as a result of the proliferation.79 However, recent evidence from various models of organ fibrosis suggests that fibroblasts may also be derived from epithelial or endothelial cells via a process called epithelial-to-mesenchymal-transition (or endothelial-to-mesenchymal transition for endothelial cells).80–87 TGFβ is the most potent inducer of EMT or EndMT and it is the primary cytokine driving fibrosis in various organs.88–90 Signaling molecules, such as AKT (Protein kinase B),91,92 integrin linked kinase,93 RhoA and beta-catenin have been implicated in TGFβ induced EMT. Importantly, as discussed in previous sections, miR-21 is rapidly inducible after TGFβ treatment,6,94 suggesting that it may play an important role in TGFβ induced pro-fibrotic effects, possibly via initiating or fostering EMT or EndMT. PTEN which is a validated target of miR-21 95,96 is a negative regulator of epithelial-to-mesenchymal transition.97,98 PTEN also negatively regulates Akt/PKB activation, which has been implicated in EMT99–104 as well as in EndMT.85,105 Consistent with this, expression of PTEN is effectively suppressed by TGFβ (and hence it is also called TEP1; TGFβ-regulated and epithelial cell-enriched phosphatase).106 All these observations suggest that miR-21 may play an important role in EMT or EndMT. Indeed, recent studies19,94 have confirmed the role of miR-21 in EMT, suggesting its crucial role in organ fibrosis.
More direct evidence for the role of miR-21 in organ fibrosis comes from the studies in mouse models of cardiac107 and pulmonary fibrosis.108 Thum et al. have shown that miRNA-21 is weakly expressed in normal myocardium where as its expression is elevated in failing myocardium.109 Further analysis revealed that miR-21 is overexpressed predominantly in cardiac fibroblasts, expression being highest in fibroblasts from the failing heart.96,107 Apoptotic response induced by miR-21 inhibition in cardiac fibroblasts is sensitive to ERK-MAPK signaling and overexpression of miR-21 led to a significant increase in ERK-MAP kinase activation suggesting that miR-21 is a mediator of ERK-MAPK signaling, which is crucial for fibroblast survival and activation, which also plays an important role in many cancers.110 Target validation experiments revealed that Sprouty1, a negative regulator of ERK-MAPK signaling, is a target of miR-21. Injection of chemically modified antisense oligonucleotides specific for miR-21 (antagomiR-21) into mice subjected to pressure overload of the left ventricle by transverse aortic constriction (TAC), normalized changes in SPRY1 expression, MAP kinase activation and reduced fibrosis.107
The role of miR-21 in organ fibrosis and the anti-fibrotic action of miR-21 inhibitors were further confirmed by a later study in bleomycin induced pulmonary fibrosis model.108 Results of this study indicate that miR-21 is highly upregulated in the lungs of mice with bleomycin-induced lung fibrosis and in the lungs of patients with idiopathic pulmonary fibrosis. miR-21 is primarily enriched in myofibroblasts in the fibrotic lungs. Likewise increased miR-21 expression in colorectal cancer tissue was mainly found in fibroblasts surrounding the cancer cells.111 As mentioned in the previous sections, the authors found that miR-21 is upregulated by TGFβ which in turn inhibited Smad7 (inhibitory Smad) leading to amplification of TGFβ signaling finally resulting in a fibrotic response in human primary fibroblasts. Sequestration of miR-21 in mouse lungs effectively attenuated bleomycin induced fibrosis suggesting a central role for miR-21 the pathogenesis of lung fibrosis. Interestingly, the chemical nature of the oligonucleotides used for miR-21 inhibition in vivo, may affect the therapeutic benefit. For example, Patrick et al.112 studied the role of miR-21 in pathological cardiac remodelling by injecting miR-21 inhibitors in mice exposed to transverse aortic constriction (TAC) for six weeks. Consistent with previous reports in reference 107 and 113 they observed enhanced cardiac miR-21 expression after TAC. However, they used short (8-nt) oligonucleotides against miR-21, which were different to the long oligonucleotide (22-nt) used by Thum et al.107,114 and failed to block the remodeling response of the heart to stress. In addition, the authors observed that miR-21, null mice still displayed cardiac hypertrophy and fibrosis in response to cardiac stress, which may be explainable by genetic compensatory mechanisms over the course of development.
The increasing information about the role of miR-21 (409 hits for “miR-21” keyword in PubMed as on 09-03-2011) suggests that miR-21 plays a very crucial role in many biological processes. Despite considerable development in understanding the transcriptional regulation of miR-21, precise mechanisms driving its upregulation are not completely understood. As discussed in previous sections, the miR-21 promoter regions have binding sites for several transcriptional factors such as AP1, STAT3, SRF, etc., but not all target genes of those factors (for example eNOS for AP1,115 miR-1 for SRF) 116 show upregulation as miR-21 in pathological conditions like heart failure. Although it could be explainable by post-transcriptional regulation of mature miR-21 expression,6 further studies are needed to ascertain the underlying mechanisms, which may offer additional level of control for pharmacological modulation of miR-21 for therapeutic reasons. Although, inhibition of miR-21 has been shown to provide significant benefit during cancer or heart failure in mice models,56,57,107 similar studies in larger animal models and other disease conditions are clearly needed to ascertain the therapeutic gain after miR-21 modulation.
This work was supported by the BMBF (# 01EO0802 to TT) and the Deutsche Forschungsgemeinschaft (DFG TH903/10-1 to TT).