miRNA regulates mRNA, which encodes proteins that modulate cellular functions and fate.
Therefore, miRNAs play important roles in physiological homeostasis in health and pathophys-iological derangement in disease. Certain miRNAs are tissue-specific—e.g., miR-1 in muscle, miR-21 in the heart, and miR-122 in the liver—whereas others are broader in distribution. The temporal expression of the tissue-specific miRNAs correlates closely with the specific physiologi-cal or pathologiphysiologi-cal status of the corresponding organs. In this section, we review the involvement of miRNAs in tissue development, cancer, cardiovascular diseases, and metabolism. The roles of miRNAs in health and disease of all organs and tissues are being extensively studied in all biomedical fields, and new knowledge is being developed on a daily basis.
3.1. miRNA Involvement in Cell and Tissue Development
Since the discovery of lin-4 and let-7 in C. elegans, there has been increasing evidence that miRNAs are engaged in vertebrate and invertebrate development, including proliferation and differentiation of embryonic stem (ES) cells, lineage commitment during embryogenesis, and maturation of multiple tissues.
In 2003, Bernstein et al. reported that disrupting the global miRNA biogenesis by ablation of Dicer in mice causes embryo death before gastrulation (47), providing the first demonstration of the essentiality of miRNA in early embryogenesis of mammals. Later studies showed that ES cells isolated from such mice have a slower proliferation rate and impaired differentiation, indicating the involvement of miRNAs in the self-renewal and pluripotency of ES cells (48).
miRNA profiling has since been performed to reveal the functions of specific miRNAs in ES cells.
For example, the miR-290 cluster is highly expressed in ES cells, and investigators have proposed that miR-290 suppresses the proteins that inhibit the expression of Oct4 (49). On the other hand, miR-21, which causes inhibition of Oct4 as a target, has a low expression in ES cells (50). The synergism of high expression of miR-290 and low expression of miR-21 in ES cells may account for the overall high level of Oct4, which is a TF necessary for the self-renewal of undifferentiated ES cells.
After lineage commitment, a highly coordinated gene-expression program directs the matu-ration of various tissues. Indeed, many studies have shown that miRNAs are regulators that fine-tune the maturation of nervous, muscle, adipose, and other tissues. miR-133b, highly expressed Annu. Rev. Biomed. Eng. 2010.12:1-27. Downloaded from www.annualreviews.org by National Chiao Tung University on 04/24/14. For personal use only.
miRNA signature:
the specific miRNA expression profile in a tissue/organ under a certain physiological or pathological condition
EC: endothelial cell
in dopaminergic neurons, regulates their maturation and function (51). miR-1 and miR-133 are present in skeletal muscle cells: miR-1 promotes their differentiation during myogenesis, and miR-133 enhances the proliferation of myoblasts (52). miR-143 is increased in adipocytes dur-ing adipogenesis. The inhibition of miR-143 effectively suppresses the differentiation process by a reduction in triglyceride accumulation and a decreased expression of adipocyte-specific genes (53). Detailed reviews of the involvement of miRNAs in the development of various tissues can be found in References 54 and 55.
3.2. miRNA and Cancer
Because miRNAs are directly involved in gene regulation, many of them have been implicated in cancer. In 2005, Lu et al. presented systematic miRNA profiling in multiple human cancer samples, showing that the changes in miRNAs correlate with developmental lineages and differentiation states of the cancers (56). This study demonstrates, for the first time, the potential of using a miRNA signature in cancer diagnosis. Since then, genome-wide profiling has been widely performed to reveal up- or downregulated miRNAs as biomarkers for specific types of cancer (see Reference 57 for review).
Studies on the role of miRNA in cancer development have identified the involvement of miR-NAs in tumorigenesis as well as metastasis, the two major processes in cancer progression. The miR-17–92 cluster, which comprises the first identified oncogenic miRNAs in mammals, is a di-rect effector of the c-myc oncogene and hence named oncomiR-1 (58). In parallel, miR-10b is the first reported metastasis-promoting miRNA; it targets homeobox D10, an antimetastatic gene (59). With the discovery of these cancer-promoting miRNAs, many cancer-suppressing miRNAs have also been identified (60). For example, miR-15a and miR-16-1 have been shown to inhibit tumorigenesis through targeting of the Bcl2 oncogene (61). Meanwhile, the list of the metastasis-suppressing miRNAs, including miR-126, miR-355, and the miR-200 family, is also expanding (62–64). A comprehensive update of currently identified miRNAs and their implications in various forms of cancer is summarized in the recent publication by Sotiropoulou et al. (65).
The important roles of miRNA in tumorigenesis and metastasis have led to the development of miRNA-based diagnostic and prognostic biomarkers as well as anticancer therapeutic agents.
Results from several trials have provided evidence to support the use of antisense miRNA to suppress oncogenic miRNA for cancer therapy (66).
3.3. miRNA in the Cardiovascular System
There is ample evidence that miRNAs also play critical roles in cardiovascular homeostasis. Dicer knockdown in endothelial cells (ECs) alters the expression of genes affecting EC biology and reduces EC proliferation and angiogenesis in vitro (67). With the knockdown of both Dicer and Drosha in ECs, capillary sprouting and tube-forming activity are significantly reduced, which may be a consequence of decreased biogenesis of let-7f and miR-27b (68). The available data indicate that the global reduction of miRNAs through the knockdown of Dicer and/or Drosha significantly affects EC functions in vitro and in vivo, suggesting the important roles of miRNAs in regulating vascular functions. Cardiac-specific deletion of Dicer is postnatal lethal, causing significant changes of miRNA expression with consequential dysregulation of muscular and ad-hesion proteins, dilated cardiomyopathy in neonates (52), and massive cardiac remodeling such as spontaneous hypertrophy (69).
Several studies have identified the involvement of specific miRNAs in cardiovascular dis-eases. van Rooij et al. have shown that more than 12 miRNAs are up- or downregulated in the Annu. Rev. Biomed. Eng. 2010.12:1-27. Downloaded from www.annualreviews.org by National Chiao Tung University on 04/24/14. For personal use only.
Antagomirs:
chemically modified antisense
oligonucleotides for silencing endogenous miRNA
myocardium of mice following transverse aortic constriction and pathological cardiac remod-eling (70). Muscle-specific miRNAs (e.g., miR-1 and miR-133) are particularly important in regulating cardiac functions. miR-133 knockout causes dilated cardiomyopathy and heart fail-ure in mice (71). miRNAs also play critical roles in regulating oxidative stress in the cardio-vascular system. It has been shown that miRNAs affect EC redox responses through the reg-ulation of the transcription factor HBP1 and the consequent expression of p47(phox) encod-ing NAD(P)H oxidase (72). Cheng et al. have demonstrated that free radicals induce miR-21 expression, which leads to the protection of cardiomyocytes from oxidative stress (73). In-creased miR-21 has also been found in balloon-injured rat carotid arteries (74). More recently, miR-221 and miR-222 have been reported to possess similar functions as those of miR-21 (75).
These studies have demonstrated the vital roles of miRNAs in regulating cardiovascular gene expression and, as a consequence, cardiovascular functions under physiological and pathological conditions.
3.4. miRNA Involvement in Metabolism
miRNAs are also regulators of metabolism and energy homeostasis. They are not only important for the differentiation of tissues involved in energy production, utilization, and storage (e.g., liver, skeletal muscle, and adipocytes), but also in the regulation of insulin release and amino acid and lipid metabolism. Insulin secretion by pancreatic β cells in mice is decreased by overexpression of miR-375 and increased by inhibition of miR-375, indicating that this miRNA negatively regulates insulin release (76). In terms of amino acid metabolism, miR-29b targets the mRNA-encoding branched-chain α-ketoacid dehydrogenase (BCKD) to prevent its translation in mammalian cells.
BCKD catalyzes the first irreversible step in branched-chain amino acid (BCAA) catabolism (77).
BCAAs (e.g., leucine, isoleucine, and valine) cannot be made de novo in mammals, and they are important in protein synthesis and nitrogen metabolism. Because leucine can stimulate insulin secretion (78), the targeting of BCKD translation by miR-29b indicates that this miRNA may also regulate insulin metabolism.
Regarding lipid metabolism, it has been found that intravenous administration of antagomirs (see Section 6.1) against miR-122 results in the degradation of miR-122 (the most abun-dant hepatic miRNA) in the mouse liver (79). Microarray analysis of liver isolated from these mouse models has shown the inhibition of several genes involved in cholesterol biosynthesis, including HMG-CoA reductase, the rate-limiting enzyme of cholesterol biosynthesis. Inhibi-tion of miR-122 in male C57BL/6 mice causes a reducInhibi-tion in plasma cholesterol level, an in-crease in hepatic β-oxidation, and a dein-crease in the synthesis of fatty acid and cholesterol in the liver; these changes are in line with the idea that miR-122 downregulates energy stor-age. Moreover, miR-122 inhibition by antisense nucleotides in a diet-induced obesity mouse model leads to a decrease in plasma cholesterol level and an improvement of liver steatosis, with reduced expression of lipogenic genes. Liver extracts from these miR-122-inhibted mice cause a 2.5-fold increase in the activity of adenosine monophosphate–activated protein kinase, which serves as an energy sensor by regulating multiple metabolic processes including energy storage, energy mobilization, and appetite (80). In addition, miR-103/107 loci are located in the introns of the genes that encode the pantothenate kinase, which is the rate-limiting en-zyme in generating coenen-zyme A (CoA) (81). Through the regulation of pantothenate kinase, miR-103/107 may modulate cellular acetyl-CoA and lipid levels. Indeed, target prediction of miR-103/107 reveals that many of its target genes are rate-limiting enzymes in metabolic pathways (82).
Annu. Rev. Biomed. Eng. 2010.12:1-27. Downloaded from www.annualreviews.org by National Chiao Tung University on 04/24/14. For personal use only.
Several methods have been developed for detecting and identifying miRNA profiles. The earlier approaches are hybridization-based methods, such as Northern blotting and microarray. The recently developed synthesis-based methods of deep-sequencing technologies have enabled the quantitative measurement of multiple miRNA samples within a single setup.
4.1. Microarray-Based Screening
Earlier studies of miRNA profiling depended on Northern blotting, RT-PCR, and cloning (83), which are labor-intensive, time-consuming, and limited in the information that they can obtain about global miRNA expression patterns. The rapid increase in the identified miRNA sequences led to the use of microarray for miRNA profiling; this method can detect thousands of miRNAs and their precursors and analyze multiple samples within a single chip. As in DNA microarray, the sequence-specific probes immobilized on the chip are used to detect miRNA levels in the sam-ples. A variety of commercial sources can provide miRNA profiling using the microarray platform containing the latest version of probes. For example, 866 human and 89 viral miRNA sequences are included in Agilent’s Human miRNA Microarray Version 3. In this platform, small RNAs iso-lated from cells or tissues are covalently labeled with fluorescent dyes and then hybridized to the immobilized probes on array chips. After the nonbinding miRNAs are washed off, the hybridized miRNAs are detected using a microarray scanner or scanning microscope to determine the fluo-rescent intensity on each probe spot, which represents the expression level of the corresponding miRNA in the cells (see References 84 and 85 for reviews).
With the use of small-scale, custom-made microarrays, early studies identified the miRNA expression profiles (i.e., miRNA signatures) during brain development (86). miRNA signatures of various developmental stages and disease progression have also been investigated using miRNA microarrays (87).
Although the miRNA microarray enables analyses of miRNA expression at the global scale with reasonable reproducibility and a medium-to-high capacity, it inherits the drawbacks of hybridization-based approaches in which the signal intensity is based on the affinity of hybridiza-tion between the miRNA and the probe. Therefore, the results are semiquantitative at best. In addition, the similarities in sequences shared by miRNAs and other small RNAs make it dif-ficult to distinguish them from one another. Furthermore, because all probes immobilized on the microarray are previously known miRNAs, this method is not suited for discovery of novel miRNAs.
4.2. High-Throughput Sequencing-by-Synthesis Technology
Several high-throughput sequencing technologies, regarded as “next-generation sequencing,”
have been developed for genome-wide nucleotide sequencing (88, 89). Compared with the ana-log nature of hybridization-based microarray, these new methods are based on the principle of sequencing-by-synthesis (SBS), which generates digital data quality. The principal method of SBS (Figure 4) is to sequence the clonal amplicons generated in vitro. The small RNA–derived complementary DNAs (cDNAs) collected from cells are ligated first with a short section of nu-cleotides (containing adaptors or tags), then immobilized on the surface of a planar substrate or micron-scale beads. The amplification involves either in situ multiple colonies (i.e., polonies) or PCR to generate clusters derived from any given single nucleotide molecule (90, 91). Because the nucleotide reads (sequences) determined by SBS are within the length range of pri-miRNA Annu. Rev. Biomed. Eng. 2010.12:1-27. Downloaded from www.annualreviews.org by National Chiao Tung University on 04/24/14. For personal use only.