microRNA

From Wikipedia, the free encyclopedia

Diagram of miRNA action with mRNA

The stem-loop secondary structure of a pre-microRNA from Brassica oleracea.

A microRNA (abbr. miRNA) is a small non-coding RNA molecule (ca. 22 nucleotides) found in plants, animals, and some viruses, which functions in transcriptional and post-transcriptional regulation of gene expression.[1] Encoded byeukaryotic nuclear DNA in plants and animals and by viral DNA in certain viruses whose genome is based on DNA, miRNAs function via base-pairing with complementary sequences within mRNA molecules, usually resulting in gene silencing via translational repression or target degradation.[2][3] The human genome may encode over 1000 miRNAs,[4][5]which may target about 60% of mammalian genes[6][7] and are abundant in many human cell types.[8] miRNAs are well conserved in eukaryotic organisms and are thought to be a vital and evolutionarily ancient component of genetic regulation.[9][10][11][12] While core components of the microRNA pathway are conserved between plants andanimals, miRNA repertoires in the two kingdoms appear to have evolved independently with different modes of function.[13] Plant miRNAs usually have perfect or near-perfect pairing with their messenger RNA targets and induce gene repression through degradation of their target transcripts.[14][15] Plant miRNAs may bind their targets in both coding regions and untranslated regions.[15] In contrast, animal miRNAs are able

to recognize their target mRNAs by as little as 6-8 nucleotides (the seed region) at the 5' end of an animal miRNA.[6][16] Combinatorial regulation is a feature of miRNA regulation. A given miRNA may have multiple different mRNA targets, and a given target might similarly be targeted by multiple miRNAs.[17][18] The first miRNAs were characterized in the early 1990s.[19] However, miRNAs were not recognized as a distinct class of biological regulators with conserved functions until the early 2000s. Since then, miRNA research has revealed multiple roles in negative regulation (transcript degradation and sequestering, translational suppression) and possible involvement in positive regulation (transcriptional and translational activation). By affecting gene regulation, miRNAs are likely to be involved in most biological processes.[20][21][22][23][24][25][26] Different sets of expressed miRNAs are found in different cell types and tissues.[27] Aberrant expression of miRNAs has been implicated in numerous disease states, and miRNA-based therapies are under investigation.[28][29][30][31] Estimates of the average number of unique messenger RNAs that are targets for repression by a typical microRNA vary, depending on the method used to make the estimate.[32] While a 2004 estimate was that the mean number of target mRNAs for a typical microRNA is only 7,[33] later estimates were higher. Krek et al.[34] found that vertebrate microRNAs target, on average, roughly 200 transcripts each. Selbach et al. [35] and Baek et al.[36] indicated that a single miRNA may repress the production of hundreds of proteins, but that this repression often is relatively mild (less than 2-fold).

History[edit]
MicroRNAs were discovered in 1993 by Victor Ambros, Rosalind Lee and Rhonda Feinbaum during a [19] study of the gene lin-14 in C. elegans development. They found that LIN-14 protein abundance was regulated by a short RNA product encoded by the lin-4 gene. A 61-nucleotide precursor from the lin4 gene matured to a 22-nucleotide RNA that contained sequences partially complementary to multiple sequences in the 3' UTR of the lin-14 mRNA. This complementarity was both necessary and sufficient to inhibit the translation of the lin-14 mRNA into the LIN-14 protein. Retrospectively, the lin-4 small RNA was the first microRNA to be identified, though at the time, it was thought to be a nematode idiosyncrasy. Only in 2000 was a second RNA characterized: let-7, which repressed lin-41, lin-14, lin-28, lin-42, and daf12 expression during developmental stage transitions in C. elegans. let-7 was soon found to be [37][38] conserved in many species, indicating the existence of a wider phenomenon.

Nomenclature[edit]
Under a standard nomenclature system, names are assigned to experimentally confirmed miRNAs before [39][40] publication of their discovery. The prefix "mir" is followed by a dash and a number, the latter often indicating order of naming. For example, mir-123 was named and likely discovered prior to mir-456. The uncapitalized "mir-" refers to the pre-miRNA, while a capitalized "miR-" refers to the mature form. miRNAs with nearly identical sequences except for one or two nucleotides are annotated with an additional lower case letter. For example, miR-123a would be closely related to miR-123b. Pre-miRNAs that lead to 100% identical mature miRNAs but that are located at different places in the genome are indicated with an additional dash-number suffix. For example, the pre-miRNAs hsa-mir-194-1 and hsa-mir-194-2 lead to an

identical mature miRNA (hsa-miR-194) but are located in different regions of the genome. Species of origin is designated with a three-letter prefix, e.g., hsa-miR-123 is a human (Homo sapiens) miRNA and oar-miR-123 is a sheep (Ovis aries) miRNA. Other common prefixes include 'v' for viral (miRNA encoded by a viral genome) and 'd' for Drosophila miRNA (a fruit fly commonly studied in genetic research). When two mature microRNAs originate from opposite arms of the same pre-miRNA, they are denoted with a -3p or -5p suffix. (In the past, this distinction was also made with 's' (sense) and 'as' (antisense)). When relative expression levels are known, an asterisk following the name indicates an miRNA expressed at low levels relative to the miRNA in the opposite arm of a hairpin. For example, miR-123 and miR-123* would share a pre-miRNA hairpin, but more miR-123 would be found in the cell.

Biogenesis[edit]

MicroRNAs are produced from either their own genes or from introns. A video of this process can be found here. The majority of the characterized miRNA genes are intergenic or oriented antisense to neighboring genes [41][41][42][43][44] and are therefore suspected to be transcribed as independent units. However, in some cases a microRNA gene is transcribed together with its host gene; this provides a means for coupled regulation [45] of miRNA and protein-coding gene. As much as 40% of miRNA genes may lie in the introns of protein [46] and non-protein coding genes or even inexons of long nonprotein-coding transcripts. These are [47][48] usually, though not exclusively, found in a sense orientation, and thus usually are regulated together

with their host genes. Other miRNA genes showing a common promoter include the 42-48% of all miRNAs originating from polycistronic units containing multiple discrete loops from which mature miRNAs [42][51] are processed, although this does not necessarily mean the mature miRNAs of a family will be homologous in structure and function. The promoters mentioned have been shown to have some similarities in their motifs to promoters of other genes transcribed by RNA polymerase II such as protein [42][52] coding genes. The DNA template is not the final word on mature miRNA production: 6% of human miRNAs show RNA editing (IsomiRs), the site-specific modification of RNA sequences to yield products different from those encoded by their DNA. This increases the diversity and scope of miRNA action beyond that implicated from the genome alone.

[46][49][50]

Transcription[edit]
miRNA genes are usually transcribed by RNA polymerase II (Pol II). The polymerase often binds to a promoter found near the DNA sequence encoding what will become the hairpin loop of the pre-miRNA. The resulting transcript iscapped with a specially modified nucleotide at the 5 end, polyadenylated with [42][47] multiple adenosines (a poly(A) tail), and spliced. Animal miRNAs are initially transcribed as part of one arm of an 80 nucleotide RNA stem-loop that in turn forms part of a several hundred nucleotides [42][47] long miRNA precursor termed a primary miRNA (pri-miRNA)s. When a stem-loop precursor is found [47] in the 3' UTR, a transcript may serve as a pri-miRNA and a mRNA. RNA polymerase III(Pol III) transcribes some miRNAs, especially those with upstream Alu sequences, transfer RNAs (tRNAs), and [53] mammalian wide interspersed repeat (MWIR) promoter units.
[42][52]

Nuclear processing[edit]
A single pri-miRNA may contain from one to six miRNA precursors. These hairpin loop structures are composed of about 70 nucleotides each. Each hairpin is flanked by sequences necessary for efficient processing. The double-stranded RNA structure of the hairpins in a pri-miRNA is recognized by a nuclear protein known as DiGeorge Syndrome Critical Region 8 (DGCR8 or "Pasha" in invertebrates), named for its association with DiGeorge Syndrome. DGCR8 associates with the enzyme Drosha, a protein that cuts [54] RNA, to form the "Microprocessor" complex. In this complex, DGCR8 orients the catalytic RNase III domain of Drosha to liberate hairpins from pri-miRNAs by cleaving RNA about eleven nucleotides from the hairpin base (two helical RNA turns into the stem). The product resulting has a two-nucleotide overhang at its 3 end; it has 3' hydroxyl and 5' phosphate groups. It is often termed as a pre-miRNA (precursor-miRNA). Pre-miRNAs that are spliced directly out of introns, bypassing the Microprocessor complex, are known as "Mirtrons." Originally thought to exist only in Drosophila and C. elegans, mirtrons have now been found in [55] mammals. Perhaps as many as 16% of pre-miRNAs may be altered through nuclear RNA editing. Most commonly, enzymes known as adenosine deaminases acting on RNA (ADARs) catalyzeadenosine to inosine (A to I) transitions. RNA editing can halt nuclear processing (for example, of pri-miR-142, leading to degradation by the ribonuclease Tudor-SN) and alter downstream processes including cytoplasmic miRNA processing and target specificity (e.g., by changing the seed region of miR[56] 376 in the central nervous system).
[56][57][58]

The RNA-induced silencing complex[edit]

Main article: RNA-induced silencing complex

The mature miRNA is part of an active RNA-induced silencing complex (RISC) containing Dicer and many [63] [64] associated proteins. RISC is also known as a microRNA ribonucleoprotein complex (miRNP); RISC with incorporated miRNA is sometimes referred to as "miRISC." Dicer processing of the pre-miRNA is thought to be coupled with unwinding of the duplex. Generally, only one strand is incorporated into the miRISC, selected on the basis of its thermodynamic instability and [65][66][67] weaker base-pairing relative to the other strand. The position of the stem-loop may also influence [68] strand choice. The other strand, called the passenger strand due to its lower levels in the steady state, is denoted with an asterisk (*) and is normally degraded. In some cases, both strands of the duplex are [69] viable and become functional miRNA that target different mRNA populations. Members of the Argonaute (Ago) protein family are central to RISC function. Argonautes are needed for miRNA-induced silencing and contain two conserved RNA binding domains: a PAZ domain that can bind the single stranded 3 end of the mature miRNA and a PIWI domain that structurally res embles ribonuclease-H and functions to interact with the 5 end of the guide strand. They bind the mature miRNA and orient it for interaction with a target mRNA. Some argonautes, for example human Ago2, cleave target transcripts directly; argonautes may also recruit additional proteins to achieve translational [70] repression. The human genome encodes eight argonaute proteins divided by sequence similarities into two families: AGO (with four members present in all mammalian cells and called E1F2C/hAgo in [64][70] humans), and PIWI (found in the germ line and hematopoietic stem cells). Additional RISC components include TRBP [human immunodeficiency virus (HIV) transactivating [71] response RNA (TAR) binding protein], PACT (protein activator of the interferon induced protein kinase (PACT), the SMN complex, fragile X mental retardation protein (FMRP), Tudor staphylococcal nucleasedomain-containing protein (Tudor-SN), the putative DNA helicase MOV10, and the RNA recognition motif [59][72][73] containing protein TNRC6B.

Mode of silencing[edit]
Gene silencing may occur either via mRNA degradation or preventing mRNA from being translated. For example, miR16 contains a sequence complementary to the AU-rich element found in the 3'UTR of many unstable mRNAs, such as TNF alpha or GM-CSF (ref Jing, Q., et al. Cell 2005 pmid = 15766526). It has been demonstrated that if there is complete complementation between the miRNA and target mRNA sequence, Ago2 can cleave the mRNA and lead to direct mRNA degradation. Yet, if there isn't complete [20] complementation the silencing is achieved by preventing translation.

miRNA turnover[edit]
Turnover of mature miRNA is needed for rapid changes in miRNA expression profiles. During miRNA maturation in the cytoplasm, uptake by the Argonaute protein is thought to stabilize the guide strand, while the opposite (* or "passenger") strand is preferentially destroyed. In what has been called a "Use it or lose it" strategy, Argonaute may preferentially retain miRNAs with many targets over miRNAs with few [74] or no targets, leading to degradation of the non-targeting molecules. Decay of mature miRNAs in Caenorhabditis elegans is mediated by the 5-to-3 exoribonuclease XRN2, [75] also known as Rat1p. In plants, SDN (small RNA degrading nuclease) family members degrade miRNAs in the opposite (3'-to-5') direction. Similar enzymes are encoded in animal genomes, but their [74] roles have not yet been described.

Several miRNA modifications affect miRNA stability. As indicated by work in the model organism Arabidopsis thaliana (thale cress), mature plant miRNAs appear to be stabilized by the addition of methyl moieties at the 3' end. The 2'-O-conjugated methyl groups block the addition of uracil (U) residues by uridyltransferase enzymes, a modification that may be associated with miRNA degradation. However, uridylation may also protect some miRNAs; the consequences of this modification are incompletely understood. Uridylation of some animal miRNAs has also been reported. Both plant and animal miRNAs may be altered by addition of adenine (A) residues to the 3' end of the miRNA. An extra A added to the end of mammalian miR-122, a liver-enriched miRNA important in Hepatitis C, stabilizes the molecule, and [74] plant miRNAs ending with an adenine residue have slower decay rates.

Cellular functions[edit]

Interaction of microRNA with protein translation process. Several (from nine documented) mechanisms of translation repression are shown: M1) on the initiation process, preventing assembling of the initiation complex or recruiting the 40S ribosomal subunit; M2) on the ribosome assembly; M3) on the translation process; M7, M8) on the degradation of mRNA. There exist other mechanisms of microRNA action on protein translation (transcriptional, transport to P-bodies, ribosome drop-off, co-translational protein degradation and others) that are not visualized here. [76] Here, 40S and 60S are light and heavy components of the ribosome, 80S is the assembled ribosome bound to mRNA, eIF4F is an translation initiation factor, PABC1 is the Poly-A binding protein, and "cap" is the mRNA cap structure needed for mRNA circularization (which can be the normal m7G-cap or artificial modified A-cap). The initiation of mRNA can proceed in a cap-independent manner, through recruiting 40S to IRES (Internal Ribosome Entry Site) located in 5UTR region. The actual work of RNA silencing is performed by RISC (RNA-induced silencing complex) in which the main catalytic subunit is one of the Argonaute proteins (AGO), and miRNA serves as a template for recognizing specific mRNA sequences.

The function of miRNAs appears to be in gene regulation. For that purpose, a miRNA is complementary to a part of one or moremessenger RNAs (mRNAs). Animal miRNAs are usually complementary to a site in the 3' UTR whereas plant miRNAs are usually complementary to coding

regions of mRNAs. Perfect or near perfect base pairing with the target RNA promotes cleavage of the [78] [79] RNA. This is the primary mode of plant miRNAs. In animals miRNAs more often have only partly the right sequence of nucleotides to bond with the target mRNA. The match-ups are imperfect. For partially complementary microRNAs to recognise their targets, nucleotides 2 7 of the miRNA (its 'seed [6][16] [80] region' ) still have to be perfectly complementary. Animal miRNAs inhibit protein translation of the [81] [79] target mRNA (this exists in plants as well but is less common). MicroRNAs that are partially complementary to a target can also speed up deadenylation, causing mRNAs to be degraded [82] sooner. While degradation of miRNA-targeted mRNA is well documented, whether or not translational repression is accomplished through mRNA degradation, translational inhibition, or a combination of the two is hotly debated. Recent work on miR-430 shows that translational repression is caused by the [83] disruption of translation initiation, independent of mRNA deadenylation. miRNAs occasionally also cause histone modification and DNA methylation of promoter sites, which [84][85] affects the expression of target genes. Nine mechanisms of miRNA action are described and assembled in a unified mathematical model: 1. Cap-40S initiation inhibition; 2. 60S Ribosomal unit joining inhibition; 3. Elongation inhibition; 4. Ribosome drop-off (premature termination); 5. Co-translational nascent protein degradation; 6. Sequestration in P-bodies; 7. mRNA Decay (destabilisation); 8. mRNA Cleavage; 9. Transcriptional inhibition through microRNA-mediated chromatin reorganization following by gene silencing. It is often impossible to discern these mechanisms using the experimental data about stationary reaction [76] rates. Nevertheless, they are differentiated in dynamics and have different kinetic signatures. Unlike plant microRNAs, the animal microRNAs target a diverse set of genes. However, genes involved in functions common to all cells, such as gene expression, have relatively fewer microRNA target sites and seem to be under selection to avoid targeting by microRNAs.
[86] [16] [76]

[77]

dsRNA can also activate gene expression, a mechanism that has been termed "small RNA-induced gene activation" or RNAa. dsRNAs targeting gene promoters can induce potent transcriptional activation of associated genes. This was demonstrated in human cells using synthetic dsRNAs termed small activating [87] [88] RNAs (saRNAs), but has also been demonstrated for endogenous microRNA. Interactions between microRNAs and complementary sequences on genes and even pseudogenes that share sequence homology are thought to be a back channel of communication regulating expression levels between paralogous genes. Given the name "competing endogenous RNAs" (ceRNAs), these microRNAs bind to "microRNA response elements" on genes and pseudogenes and may provide another [89] explanation for the persistence of non-coding DNA.

Evolution[edit]
MicroRNAs are significant phylogenetic markers because of their astonishingly low rate of [90] evolution. MicroRNAs origin as a regulatory mechanism developed from previous RNAi machinery [91] which was initially used as a defense against exogenous genetic material such as viruses. Their origin may have permitted the development of morphological innovation, and by making gene expression more [92] specific and 'fine-tunable', permitted the genesis of complex organs and perhaps, ultimately, complex [93] life. Indeed, rapid bursts of morphological innovation are generally associated with a high rate of [90][92] microRNA accumulation. New microRNAs are created in multiple different ways. Novel microRNAs can originate from the random formation of hairpins in "non-coding" sections of DNA (i.e. introns or intergene regions), but also by the [94] duplication and modification of existing microRNAs. MicroRNAs can also form from inverted duplications of protein-coding sequences, which allows for the creation of a foldback hairpin [95] structure. The rate of evolution (i.e. nucleotide substitution) in recently originated microRNAs is comparable to that elsewhere in the non-coding DNA, implying evolution by neutral drift; however, older microRNAs have a much lower rate of change (often less than one substitution per hundred million [93] years), suggesting that once a microRNA gains a function it undergoes extreme purifying [94] selection. Additionally, different regions within an miRNA gene seem to be under different evolutionary pressures, where regions that are vital for processing and function have much higher levels of [96] [93] conservation. At this point, a microRNA is rarely lost from an animal's genome, although microRNAs [94] that are more recently derived (and thus presumably non-functional) are frequently lost. In Arabidopsis thaliana, the net flux of miRNA genes has been predicted to be between 1.2 and 3.3 genes per million [97] years. This makes them a valuable phylogenetic marker, and they are being looked upon as a possible [98] solution to such outstanding phylogenetic problems as the relationships of arthropods. MicroRNAs feature in the genomes of most eukaryotic organisms, from the brown algae to the animals. However, the difference in how these microRNAs function and the way they are processed suggests that [100] microRNAs arose independently in plants and animals. Across all species, in excess of 5000 had [101] been identified by March 2010. Whilst short RNA sequences (50 hundreds of base pairs) of a [102] broadly comparable function occur in bacteria, bacteria lack true microRNAs.
[99]

Experimental detection and manipulation of miRNA[edit]

While researchers have focused on the study of miRNA expression in physiological and pathological processes, various technical variables related to microRNA isolation have emerged. The stability of the [103] stored miRNA samples has often been questioned. MicroRNAs are degraded much more easily than mRNAs, partly due to their length, but also because of the ubiquitously present RNases. This makes it necessary to cool samples on ice and use RNase-free equipment whenever working with [104] microRNAs. MicroRNA expression can be quantified in a two-step polymerase chain reaction process of modified RTPCR followed by quantitative PCR. Variations of this method achieve absolute or relative [105] quantification. miRNAs can also be hybridized to microarrays, slides or chips with probes to hundreds or thousands of miRNA targets, so that relative levels of miRNAs can be determined in different [106] samples. MicroRNAs can be both discovered and profiled by high-throughput sequencing methods [107] (MicroRNA Sequencing). The activity of an miRNA can be experimentally inhibited using a locked

nucleic acid (LNA) oligo, a Morpholino oligo or a 2'-O-methyl RNA oligo. Additionally, a specific miRNA can be silenced by a complementary antagomir. MicroRNA maturation can be inhibited at several [111] points by steric-blocking oligos. The miRNA target site of an mRNA transcript can also be blocked by a [112] [113] [114] steric-blocking oligo. For the in situ detection of miRNA, LNA or Morpholino probes can be used. The locked conformation of LNA results in enhanced hybridization properties and increases [115] sensitivity and selectivity, making it ideal for detection of short miRNA. High-throughput quantification of miRNAs is often difficult and prone to errors, for the larger variance (compared to mRNAs) that comes with the methodological problems. mRNA-expression is therefore often [116][117] analyzed as well to check for miRNA-effects in their levels (e. g. in ). To pair mRNA- and miRNA[118][119] data, databases can be used which predict miRNA-targets based on their base sequence. While this is usually done after miRNAs of interest have been detected (e. g. because of high expression levels), ideas for analysis tools that integrate mRNA- and miRNA-expression information have been [120][121] proposed.

[108][109]

[110]

miRNA and disease[edit]

Just as miRNA is involved in the normal functioning of eukaryotic cells, so has dysregulation of miRNA [122] been associated with disease. A manually curated, publicly available database,miR2Disease, [123] documents known relationships between miRNA dysregulation and human disease.

miRNA and inherited diseases[edit]

A mutation in the seed region of miR-96 causes hereditary progressive hearing loss. A mutation in the [125] seed region of miR-184 causes hereditary keratoconus with anterior polar cataract. Deletion of the [126] miR-17~92 cluster causes skeletal and growth defects.
[124]

miRNA and cancer[edit]

Role of miRNA in a cancer cell

The first human disease known to be associated with miRNA deregulation was chronic lymphocytic [122] leukemia and later many miRNAs have been found to have links with some types

of cancer and are sometimes referred to as "oncomirs". MicroRNA-21 is involved in several cancer-types such as glioblastoma and astrocytoma was one of the first microRNAs to be identified as an [129] oncomir. A study of mice altered to produce excess c-Myc a protein with mutated forms implicated in several cancers shows that miRNA has an effect on the development of cancer. Mice that were engineered to produce a surplus of types of miRNA found in lymphoma cells developed the disease within 50 days and [127] died two weeks later. In contrast, mice without the surplus miRNA lived over 100 days. Leukemia can be caused by the insertion of a viral genome next to the 17-92 array of microRNAs leading to increased [130] expression of this microRNA. Another study found that two types of miRNA inhibit the E2F1 protein, which regulates cell proliferation. miRNA appears to bind to messenger RNA before it can be translated to proteins that switch genes on [131] and off. By measuring activity among 217 genes encoding miRNA, patterns of gene activity that can distinguish types of cancers can be discerned. miRNA signatures may enable classification of cancer. This will allow doctors to determine the original tissue type which spawned a cancer and to be able to target a treatment [132] course based on the original tissue type. miRNA profiling has already been able to determine whether [128] patients with chronic lymphocytic leukemia had slow growing or aggressive forms of the cancer. Transgenic mice that over-express or lack specific miRNAs have provided insight into the role of small [133] RNAs in various malignancies. Much work has also been done on the role of microRNAs in establishing and maintaining cancer stem cells that are especially resistant to chemotherapy and often [134] responsible for relapse. A novel miRNA-profiling based screening assay for the detection of early-stage colorectal cancer has been developed and is currently in clinical trials. Early results showed that blood plasma samples collected from patients with early, resectable (Stage II) colorectal cancer could be distinguished from those of sex-and age-matched healthy volunteers. Sufficient selectivity and specificity could be achieved using small (less than 1 mL) samples of blood. The test has potential to be a cost-effective, non-invasive [135][136] way to identify at-risk patients who should undergo colonoscopy. Another role for miRNA in cancers is to use their expression level as a prognostic, for example one study on NSCLC samples found that low miR-324a levels could serve as a prognostic indicator of poor [137] survival, another found that either high miR-185 or low miR-133b levels correlated with metastasis and [138] poor survival in colorectal cancer. Optimal treatment for cancer involves accurately identifying patients for risk-stratified therapy. Those with a rapid response to initial treatment may benefit from truncated treatment regimens, thus the need for more accurate measures of disease response. Cell-free miRNA are highly stable in blood, are overexpressed in cancer, and are quantifiable within the diagnostic laboratory. In classical Hodgkin Lymphoma, plasma miR-21, miR-494, and miR-1973 are promising disease response [139] biomarkers. Circulating miRNAs have the potential to greatly assist clinical decision making and aid interpretation of positron emission tomography combined with computerized tomography. A further advantage is they can also be performed at each consultation to assess disease response and detection of early relapse.

[122][127][128]

Recent studies have miR-205 targeted for inhibiting the metastatic nature of breast cancer. Five members of the microRNA-200 family (miR-200a, miR-200b, miR-200c, miR-141 and miR-429) are down [141] regulated in tumour progression of breast cancer.

[140]

miRNA, DNA repair and cancer[edit]

Individuals with an inherited deficiency in DNA repair capability are at increased risk of cancer (also see DNA repair-deficiency disorder). If repair is deficient, damage tends to accumulate in DNA. Such DNA damage can cause mutational errors during DNA replication due to error-prone translesion synthesis. Accumulated DNA damage can also cause epigenetic alterations due to errors during DNA [143][144] repair. Such mutations and epigenetic alterations can give rise to cancer (see malignant neoplasms). Germ line mutations in DNA repair genes cause only 25% of colon cancer cases. However, altered expression of microRNAs, causing DNA repair deficiencies, are frequently associated with cancers and may be an important causal factor for these cancers. Among 68 sporadic colon cancers with reduced expression of the DNA mismatch repair protein MLH1, most were found to be deficient due to epigenetic methylation of the CpG island of the MLH1 [146] gene. However, up to 15% of the MLH1-deficiencies in sporadic colon cancers appeared to be due to [147] over-expression of the microRNA miR-155, which represses MLH1 expression. In 2966% of glioblastomas, DNA repair is deficient due to epigenetic methylation of the MGMT gene, which reduces protein expression of MGMT. However, for 28% of glioblastomas, the [148] MGMT protein is deficient but the MGMT promoter is not methylated. In the glioblastomas without methylated MGMT promoters, the level of microRNA miR-181d is inversely correlated with protein expression of MGMT and the direct target of miR-181d is the MGMT mRNA 3UTR (the three prime [148] untranslated region of MGMT mRNA). Thus, in 28% of glioblastomas, increased expression of miR181d and reduced expression of DNA repair enzyme MGMT may be a causal factor. HMGA proteins (HMGA1a, HMGA1b and HMGA2) are implicated in cancer, and expression of these proteins is regulated by microRNAs. HMGA expression is almost undetectable in differentiated adult tissues but is elevated in many cancers. HGMA proteins are polypeptides of ~100 amino acid residues characterized by a modular sequence organization. These proteins have three highly positively-charged regions, termed AT hooks, that bind the minor groove of AT-rich DNA stretches in specific regions of DNA. Human neoplasias, including thyroid, prostatic, cervical, colorectal, pancreatic and ovarian [150] carcinoma, show a strong increase of HMGA1a and HMGA1b proteins. Transgenic mice with HMGA1 targeted to lymphoid cells develop aggressive lymphoma, showing that high HMGA1 expression is not only associated with cancers, but that the HMGA1 gene can act as an oncogene to cause [151] [152] cancer. Baldassarre et al., showed that HMGA1 protein binds to the promoter region of DNA repair gene BRCA1 and inhibits BRCA1 promoter activity. They also showed that while only 11% of breast tumors had hypermethylation of the BRCA1 gene, 82% of aggressive breast cancers have low BRCA1 protein expression, and most of these reductions were due to chromatin remodeling by high levels of HMGA1 protein. HMGA2 protein specifically targets the promoter of ERCC1, thus reducing expression of this DNA repair [153] gene. ERCC1 protein expression was deficient in 100% of 47 evaluated colon cancers (though the [154] [155] extent to which HGMA2 was involved is not known). Palmieri et al. showed that, in normal tissues, HGMA1 and HMGA2 genes are targeted (and thus strongly reduced in expression) by miR-15, miR-16,
[148][149] [145] [142]

miR-26a, miR-196a2 and Let-7a. However, each of these HMGA-targeting miRNAs are drastically reduced in almost all human pituitary adenomas studied, when compared with the normal pituitary gland. Consistent with the down-regulation of these HMGA-targeting miRNAs, an increase in the HMGA1 and HMGA2-specific mRNAs was observed. Three of these microRNAs (miR-16, miR-196a and Let[156][157] 7a) have methylated promoters and therefore low expression in colon cancer. For two of these, miR-15 and miR-16, the coding regions are epigenetically silenced in cancer due to histone deacetylase [158] activity. When these microRNAs are expressed at a low level, then HMGA1 and HMGA2 proteins are expressed at a high level. HMGA1 and HMGA2 target (reduce expression of) BRCA1 and ERCC1 DNA [159] repair genes. Thus DNA repair can be reduced, likely contributing to cancer progression. In contrast to the previous example, where under-expression of miRNAs indirectly caused reduced expression of DNA repair genes, in some cases over-expression of certain miRNAs may directly reduce [160] expression of specific DNA repair proteins. Wan et al. referred to 6 DNA repair genes that are directly targeted by the miRNAs indicated: ATM (miR-421), RAD52 (miR-210, miR-373), RAD23B (miR-373), MSH2 (miR-21), BRCA1 (miR-182) and P53 (miR-504, miR-125b). Three of these miRNAs (miR-21, miR[157] 182, miR-125b) are among those identified by Schnekenburger and Diederich as over-expressed in colon cancer through epigenetic hypomethylation. Over expression of any one of these miRNAs can cause reduced expression of its target DNA repair gene.

miRNA and heart disease[edit]

The global role of miRNA function in the heart has been addressed by conditionally inhibiting miRNA maturation in the murine heart, and has revealed that miRNAs play an essential role during its [161][162] development. miRNA expression profiling studies demonstrate that expression levels of specific miRNAs change in diseased human hearts, pointing to their involvement [163][164][165] incardiomyopathies. Furthermore, studies on specific miRNAs in animal models have identified distinct roles for miRNAs both during heart development and under pathological conditions, including the regulation of key factors important for cardiogenesis, the hypertrophic growth response, and cardiac [162][166][167][168][169][170] conductance.

miRNA and the nervous system[edit]

miRNAs appear to regulate the nervous system. Neural miRNAs are involved at various stages of synaptic development, including dendritogenesis (involving miR-132, miR-134 and miR124),synapse formation and synapse maturation (where miR-134 and miR-138 are thought to be [172] [173][174] involved). Some studies find altered miRNA expression in schizophrenia.
[171]

miRNA and obesity[edit]

miRNAs play crucial roles in the regulation of stem cell progenitors differentiating [175] into adipocytes. Studies to determine what role pluripotent stem cells play in adipogenesis, were [176] examined in the immortalized human bone marrow-derived stromal cell line hMSC-Tert20. Decreased expression of miR-155,miR-221,and miR-222, have been found during the adipogenic programming of both immortalized and primary hMSCs, suggesting that they act as negative regulators of differentiation. Conversely, ectopic expression of the miRNAs 155,221, and 222 significantly inhibited adipogenesis and repressed induction of the master regulators PPAR and CCAAT/enhancer-binding protein alpha [177] (CEBPA). This paves the way for possible obesity treatments on the genetic level.

Another class of miRNAs that regulate insulin resistance, obesity, and diabetes, is the let-7 family. Let-7 is known to accumulate in human tissues during the course of aging. When let-7 was ectopically overexpressed to mimic accelerated aging, mice became insulin-resistant, and thus more prone to high [178] fat diet-induced obesity and diabetes. In contrast when let-7 was inhibited by injections of let-7specific antagomirs, mice become more insulin-sensitive, and remarkably resistant to high fat dietinduced obesity and diabetes. Not only could let-7 inhibition prevent obesity and diabetes, it could also [179] reverse and cure diabetes. These experimental findings suggest that let-7 inhibition could represent a new therapy for obesity and type 2 diabetes.

miRNA and non-coding RNAs[edit]

When the human genome project mapped its first chromosome in 1999, it was predicted the genome would contain over 100,000 protein coding genes. However, only around 20,000 were eventually [180] identified (International Human Genome Sequencing Consortium, 2004). Since then, the advent of bioinformatics approaches combined with genome tiling studies examining the [181] [182] transcriptome, systematic sequencing of full length cDNA libraries, and experimental [183] validation (including the creation of miRNA derived antisense oligonucleotides called antagomirs) have [184] revealed that many transcripts are non protein-coding RNA, including several snoRNAs and miRNAs.

miRNA and viruses[edit]

The expression of transcription activators by human herpesvirus-6 DNA is believed to be regulated by [185] viral miRNA.

MiRNAs involved in cancer cell metabolism

The biogenesis of miRNAs is tightly associated with their action mechanism (Figure1). Most miRNAs derived from independent transcription units [13,14] and are encoded by a bewildering array of genes. Their transcription is typically performed by RNA polymerase II, with transcripts capped and polyadenylated. The resulting primary or pri-miRNA transcript extends both 5 and 3 from the miRNA sequence. The sequential processing reaction excises the stem-loop from the remainder of the transcript to create a pre-miRNA product, which occurs in the nucleus and is mostly carried out by a nuclear member of the RNase III family (Drosha). The following step excises the terminal loop from the pre-miRNA stem to create a mature miRNA duplex of approximately 22bp length, which is carried out by the canonical Dicer enzyme in the cytoplasm. Either of the strands becomes stably associated with RNA-induced silenced complex (RISC), which can be called miRISC complex[15,16]. The miRISC complex acts as a regulator of target gene by specially recognizing and regulating particular mRNAs to inhibit target genes [17].

Figure 1. Biological functions of miRNA. The first step is the nuclear cleavage of the pri-miRNA, with a ~60-70 nt stem loop intermediate liberated, known as the miRNA precursor, or the pre-miRNA. Then this pre-miRNA is actively transported from the nucleus to the cytoplasm by Ran-GTP and export receptor. One end of the mature miRNA was cut by Drosha in nuclear and the other end is processed in the cytoplasm by the enzyme Dicer. Either of the strands becomes stably associated with RNA-induced silenced

complex (RISC), which can be called miRISC complex. The miRISC complex inhibits the target genes by (A) repressing initiation at the cap recognition, (B) inducing deadenylation of mRNA and thereby inhibiting circularization of mRNA, ( C) inducing ribosomes to drop off prematurely thus repressing the translation initiation and (D) promoting mRNA degradation. A shift in glucose metabolism from oxidative phosphorylation to aerobic glycolysis was a key biochemical hallmark of tumor cells [18,19]. The altered metabolism was called Warburg phenomenon, which consists of an increase in glycolysis maintained in conditions of high oxygen tension and gives rise to enhanced lactate production [20,21]. Metabolic shift in cancer cells seems to be influenced by oncogene and tumor suppressor networks [22]. Whats more, most of these tumor suppressors are miRNA targets. For example, phosphatidylinositol 3-kinase, a lipid kinase that regulates the levels of phosphorylated phosphatidylinositol at the plasma membrane, plays a key role in cancer cell metabolism, which is targeted by miR-320, miR123a, miR-422, miR-506 and miR-136. There are several lines of evidence that many key molecules in cell metabolism are miRNA targets, thus giving a clue that miRNA regulates cell metabolism. Since miRNAs regulate a substantial fraction of genes in animal genomes, Tibiche and Wang systematically analyzed the human metabolic network by integrating miRNA target genes into the network [23]. They performed randomization tests to determine whether a multiple-gene-node is significantly regulated by miRNAs and defined 79 multiple-gene-nodes as miRNA targets. They merged the miRNA targets of single-gene-nodes with the multiple-gene-nodes, and found that 238 (22%) nodes are miRNA targets. The functional association analysis of miRNAs and metabolic pathways uncovered that miRNAs predominantly regulate central metabolic pathways such as amino acid biosynthesis, certain sugar and lipid metabolism (Figure2).

Figure 2. The main miRNAs involved in metabolism of glucose, lipid and amino acid, as well as metabolismassociated oncogenic signaling pathways . Among the total 60 miRNAs mentioned in the text, more than 20 miRNAs, including miR375, miR-133, miR-199a, miR-138, etc., involve in glucose metabolism. And miR-14, miR-27a, miR-34a, miR-146, miR-335, miR-370, miR-122 and miR-33a/b function on lipid metabolism, including participating in controlling Acetyl-CoA and plasma cholesterol. While several miRNAs (miR-23b*, miR-29a/b, miR-277 etc.) play functions in amino acid metabolism mainly through regulating acyltransferase and -ketoacid dehydrogenase. In addition, about 29% of the mentioned miRNAs (miR-125b, miR-504, miR-25, miR30d, etc.) participate in metabolism-associated oncogenic signaling pathways.

Regulation of metabolic activity by miRNAs

MiRNAs regulate cell metabolic processes through complicated mechanisms, including directly targeting key molecules (transporters or enzymes / kinases) of metabolic processes and regulating multiple oncogenic signaling pathways (Figure3). MiRNAs could directly modulate the expression of metabolic transporters or enzyme activities. In addition, MiRNAs also play pivotal roles in the expression level of transcription factors and oncogenes or tumor suppressors, including p53, c-Myc, AMPK and AKT signaling pathway.

Figure 3. MicroRNAs regulate cell metabolism by targeting key metabolic enzymes and multiple oncogenic signaling pathways.MiRNAs could regulate cell metabolism by modulating the expression of metabolic transporters (like GLUT) or enzymes (HK2, ALDOA and PDK1) and acting on p53, c-Myc and AKT/mTOR signaling pathways. The steps regulated by miRNAs are indicated by red circular arrows, and the related miRNAs are listed in the bracket. FASN, fatty acid synthase; GLUT, glucose transporter; HIF, hypoxia-inducible factor; LAT1, L-type amino acid transporter 1; LDH-A, lactate dehydrogenase isoform A; MCT, monocarboxylate transporter; PDH, pyruvate dehydrogenase; PDK, pyruvate dehydrogenase kinase; PI3K, phosphatidylinositol 3kinase. The molecular mechanisms driving the Warburg effect in cancer cells were taken as an example to explain miRNA regulation in energy metabolism. As shown in Figure3, several miRNAs affect gene transcription and expression of glucose transporters (GLUTs) which are responsible for transporting glucose into cytoplasm. In the initial step of glucose metabolism, glucose could be transported over a plasma membrane by GLUT3 or GLUT4 which is a target of miR-133 [24] or miR-195-5p [25]. Thus, miRNAs could directly regulate intracellular glucose levels. In the following step, the hexokinase 2 (HK2), the first rate-limiting enzyme of glycolysis, is among the top list of genes predicted and potentially regulated by multiple miRNAs including miR-143 [26]. Along the glycolysis reaction chain, fructose 1,6-bisphosphate is broken down into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, which is catalyzed by aldolase A (Aldo A) in the reversible aldol reaction. While the enzyme Aldo A is down-regulated by miR-15a/16-1cluster [27]. And the specific miRNAs in glucose metabolism will be summarized into 4 subtitles as follows, including miRNA effects on glucose uptake, glycolysis, tricarboxylic acid (TCA) cycle and insulin regulation. On the other hand, aerobic glycolysis in tumor cells is driven by multiple miRNA-involved oncogenic signaling pathways. For example, AKT, a cardinal node in diverse signaling cascades, is regulated by miR21 [28], which stimulates glycolysis by directly regulating glycolytic enzymes and activating downstream mammalian target of rapamycin (mTOR) activity. The regulation of several specifically signaling pathways involved in cancer cell metabolism by miRNAs will be introduced in detail in the second section of this review. Therefore, mRNA dysregulation in any step of metabolic processes contributes to metabolic abnormalities and even cancer development.

MiRNAs regulate glucose metabolism MiRNAs affect glucose uptake

GLUTs (or SLC2A) are a wide group of membrane proteins that facilitate the transport of glucose over a plasma membrane in most mammalian cells. To date, 14 members of GLUTs have been identified [29]. The amounts of the GLUT1, GLUT2, and GLUT3 transcripts were elevated in most cancer tissues, while mRNA levels of GLUT4 and GLUT5 were below sensitivity in these cancer tissues. The potential effects of the GLUTs level seem to facilitate accelerated metabolism, high glucose requirements, and increased glucose uptake in malignant cells. Several factors have been implicated in the regulation of their expressions. Hormonal, for example, ovarian hormones, particularly estrogen, could provide a mechanism of GLUT regulation [30]. In addition, hypoxic also drives GLUT expression [31] as well as metabolic-stress-induced signaling pathways, such as adenosine monophosphate-activated protein kinase (AMPK), triggering upregulation of GLUT receptors [32]. MiRNAs could regulate glucose uptake via altering the GLUTs expressions. MiR-133 has been confirmed to regulate the expression of GLUT4 by targeting KLF15 in a rat model [31]. A study in renal cell carcinoma

demonstrated that down-regulated miR-199a, miR-138, miR-150 and miR-532-5p were correlated with an increased expression of GLUT-1, whereas an increased expression of miR-130b, miR-19a, miR-19b and miR301a can result in the down-regulation of GLUT-1 [32]. MiR-195-5p has been identified as a direct regulator of GLUT3 by targeting GLUT3 3-untranslated region in bladder cancer T24 cells [33]. Interestingly, miR-19a and miR-133a are altered in colorectal carcinoma [34], and their roles in regulating GLUT expression might explain the disordered metabolism in colorectal carcinoma. In addition, miR-130b is highly down-regulated in pancreatic tumors, and its role in regulating GLUT-1 expression might explain the increased glucose uptake in pancreatic adenocarcinoma [35].

Functions of miRNAs on glycolysis

Studies show that miRNAs regulate the irreversible steps in glycolysis, especially the key enzymes[33]. For example, miR-143, as an essential regulator of glycolysis, modulates glycolysis via targeting HK2 [12], which phosphorylates glucose to produce glucose 6-phosphate, thus committing glucose to the glycolytic pathway. Recently new protein targets of miRNAs have been identified by sensitive mass spectrometric studies. The oxysterol-binding-protein-related-protein 8 has been revealed as a target of miR-143 by quantitative mass spectrometry analysis [34]. For example, miR-155 could repress miR-143 thereby upregulating the expression of HK2 at the post-transcriptional level, except by activating the signal transducer and activator of transcription 3, a transcriptional activator for HK2 [35]. Besides, miR-143 inhibits the expression of HK2 both in primary keratinocytes and in head and neck squamous cell carcinoma-derived cell lines [36]. Whats more, HK2 has been validated as a miR-143 target and thus miR-143 could affect glucose metabolism in colon cancer cells [37]. Likewise, miR-143 has also been identified as an essential regulator of cancer glycolysis via targeting HK2 in human lung cancer [12]. Interestingly, the above articles were published almost at the same time. These reports all illustrated that miR-143 targets HK2 to regulate glucose metabolism in cancer cells, and it is a potential cancer therapeutic target. Except for targeting the irreversible rate-limiting steps, miRNAs also regulate other important intermediate steps in the glycolysis pathway. The enzyme Aldo A catalyzes a reversible aldol reaction in which fructose 1,6-bisphosphate is broken down into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. In this process, miR-122 was predicted to target Aldo A [26,38], and the miR-15a/16-1 cluster could reduce the levels of Aldo A [27]. Thus miR-122 and miR-15a/16-1 cluster are involved in glycolysis in cancer cells.

MiRNAs as biomarkers for human cancer

By targeting and controlling the expression of mRNA, miRNAs can control highly complex signal transduction pathways and multiple metabolic processes, which are usually involved in different oncogenic pathways [92]. The knowledge that miRNA expression is frequently dysregulated in cancer has uncovered an entirely new repertoire of molecular factors upstream of gene expression, with exciting potential as novel biomarkers and therapeutic targets in cancer [93]. Exploiting the unique characteristics of these molecules including their stability, tissue specificity, ease of detection and manipulation, will bring clinicians ever closer to achieving the goal of individualized cancer treatment [94]. On the one hand, miRNAs are produced in a tissue-specific manner, and changes in miRNA within a tissue type can be correlated with disease status. The tissue concentrations of specific miRNAs have been associated with tumor invasiveness, metastatic potential, and other clinical characteristics for several types of cancers, including chronic lymphocytic leukemia, and breast, colorectal, hepatic, lung, pancreatic, and prostate cancers [95]. On the other hand, there has been an accumulating body of evidence to support circulating miRNAs as non-invasive, sensitive biomarkers of disease states, particularly cancers (breast, lung, pancreas, ovarian, and prostate)[96]. For example, miR-9 and miR-9* (expressed from the 3' mature sequence), mostly neuronal and thus expressed in central nervous system tumors but absent in other tumors, present their potential as tumor markers [97]. In addition, the reduced levels of miR-126, members of the miR-17-92 cluster, inflammation-related miR-155, and smooth muscle-enriched miR-145 in patients with coronary artery disease compared with healthy controls [98]. Whats more, published data showed that plasma miR-29a and miR-92a have strong potential as novel noninvasive biomarkers for early detection of colorectal carcinoma [99]. Furthermore, since they are abundant in blood, easy to measure, highly stable and disease associated, serum microRNAs are attractive disease biomarkers [100]. There have been over 200 publications on circulating miRNA in cancers including prostate, breast, colon, lung, ovarian and leukemia since 2008. Considering the sources of variation, state of microRNA in plasma and origin and implications

for disease specificity, miRNA expression profiles of potential patients could be assessed by measuring circulating miRNAs in patient serum. This profile could be hopefully used for early detection of cancer.

Conclusion and perspective

MiRNAs are important regulators of numerous aspects of metabolic homeostasis, physiology and disease. In general, miRNAs could mainly have two ways to regulate cellular metabolism. MiRNAs could regulate transcription factors or signaling proteins, which in turn regulate metabolic enzymes. Alternatively, miRNAs could regulate the production of certain metabolites by directly regulating the genes that encode metabolic enzymes [101]. In addition, miRNAs could regulate mRNAs through chromatin remodeling [102]. The emergence of miRNAs as important regulators of metabolism has garnered much interest not only from a scientific point of view but also from a clinical perspective. The function of miRNAs on cellular metabolism reveals molecular strategies for controlling metabolic flux by miRNAs in living organisms, thus lighting up one aspect of miRNA therapeutics. MiRNAs are promising in the diagnosis of cancer, drug target identification and clinical treatment in the future (Figure4). The use of miRNAs, such as oligonucleotide complementary[103] or antisense oligonucleotides [104] in miRNA inhibition, to suppress cell metabolism altering will hopefully lead to a new therapeutic strategy for malignant cancer [105,106]. For example, endothelial miR-126 is deregulated in patients with type 2 diabetes, which may ultimately lead to novel biomarkers for risk estimation and classification and could be exploited for miRNA-based therapeutic interventions of vascular complications associated with this disease [107].

Figure 4. MiRNA-based diagnosis strategies for cancer. The workflow of detecting cancer based on miRNA profiling is included sample (serum or tissue) collection, miRNA expression profiling, data analysis of miRNA expression level and cancer risk assessment. The level of the at least one miRNA gene product can be measured using a variety of techniques (microRNA chip, quantitative or semi-quantitative RT-PCR, northern blot analysis, solution hybridization detection etc.) to provide a profile for the test sample. The level of at least one miRNA gene product in a test sample from the subject is compared to that in a control sample. A significantly increased or decreased alteration in the level of the miRNA gene product in the detected sample is indicative of the subject either having or being at risk for developing a cancer. So far, a variety of new strategies to identify and characterize the targets of individual miRNAs have been developed. Because miRNAs can also regulate other non-coding RNAs, these interactions will increase the complexity of gene regulation. Moreover, cost-effective miRNA profiling strategies and larger studies are needed to determine its advantage for cancer diagnosis. Additionally, a new class of miRNA-based drugs that are capable of targeting molecules outside the range of traditional medicinal chemistry, their clinical implementation will require improvements in drug composition and delivery. Since these challenges lie on the way, molecular strategies for cancer therapy by miRNAs are still in their infancy. Nevertheless, the successful development of miRNA biology technologies could ultimately translate our understanding of miRNA functions in cancer into strategies for the control of cancer.

Stefanie Sassen,

Eric A. Miska,2 and Carlos Caldas1

Author information Article notes Copyright and License information This article has been cited by other articles in PMC.

Abstract
Go to:

The small RNA revolution

Small ribonucleic acid (RNA) can act as a specific regulator of gene expression. This discovery has been an exciting breakthrough in Biological Sciences of the past decade, culminating in last years Nobel Prize in Physiology or Medicine awarded to Andrew Fire and Craig Mello. Building on previous work mainly in plants [50], Fire et al. [23] discovered that exogenous double-stranded RNA can be used to specifically interfere with gene function. This phenomenon was called RNA interference (RNAi). They also speculated that organisms might use double-stranded RNA naturally as a way of silencing genes. It was then shown that RNA interference was mediated by 22 nucleotide single-stranded RNAs termed small interfering RNAs (siRNAs) derived from the longer double-stranded RNA precursors [87]. The small interfering RNAs were found to repress genes by eliminating the corresponding messenger RNA transcripts, and thus, preventing protein synthesis. Over the following years, many new small functional RNAs have been found. RNA is usually thought of as messenger RNA that serves as the template for translation of genes into proteins. In contrast, functional or non-coding RNA molecules are transcribed from a DNA sequence, but not translated into protein. The encoding DNA sequence is often referred to as an RNA gene. Functional RNA genes in the human genome include transfer RNA (tRNA), ribosomal RNA (rRNA), and various other small non-coding RNAs. Several hundred genes in our genome encode small functional RNA molecules collectively called microRNAs (miRNAs). Precursors of these miRNA molecules form structures of double-stranded RNA that can activate the RNA interference machinery. MicroRNAs downregulate gene expression either by degradation of messenger RNA through the RNA interference pathway or by inhibiting protein translation. The first miRNA was discovered in 1993 by Victor Ambros and colleagues Rosalind Lee and Rhonda Feinbaum [42]. A genetic screen in the roundworm Caenorhabditis elegans, a millimeter-long animal used as a model organism in biological research, identified genes involved in developmental timing [42]. Surprisingly, one of the genes, termed lin-4, did not encode a protein but instead a novel 22-nucleotide small RNA. Seven years later, Reinhart et al. [70] discovered a second 22-nucleotide small RNA of this type, let-7, a gene also involved in C. elegans developmental timing. The lin-4 and let-7 small regulatory RNAs soon became very exciting for two reasons. Firstly, homologs of the let-7 gene were identified in other animals including humans [65]. The conservation of let-7 across species suggested an important and fundamental biological role for this small RNA. Secondly, the mechanism of RNA interference (RNAi) was discovered at that time, and it became clear that miRNA and RNAi pathways were intricately linked and shared common components. Within the following year, more than 100 additional small regulatory RNAs similar to lin-

4 and let-7 were identified in worms, the fruit fly Drosophila, and in humans [38, 40, 41]. These small non-coding RNAs were named microRNAs (miRNAs) [38, 40, 41]. Subsequently, many more short regulatory RNAs were identified in almost all multicellular organisms, including flowering plants, worms, flies, fish, frogs, mammals [38, 40, 41, 48, 71], and in single cellular algae and DNA viruses [66, 75]. To date, more than 500 human miRNAs have been experimentally identified. Computational predictions of miRNA targets suggest that up to 30% of human protein coding genes may be regulated by miRNAs [46, 68]. This makes miRNAs one of the most abundant classes of regulatory genes in humans. MicroRNAs are now perceived as a key layer of post-transcriptional control within the networks of gene regulation. MicroRNAs are sequentially processed from longer precursor molecules that are encoded by the miRNA genes [1] (Fig. 1). MiRNA genes are referred to by the same name (termed mir) written in italics to distinguish them from the corresponding mature miRNA (termed miR) followed by a number, e.g.,mir-1 or miR-1. The encoding DNA sequence is much longer than the mature miRNA. Two ribonuclease enzymes, Drosha and Dicer, subsequently process the primary transcripts (or pri-miRNA) to generate mature miRNAs. The primary transcripts contain one or more stem-loop structures of about 70 bases. Stem-loops are doublestranded RNA structures consisting of a nucleotide sequence that can fold back on itself to form a double helix with a region of imperfect base pairing that forms an open loop at the end (Fig. 1a). The ribonuclease Drosha excises the stem-loop structure to form the precursor miRNA (or pre-miRNA) [43]. After export into the cytoplasm, the pre-miRNA is cleaved by the ribonuclease Dicer to generate a short RNA duplex [6, 28]. After untwisting, one RNA strand becomes the mature single-stranded miRNA, while the complementary strand, termed miRNA*, is usually rapidly degraded (Fig. 1b).

Fig. 1 The biogenesis and function of miRNAs. a Primary miRNAs (pri-miRNA) are transcribed from longer encoding DNA sequences (miRNA genes). The pri-miRNA contains one or more stem-loop structures of about 70 bases. In the nucleus, the ribonuclease enzyme Drosha ... MicroRNAs recognize their targets based on sequence complementarity [10]. The mature miRNA is partially complementary to one or more messenger RNAs. In humans, the

complementary sites are usually within the 3-untranslated region of the target messenger RNA. To become effective, the mature miRNA forms a complex with proteins, termed the RNA-induced silencing complex. The miRNA incorporated into the silencing complex can bind to the target messenger RNA by base pairing. This base pairing subsequently causes inhibition of protein translation and/or degradation of the messenger RNA (Fig. 1c). The potential mechanisms underlying this process were recently reviewed [30, 67]. Protein levels of the target gene are consequently reduced, whereas messenger RNA levels may or may not be decreased. In humans, miRNAs mainly inhibit protein translation of their target genes and only infrequently cause degradation or cleavage of the messenger RNA [1]. The biological role and in vivo functions of most mammalian miRNAs are still poorly understood. In invertebrates, miRNAs regulate developmental timing (e.g., lin-4), neuronal differentiation, cell proliferation, growth control, and programmed cell death [9, 33, 42]. In mammals, miRNAs have been found to play a role in embryogenesis and stem cell maintenance [7], hematopoietic cell differentiation [17], and brain development [59, 60]. To date, knowledge of human miRNAs has been primarily descriptive. MicroRNA expression has been found to be deregulated in a wide range of human diseases including cancer. However, it remains uncertain whether altered miRNA expression is a cause or consequence of pathological processes. The underlying mechanisms of why and how miRNAs become deregulated are largely unknown. Although bioinformatics approaches can predict thousands of genes that are potentially targeted and regulated by miRNAs based on sequence complementarity, only very few miRNA target genes have been functionally validated. Our group is currently investigating the role of miRNAs in mammary gland development and breast cancer pathogenesis. A comparison of miRNA and gene expression identified miRNAs that classify molecular breast cancer subtypes [8]. As cancer is ultimately a consequence of disordered gene expression, miRNAs have been suggested to contribute to the development of cancer [11]. This review will focus on the connection between human miRNA biology and different aspects of carcinogenesis. Various techniques available to investigate miRNAs will also be discussed. Go to:

MicroRNAs and cancer

Three important observations early in the history of miRNAs suggested a potential role in human cancer. Firstly, the earliest miRNAs discovered in the roundworm C. elegans and the fruit fly Drosophilawere shown to control cell proliferation and apoptosis [9, 42]. Their deregulation may therefore contribute to proliferative diseases such as cancer. Secondly, when human miRNAs were discovered, it was noticed that many miRNA genes were located at fragile sites in the genome or regions that are commonly amplified or deleted in human

cancer [14]. Thirdly, malignant tumors and tumor cell lines were found to have widespread deregulated miRNA expression compared to normal tissues [12, 24, 52]. The question remained whether the altered miRNA expression observed in cancer is a cause or consequence of malignant transformation.
MicroRNAs as causal cancer genes at genomic breakpoints

Five years ago, the first direct evidence for an involvement of miRNAs in cancer was reported [13]. Calin et al. studied a well-known deletion on chromosome 13, which is the most frequent chromosomal abnormality in chronic lymphocytic leukemia (CLL). This deletion had long been suspected to contribute to leukemogenesis. However, extensive studies had failed to identify a causal gene. Calin et al. [13] found that two miRNA genes, mir-15 and mir-16, were located within this 30-kb deletion. They subsequently analyzed the expression of miR-15 and miR-16 in blood samples from patients with CLL. Both miRNAs were absent or downregulated in the majority (68%) of cases when compared to normal tissue or lymphocytes. This finding suggested that these two miRNAs were causally involved in the pathogenesis of chronic lymphocytic leukemia. In 2005, three reports provided the first mechanistic insight into how miRNAs might contribute to carcinogenesis. Two independent studies described the relationship between a miRNA cluster, mir-17-92, and the Myc oncogenic pathway [27, 63]. A third report demonstrated an interaction between let-7miRNA and the RAS proto-oncogene [32].
The mir-17-92 clustersmall RNAs with oncogenic potential

A cluster of six miRNAs, the mir-17-92 cluster, was found to be located within a region on chromosome 13 that is commonly amplified in human B-cell lymphomas [64]. He et al. [27] demonstrated that the miRNAs from the mir-17-92 cluster were overexpressed in lymphoma cell lines carrying this amplification, and expression levels correlated with gene copy number of the mir-17-92 locus [27]. Further, the miR-17-92 primary transcript was found to be overexpressed in tumor samples from lymphoma patients. To test their hypothesis that mir-17-92 actively contributes to lymphomagenesis, the authors took advantage of a mouse model of human B-cell lymphoma. These mice develop lymphomas due to an overexpression of the Myc oncogene. The Myc oncogene encodes the transcription factor c-Myc that regulates cell proliferation, growth, and apoptosis, and overexpression of c-Myc is common in cancer. He et al. [27] demonstrated that additional expression of the mir-17-92 cluster accelerated c-Myc-induced tumorigenesis in mice. The authors therefore suggested that mir-17-92 was the first potential non-coding oncogene, referred to as oncomir-1.

The cellular function of miR-17-92 was not identified in these experiments. Nevertheless, the pathology of the tumors indicated lower rates of apoptosis as compared to tumors with Myc overexpression alone. Three recent studies contributed towards our understanding of the oncogenic potential of miR-17-92. Two reports demonstrated an anti-apoptotic effect of miR-17-92 through various pathways that promote cell proliferation and growth [55, 76]. A third study identified mir-17-92 as a mediator of angiogenesis in tumors induced by the oncogene c-Myc [19]. ODonnell et al. [63] independently identified the same cluster of miRNAs, mir-17-92, to be regulated by the transcription factor c-Myc. The transcription factor Myc induces expression of E2F1 growth factor. The mir-17-92 cluster which is also induced by c-Myc does, in contrast, inhibit E2F1 expression. The authors therefore suggested a novel regulatory mechanism by which c-Myc fine-tunes gene expression by activating the transcription of target genes and by simultaneously inducing inhibitory miRNAs that reduce their translation. The example of the mir-17-92 cluster highlights that a distinction between oncogenic and tumor suppressor miRNAs is likely to be an oversimplification. The same miRNAs may have oncogenic or tumor suppressor activity depending on the context and the cell type they are expressed in. A single miRNA may regulate various unrelated target genes and thereby control opposing activities such as cellular proliferation and apoptosis. The ultimate function of a miRNA may depend on the tissue type they are expressed in and what target genes are present.
MicroRNAs with tumor suppressor potential

The let-7 family of miRNAs was the first group of miRNAs shown to regulate expression of a proto-oncogene, the RAS protein. RAS proteins are membrane-associated signaling proteins that regulate cell growth and differentiation. A miRNA that controls expression of these potentially oncogenic proteins would be predicted to possess tumor suppressor activity. Mutations in the RAS oncogene are present in approximately 1530% of all human cancers, and overexpression of the RAS oncogene is common in lung cancer. Johnson et al. [32] showed that overexpression of RAS protein in lung cancer tissue correlated with reduced expression of let-7 miRNA. They experimentally confirmed that let-7 can inhibit RAS expression in human cancer cell lines. Loss or reduction of let-7 in lung cancer leads to RAS overexpression, thus, promoting cellular growth and contributing to tumorigenesis. The authors therefore suggested that let-7 acts as tumor suppressor [32]. Another group independently reported reduced expression of let-7 in lung cancers and found that this correlated with a poor prognosis [77].

Global loss of miRNA expression in cancer

A global decrease in miRNA levels has been observed in human cancers, indicating that small RNAs may have an intrinsic function in tumor suppression. Lu et al. [52] were the first to show that the expression levels of many miRNAs were significantly reduced in cancers compared to the corresponding normal tissues. They analyzed a total of 217 human and mouse miRNAs across 334 human cancers, cancer cell lines, and normal tissues. Cancers had significantly reduced global miRNA expression. Poorly differentiated tumors had lower miRNA levels compared with more-differentiated tumors. The authors hypothesized that miRNAs can function to drive terminal differentiation and prevent cell division. Global changes in miRNA expression may reflect the degree of cell differentiation [52]. A recent study examined the expression of 241 human miRNAs in a comprehensive panel of human cancer cell lines, the NCI-60 panel, and in normal tissues [24]. The authors confirmed the finding that most miRNAs were expressed at lower levels in human tumorderived cell lines compared with the corresponding normal tissue [24]. Until recently, considerable uncertainty remained as to whether the altered miRNA expression observed in cancer was a cause or consequence of malignant transformation. Earlier this year, a study by Kumar et al. [37] proved for the first time that widespread reduction in miRNA expression does, indeed, promote tumorigenesis. The authors globally reduced the production of mature miRNAs through a knockdown of the miRNA-processing enzymes Drosha and Dicer in cell lines. The mouse and human cancer cells consequently showed decreased steady-state miRNA levels. These cells with global miRNA loss showed enhanced cellular growth in vitro [37]. When injected into nude mice, these cells generated faster growing and more invasive tumors compared to controls. To assess the effect of global miRNA loss in vivo, the authors deleted the miRNA-processing enzyme Dicer in a mouse model of lung cancer. The Dicer mutant mice who had impaired miRNA processing developed an increased tumor burden, with an expansion in tumor number and tumor size, as well as tumors which were less well differentiated compared to controls [37]. Overall, these data clearly suggest that global miRNA loss enhances tumorigenesis. Kumar et al. demonstrated that loss of miRNAs leads to upregulation of proto-oncogenes such as RAS and c-Myc. However, it remains to be elucidated whether loss of all miRNAs is necessary or whether reduction of a subgroup of key tumor suppressor miRNAs, such as let-7, is the event that promotes malignant transformation.
MicroRNAs in the p53 tumor suppressor network

Transcriptional networks are often deregulated in cancer cells and may lead to altered transcription of miRNA genes. Two recent studies identified a miRNA, miR-34, to be regulated by the p53 transcription factor [16, 26]. The p53 protein, also called the guardian of the genome, regulates the cellular response to stress and cancer-initiating events such as

DNA damage. He et al. [26] found that a miRNA, miR-34, is directly activated by the transcription factor p53 after DNA damage. Expression of miR-34 induces cell cycle arrest and thereby acts together with other effectors of the p53 tumor suppressor network to inhibit inappropriate cell proliferation. Another group independently demonstrated that miR-34 is upregulated by p53 upon DNA damage and promotes apoptosis [16]. Together, these data indicate that altered expression of miRNAs is not simply a secondary event that reflects the less differentiated state of cancer cells. In contrast, at least in some cases, miRNA expression is specifically driven by tumor suppressors and oncogenes.
MicroRNAs with a role in tumor invasion and metastasis

Transcriptional networks may drive miRNA expression in cancers. Recent work from Ma et al. [54] suggested a model by which a pleiotropic transcription factor, Twist, induces expression of a specific miRNA, which suppresses its direct target and in turn activates a pro-metastatic gene, leading to tumor cell invasion and metastasis. The expression of miR10b induced by the transcription factor Twist promoted cell migration and invasion in mouse and human breast cancer cells. Furthermore, the expression level of miR-10b in primary human breast carcinomas correlated with clinical progression [54]. These findings, if confirmed, suggest that specific miRNAs may have a role beyond the tumor-initiating event and directly participate in tumor progression and metastasis.
Regulation of miRNAs in cancerwho regulates the regulators?

In few cases, the underlying cause of miRNA deregulation in cancer is clear. As discussed above, the overexpression of miR-17-92 correlates with amplification of its gene locus [27]. Similarly, decreased expression of miR-15 and miR-16 is associated with a corresponding chromosomal deletion [13]. Transcriptional or epigenetic regulation of miRNAs has been recently reported [53, 73]. The transcription of a miRNA gene, mir-124a, was shown to be inactivated by hypermethylation of its promoter in various human tumors. This process of epigenetic silencing is a wellknown mechanism to inactivate protein-coding genes in cancer cells and may similarly apply to miRNAs. The miRNA genemir-127 is usually expressed in normal cells but not in cancer cells. Saito et al. [73] demonstrated that miR-127 was highly induced in cultured human cancer cells after treatment with demethylating drugs, suggesting that it is subject to epigenetic silencing through promoter hypermethylation. A novel mechanism of miRNA regulation was suggested by Mayr et al. [56] and Lee and Dutta [44]. They demonstrated that miRNA function could be regulated through loss of miRNA binding sites in the target gene. Both groups independently demonstrated that chromosomal translocations in a known oncogene, high mobility group A2 (Hmga2), led to

loss of the let-7 miRNA binding sites in its messenger RNA. Disrupted repression of Hmga2 by let-7 promoted oncogenic transformation and growth in mammalian cells. These two studies provide the first evidence that disrupting the interaction of a single miRNA and its target can produce an abnormal phenotype in mammalian cells [44, 56]. In addition, there is evidence that miRNAs are regulated indirectly through control of their processing enzymes. Thomson et al. [81] showed that a downregulation of miRNAs in human cancer was not associated with reduced levels of the primary miRNA transcripts. The authors therefore suggested regulation of miRNAs during subsequent processing steps, e.g., through altered function of the enzyme Drosha [81].
MicroRNA profilingimplications for cancer diagnosis

Lu et al. [52] asked the question whether global miRNA expression profiles could classify human cancer. MicroRNA expression profiles clearly differentiated human cancers according to their developmental origin. Cancers of epithelial and hematopoietic origin had distinct miRNA profiles. A subgroup of gastrointestinal tumors, which arise from endoderm, was distinguished by miRNA expression patterns. Furthermore, tumors within a single cell lineage such as acute lymphoblastic leukemia were further differentiated according to their underlying genetic abnormality into BCR/ABL-positive tumors, T-cell tumors, and those with MLL gene rearrangement [52]. Finally, the authors applied the miRNA expression profiles they had established to an independent series of 17 poorly differentiated tumors of unknown origin. Based on the differential expression of 217 miRNAs, a correct diagnosis could be established in 12 out of 17 of the tumors. In contrast, gene expression profiling based on 16,000 messenger RNAs did not accurately classify the tumors [52]. This has potential important clinical implications. If miRNAs prove useful for clinical diagnosis, their key advantage might be their high stability. In contrast to most messenger RNAs, they are long-lived in vivo [49] and very stable in vitro [78], which might allow analysis of paraffin-embedded samples for routine diagnostic applications. Go to:

MicroRNAsnovel therapeutic targets?

Regulatory RNAs may also have therapeutic applications by which disease-causing miRNAs could be antagonized or functional miRNAs restored. The most intuitive choice of molecules to correct altered miRNAmessenger RNA interactions are RNA oligonucleotides. These oligonucleotides need to be chemically modified to allow for stability in serum and cellular uptake. Modified antisense oligonucleotides are already being developed to utilize the

intrinsic RNAi pathway for delivery of gene therapy. If the delivery problem can be overcome, then miRNA therapies may also be possible. Two studies have successfully applied 2-O-Methyl-modified antisense RNAs to inhibit miRNA function in cultured cells [29, 57]. Recent work by Krutzfeldt et al. [36] demonstrated that modified cholesterol-conjugated antisense RNAs designated antagomirs could effectively inhibit miRNA function in vivo in the adult mouse. The authors applied three daily intravenous injections of antagomirs and achieved effective inhibition of four miRNAs over a period of weeks in most tissues except brain [36]. A novel approach was recently reported by Ebert et al. [21]. They developed miRNA inhibitors that can be transiently expressed in cultured mammalian cells. These competitive inhibitors termed miRNA sponges derepressed miRNA targets at least as strongly as chemically modified antisense oligonucleotides [21]. A different approach was taken by Tsuda et al. [83]. The authors designed synthetic miRNAs to target overexpressed tumor proteins, such as HER-2 protein. A synthetic miRNA targeting HER-2 messenger RNA successfully inhibited HER-2 protein expression in ovarian cancer cells [83]. Together, these studies hold some promise of miRNAs as future therapeutic targets. One limitation of antisense RNA therapies is the restricted number of cells that can be targeted. Any approach to knock down a particular miRNA with antisense oligonucleotides will only result in partial knockdown. This may represent a limitation for cancer therapies. It remains to be seen whether indirectly mediated bystander effects on cancer cells that have not been directly targeted may partly overcome this limitation. In contrast, a partial effect on function may be of therapeutic value in neurodegenerative diseases, such as Parkinsons or Alzheimers disease. A partial restoration of dopamine production by antisense therapy might result in a significant clinical improvement in Parkinson patients. Similarly, a partial reduction of the disease-causing proteins in Alzheimers disease may lead to a clinical improvement and might be achievable by RNA based or miRNA gene therapy. Go to:

Techniques and approaches to study miRNAs

All known miRNAs are registered in a public web-based registry, the miRBase database that provides up-to-date information on all published miRNAs [25]. Novel miRNA genes can be discovered by bioinformatics approaches searching for evolutionary conserved stemloop structures in the genome (reviewed in [3, 5]). Experimentally, miRNAs are discovered by cloning all small RNAs from a certain tissue type or developmental stage and subsequent sequencing to identify the subgroup of small RNAs that fulfill the criteria for miRNAs [38, 51]. Both computational and experimental approaches indicate that many more miRNAs are likely to be identified [4, 5], which is reflected by the rapidly increasing number

of annotated miRNAs which increased from less than 300 to more than 4,000 over the past 4 years [58].
MicroRNA expression studies

Northern blot analysis is a well-established technique for studying messenger RNA expression and was soon adapted to detect miRNAs in cells or tissues [42, 84]. Subsequently, conventional DNA microarray technology was modified to form miRNA microarrays, allowing for the detection of multiple miRNAs simultaneously across various samples [15, 60, 62, 82]. Lu et al. [52] developed a novel microarray strategy to improve probe specificity, which is critical due to the short nature of mature miRNAs. They performed hybridization in solution using polystyrene capture beads that are coupled to oligonucleotide probes complementary to the miRNAs of interest. The solution hybrids are then analyzed using a multicolor flow cytometer measuring bead color, denoting miRNA identity, and labeling intensity, denoting miRNA abundance [52]. In parallel to microarray platforms, commercial assays for quantitative reverse transcriptase polymerase chain reaction (RT-PCR) have become available. These allow for the analysis of miRNAs in small tissue samples or even single cells [79], as well as validation of microarray data. In addition to mature miRNAs, these quantitative RT-PCR assays can be applied to analyze miRNA precursors and primary transcripts [31]. In situ hybridization for the detection of mature miRNAs has recently become possible by using special high-affinity locked nucleic acid (LNA)-modified DNA oligonucleotide probes and holds promise for the application on human formalin-fixed and paraffin embedded tissue [35, 61].
Functional characterization of miRNAs

Various strategies have been used to investigate the function of specific miRNAs. In worms and flies, loss-of-function mutants for specific miRNAs or miRNA families allow us to draw conclusions regarding possible physiological functions of miRNAs from the resulting abnormal phenotype [34, 39,86]. The knockdown of miRNAs or pre-miRNAs using modified antisense oligonucleotides has proven particularly useful in cell lines [29, 45, 57]. LNA-modified antisense oligonucleotides have been successfully utilized to knock down specific miRNAs in cultured cells [22]. This approach allowed identification of a crucial role for a miRNA, miR-223, in granulocytic differentiation [22]. In addition, the modified antisense RNAs (antagomirs) described by Krutzfeldt et al. [36], which inhibit miRNA function in the adult mouse, may provide a potential research tool to study miRNA function in vivo.

In mammals, induced defects in miRNA biogenesis are a useful tool for investigating the biological roles of miRNAs, as loss-of-function mutants are not available for most miRNA genes. Dicer knockout mouse models have revealed essential roles for miRNAs in murine organogenesis [88]. A recent study utilized a combined knockdown of the miRNAprocessing enzymes Drosha, Dicer1, and DGCR8 to study the consequences of a global decrease in mature miRNAs in cancer cell lines and in a mouse model for lung cancer [37]. Earlier this year, four independent groups have, for the first time, deleted genes for single miRNAs in mice [72, 80, 85, 88]. Two of the groups deleted the same DNA sequence for mir-155 and described severe immune defects [72, 80]. Mice lacking miR-155 showed impaired function of B and T lymphocytes and dendritic cells [72]. In particular, T helper cell differentiation and the germinal center reaction to produce a T-cell-dependent antibody response were defective [80]. Together, these two studies demonstrated a key role for miR155 in normal immune function. The two other groups deleted different miRNAs, miR-1-2 and miR-208, and reported cardiac defects. Mice lacking miR-208 showed inadequate cardiac growth in response to stress [85], while mice lacking miR-1-2 had defects in cardiac morphogenesis and electrical conduction [88].
MicroRNA target sites

A validated biochemical strategy for identifying miRNA targets would be highly desirable. Two groups have recently reported promising approaches to experimentally identify miRNA targets. Both approaches apply biochemical methods to purify the effector complexes of miRNAs associated with proteins and bound messenger RNA targets [2, 20]. An increasing number of sophisticated bioinformatics approaches are being developed to predict putative miRNA target genes [3, 10, 69, 74]. This is based on the fact that miRNA target recognition is at least partly based on simple sequence complementarity. Interestingly, exact base pairing between miRNAs and their targets commonly appears to be required only in the first six to eight bases from the 5 end of the miRNA. The short nature of this designated seed region allows a single miRNA to act on up to a hundred different target sites, and all human miRNAs together may regulate up to one third of protein coding genes [10, 47]. A different approach to discovering miRNA target genes is to knock out or overexpress a particular miRNA and use conventional microarrays to identify genes that show changes in expression. This approach is based on the observation that some miRNAs can also downregulate messenger RNA levels in addition to downregulating protein levels of their target genes [49]. Experimental validation of miRNA target sites has been limited to date. A common approach has been to express a miRNA in vivo while simultaneously expressing and monitoring the target messenger RNA linked to a reporter gene, i.e., Luciferase [10, 18, 55, 56, 76]. The fact that a single miRNA can regulate multiple targets

and a particular target may be regulated by various miRNAs suggests a highly complex network of miRNA-target interactions, which is only beginning to be unraveled. Go to:

Conclusions
Over recent years, miRNAs have emerged as major players in the complex networks of gene regulation and have been implicated in various aspects of human disease. Only 5 years after the first study reported a direct involvement of miRNAs in cancer, these small RNAs have already significantly improved our understanding of carcinogenesis. In addition to proteincoding oncogenes and tumor suppressor genes, we will have to take into account miRNAs and their regulatory networks if we aim to understand the complex processes underlying malignant transformation.