miRNA Processing Machinery Research Articles

A role for the cancer-associated miR-106b~25 cluster in neuronal stem cells

A role for the cancer-associated miR-106b~25 cluster in neuronal stem cells

Impaired MicroRNA Processing Facilitates Breast Cancer Cell Invasion by Upregulating Urokinase-Type Plasminogen Activator Expression

Global mature microRNA (miRNA) expression is downregulated in cancers, and impaired miRNA processing enhances cancer cell proliferation. These findings indicate that the miRNA system generally serves as a negative regulator during cancer progression. In this study, we investigated the role of the miRNA system in cancer cell invasion by determining the effect of damaging miRNA processing on invasion-essential urokinase-type plasminogen activator (uPA) expression in breast cancer cells. Short hairpin RNAs specific for Drosha, DGCR8, and Dicer, key components of miRNA processing machinery, were introduced into 2 breast cancer cell lines with high uPA expression and 2 lines with poor uPA expression. Knockdown of Drosha, DGCR8, or Dicer led to even higher uPA expression in cells with high uPA expression, while it was unable to increase uPA level in cells with poor uPA expression, suggesting that the miRNA system most likely impacts uPA expression as a facilitator. In cells with high uPA expression, knockdown of Drosha, DGCR8, or Dicer substantially increased in vitro invasion, and depleting uPA abrogated enhanced invasion. These results thus link the augmented invasion conferred by impaired miRNA processing to upregulated uPA expression. uPA mRNA was a direct target of miR-193a/b and miR-181a, and a higher uPA level in cells with impaired miRNA processing resulted from less mature miR-193a/b and miR-181a processed from their respective primary miRNAs. Importantly, the levels of mature miR-193a, miR-193b, and miR-181a, but not their respective primary miRNAs, were lower in high uPA-expressing cells compared to cells with low uPA expression, and this apparently attributed to lower Drosha/DGCR8 expression in high uPA-expressing cells. This study suggests that less efficient miRNA processing can be a mechanism responsible for reduced levels of mature forms of tumor-suppressive miRNAs frequently detected in cancers.

The NK Cell MicroRNA Transcriptome Defined by Next-Generation Sequencing Identifies IL-15-Signaled Alterations In Mature MiR-223 Expression, and MiR-223 as a Potential Regulator of Murine Granzyme B

AbstractAbstract 104Natural killer (NK) cells are innate lymphocytes important for early host defense against infectious pathogens and surveillance against malignant transformation. Resting murine NK cells regulate the translation of effector molecule mRNAs (e.g. granzyme B, GzmB) through unclear molecular mechanisms. MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate the translation of their mRNA targets, and are therefore candidates mediating this control process. While the expression and importance of miRNAs in T and B lymphocytes has been established, little is known about miRNAs in NK cells. Here, we utilized two next-generation sequencing platforms, Illumina GA and SOLiD, to define the miRNA transcriptomes of resting and IL-15-activated primary murine NK cells. Using Illumina GA sequencing, we identified expression of 297 mature miRNA and 64 miRNA* sequences in NK cells at read counts of 1 to 507,369. The 5 miRNAs with the highest abundance (percent normalized read count) in resting NK cells were miR-21 (31.6%), miR-16 (8.3%), miR-142-5p (6.5%), miR-142-3p (3.8%), miR-24 (3.7%). Detection of known miRNA was similar using SOLiD sequencing in both resting (r2=0.79) and IL-15-activated (r2=0.75) NK cells, with most of the discordant detection arising in miRNAs with low expression (1-10 read counts). These results were further validated using real-time RT-qPCR and microarray profiling. A bioinformatics pipeline was developed that identified 37 novel miRNA genes that encoded 26 novel mature miRNA sequences, including miRNAs processed from snoRNAs. Of 84 miRNAs with significantly altered expression levels (Illumina, normalized read counts) following IL-15-activation, miR-223 demonstrated the greatest fold decrease (2.3, P=1.03E-10). The decrease in mature miR-223 following 24 hours of IL-15 activation was confirmed in SOLiD sequencing (7.7 fold-decrease, P=1.0E-10), qPCR (2.5 fold-decrease, P=0.01), and microarrays (4.6 fold-decrease, P=0.003). IL-15 induced reduction in mature miR-223 was time-dependent with fold decreases of 1.5 (NS) at 8 hours, 2.2 (P=0.003) at 24 hours, and 8.1 (P=0.012) at 48 hours, assessed by real-time qPCR. Decreases in mature miR-223 expression early following activation likely occur via regulation of the miRNA processing machinery, since primary and precursor miR-223 transcripts exhibited increased expression after 8 hours (2.1±0.3 fold increase, P=0.03) and 24 hours (2.5±0.3 fold increase, P=0.03) of IL-15-activation. MiR-223 is computationally predicted to bind to the murine GzmB 3'UTR sequence, and we therefore evaluated the ability of miR-223 to target the murine GzmB 3'UTR in vitro. Overexpression of miR-223 in 293T cells decreased luciferase protein signal controlled by the GzmB 3'UTR by 44±2% (P=0.002) without affecting negative controls (perforin 3'UTR or no 3'UTR). Moreover, mutating the miR-223 predicted binding site within the murine GzmB 3'UTR abrogated miR-223 mediated regulation. These data suggest that miR-223 directly targets the murine GzmB 3'UTR, and thereby contributes to tonic suppression of GzmB mRNA translation in resting NK cells. Thus, the sequenced and validated miRNA transcriptome provides a valuable framework for further elucidation of miRNA expression and function in NK cell biology. Disclosures:No relevant conflicts of interest to declare.

Downregulation of Dicer enhances tumor cell proliferation and invasion

miRNAs are non-coding, single-stranded RNAs that regulate target gene expression by repressing translation or promoting RNA cleavage. Dicer is an essential component of the miRNA processing machinery. To identify a role for miRNAs in tumorigenesis, we designed an adenovirus expressing small hairpin RNA (shRNA) to silence Dicer and globally suppress the maturation of miRNAs. We identified that the impairment of miRNA processing conferred an enhanced proliferative activity and invasive ability on each of three tumor cell lines in vitro. Inhibition of Dicer was associated with activation of p-Akt and enhanced expression of the cell cycle associating molecules, cyclin A and PCNA, as well as MMP-2 and MMP-9, proteins involved in tumor cell invasion. Adenoviral gene silencing of Dicer in subcutaneous MCF-7 xenografts significantly increased tumor growth in vivo compared to tumors infected with non-loading adenovirus. Increased tumor growth was associated with p-Akt activation and upregulation of cyclin A, PCNA MMP-2 and MMP-9. These findings demonstrate that global reduction of miRNA processing by silencing Dicer enhances tumor proliferation and invasion, and the p-Akt pathway may contribute to this phenotype via the downstream molecules, cyclin A, PCNA, MMP-2 and MMP-9.

A MicroRNA Targeting Dicer for Metastasis Control

Although specific microRNAs (miRNAs) can be upregulated in cancer, global miRNA downregulation is a common trait of human malignancies. The mechanisms of this phenomenon and the advantages it affords remain poorly understood. Here we identify a microRNA family, miR-103/107, that attenuates miRNA biosynthesis by targeting Dicer, a key component of the miRNA processing machinery. In human breast cancer, high levels of miR-103/107 are associated with metastasis and poor outcome. Functionally, miR-103/107 confer migratory capacities in vitro and empower metastatic dissemination of otherwise nonaggressive cells in vivo. Inhibition of miR-103/107 opposes migration and metastasis of malignant cells. At the cellular level, a key event fostered by miR-103/107 is induction of epithelial-to-mesenchymal transition (EMT), attained by downregulating miR-200 levels. These findings suggest a new pathway by which Dicer inhibition drifts epithelial cancer toward a less-differentiated, mesenchymal fate to foster metastasis.

The tumor suppressors p53, p63, and p73 are regulators of microRNA processing complex.

The tumor suppressors p53, p73, and p63 are known to function as transcription factors. They promote either growth arrest or apoptosis, depending upon the DNA damage. A number of microRNAs (miRNAs) have been shown to function as transcriptional targets of p53 and they appear to aid p53 in promoting growth arrest and apoptosis. However, the question of p53/p63/p73 regulating the miRNA processing complex has not been addressed in depth so far. Comparative/computational genomic analysis was performed using Target scan, Mami, and Diana software to identify miRNAs that regulate the miRNA processing complex. Here, I present evidence for the first time that the tumor suppressors p53, p63, and p73 function as both positive and negative regulators of the miRNA processing components. Curated p53-dependent miRNA expression data was used to identify p53-miRs that target the components of the miRNA-processing complex. This analysis suggests that most of the components (mRNAs' 3′UTR) of the miRNA processing complex are targeted by p53-miRs. Remarkably, this data revealed the conserved nature of p53-miRs in targeting a number of components of the miRNA processing complex. p53/p73/p63 appears to regulate the major components of the miRNA processing, such as Drosha-DGCR8, Dicer-TRBP2, and Argonaute proteins. In particular, p53/p73/p63 appears to regulate the processing of miRNAs, such as let-7, miR-200c, miR-143, miR-107, miR-16, miR-145, miR-134, miR-449a, miR-503, and miR-21. Interestingly, there seems to be a phenotypic similarity between p63−/− and dicer−/− mice, suggesting that p63 and dicer could regulate each other. In addition, p63, p73, and the DGCR8 proteins contain a conserved interaction domain. Further, promoters of a number of components of the miRNA processing machinery, including dicer and P2P-R, contain p53-REs, suggesting that they could be direct transcriptional targets of p63/p73/p53. Together, this study provides mechanistic insights into how p53, p63, and p73 regulate the components of the miRNA processing; and how p53, TA-p63, and TA-p73 regulated miRNAs inhibit tumorigenesis, EMT, metastasis, and cancer stem cell proliferation.

Abstract 2068: Profile, target genes and regulation of microRNAs in ovarian carcinoma tumor progression

Abstract MicroRNAs (miRNAs) are small non-coding RNAs, that exert their regulatory effect post-transcriptionally by binding the 3’-UTR of their target mRNA and inhibiting gene translation to protein. Depending on whether miRNAs target oncogenes or tumor suppressor genes, they may be referred to as tumor suppressors or oncogenes respectively. Cellular miRNA expression is tightly regulated. One of the post-transcriptional regulatory mechanisms involves changes in expression of miRNA machinery proteins, i.e., Dicer, and miRISC components such as the Argonaute (Ago) family members. Numerous studies, using different profiling approaches, have unraveled that miRNA expression is deregulated in various human cancers. Ovarian cancer is the leading cause of death from gynecological cancers in western countries. The disease is asymptomatic in the early stages, and is usually diagnosed at an advance stage. Primary solid tumors, solid metastases, and effusions to the peritoneal and (ascites) pleural cavities characterize the tumor as it progresses. The aim of the study was to characterize the difference in miRNA expression pattern between primary ovarian solid carcinomas and effusion-derived cells, using fresh-frozen samples. We also assessed changes in regulation of miRNA by evaluating the expression of the machinery proteins at these two sites. Using microRNA-array platforms, we identified three sets of miRNAs: one set is highly expressed in both primary solid carcinomas and effusions. The second set is relatively elevated in effusions, and the third set is relatively reduced in effusions. The most significant miRNAs were validated by real-time PCR. Our results show concordance between the training and the independent test cohorts for the reduced miR-145 and miR-214 and for the elevated let-7f, miR-182, miR-210, miR-200c, miR-222 and miR-23a in effusions. Using in-silico target prediction programs we identified potential target genes for the miRNAs of interest listed above, we investigated the changes of those genes in our cohort. We analyzed the expression levels of Zeb1, a confirmed target of miR-200c as well as c-Myc, that was found to be a predicted target of miR-200c. In addition, we analyzed Pak1 and PTEN, both predicted targets of miR-222. We found inverse correlations between the expression levels of the indicated miRNAs and of the predicted target genes. We further analyzed the miRNA processing machinery genes that regulate miRNA generation and action. We found significantly higher expression of Ago1, Ago2 and Dicer in effusion-derived cells compared to primary carcinoma tumors. These alterations in expression levels indicate on difference in miRNA regulation between the two sites. In summary, miRNA expression profiles and the changes in miRNA processing machinery genes reveal a new level of regulatory elements in ovarian carcinoma tumor progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2068.

Alternative Processing of Primary microRNA Transcripts by Drosha Generates 5′ End Variation of Mature microRNA

BackgroundIt is generally believed that the miRNA processing machinery ensures the generation of a mature miRNA with a fixed sequence, particularly at its 5′ end. However, we and others have recently noted that the ends of a given mature miRNA are not absolutely fixed, but subject to variation. Neither the significance nor the mechanism behind the generation of such miRNA polymorphism is understood. miR-142 is an abundantly expressed miRNA in hematopoietic cells and exhibits a high frequency of 5′ end polymorphism.Methodology/Principal FindingsHere we show that a shift in the Drosha processing of pri-miRNA generates multiple forms of miR-142s in vivo with differing 5′ ends that might target different genes. Sequence analysis of several pre-miRNA ends cloned from T cells reveals that unlike many other pri-miRNAs that are processed into a single pre-miRNA, pri-miR-142 is processed into 3 distinct pre-miR-142s. Dicer processing studies suggest that each of the 3 pre-miR-142s is processed into a distinct double-stranded miRNA, giving rise to 4 mature miRNA variants that might regulate different target gene pools.Conclusions/SignificanceThus, alternative Drosha processing might be a novel mechanism for diversification of the miRNA target gene pool.

Sequence features associated with microRNA strand selection in humans and flies

BackgroundDuring microRNA (miRNA) maturation in humans and flies, Drosha and Dicer cut the precursor transcript, thereby producing a short RNA duplex. One strand of this duplex becomes a functional component of the RNA-Induced Silencing Complex (RISC), while the other is eliminated. While thermodynamic asymmetry of the duplex ends appears to play a decisive role in the strand selection process, the details of the selection mechanism are not yet understood.ResultsHere, we assess miRNA strand selection bias in humans and fruit flies by analyzing the sequence composition and relative expression levels of the two strands of the precursor duplex in these species. We find that the sequence elements associated with preferential miRNA strand selection and/or rejection differ between the two species. Further, we identify another feature that distinguishes human and fly miRNA processing machinery: the relative accuracy of the Drosha and Dicer enzymes.ConclusionOur result provides clues to the mechanistic aspects of miRNA strand selection in humans and other mammals. Further, it indicates that human and fly miRNA processing pathways are more distinct than currently recognized. Finally, the observed strand selection determinants are instrumental in the rational design of efficient miRNA-based expression regulators.

MiRNA in the Regulation of Skeletal Muscle Adaptation to Acute Endurance Exercise in C57Bl/6J Male Mice

MicroRNAs (miRNAs) are evolutionarily conserved small non-coding RNA species involved in post-transcriptional gene regulation. In vitro studies have identified a small number of skeletal muscle-specific miRNAs which play a crucial role in myoblast proliferation and differentiation. In skeletal muscle, an acute bout of endurance exercise results in the up-regulation of transcriptional networks that regulate mitochondrial biogenesis, glucose and fatty acid metabolism, and skeletal muscle remodelling. The purpose of this study was to assess the expressional profile of targeted miRNA species following an acute bout of endurance exercise and to determine relationships with previously established endurance exercise responsive transcriptional networks. C57Bl/6J wild-type male mice (N = 7/group) were randomly assigned to either sedentary or forced-endurance exercise (treadmill run @ 15 m/min for 90 min) group. The endurance exercise group was sacrificed three hours following a single bout of exercise. The expression of miR- 181, 1, 133, 23, and 107, all of which have been predicted to regulate transcription factors and co-activators involved in the adaptive response to exercise, was measured in quadriceps femoris muscle. Endurance exercise significantly increased the expression of miR-181, miR-1, and miR-107 by 37%, 40%, and 56%, respectively, and reduced miR-23 expression by 84% (P≤0.05 for all), with no change in miR-133. Importantly, decreased expression of miRNA-23, a putative negative regulator of PGC-1α was consistent with increased expression of PGC-1α mRNA and protein along with several downstream targets of PGC-1α including ALAS, CS, and cytochrome c mRNA. PDK4 protein content remains unaltered despite an increase in its putative negative regulator, miR-107, and PDK4 mRNA expression. mRNA expression of miRNA processing machinery (Drosha, Dicer, and DGCR8) remained unchanged. We conclude that miRNA-mediated post-transcriptional regulation is potentially involved in the complex regulatory networks that govern skeletal muscle adaptation to endurance exercise in C57Bl/6J male mice.

Neuron‐specific RNA interference using lentiviral vectors

Viral vectors have been used in several different settings for the delivery of small hairpin (sh) RNAs. However, most vectors have utilized ubiquitously-expressing polymerase (pol) III promoters to drive expression of the hairpin as a result of the strict requirement for precise transcriptional initiation and termination. Recently, pol II promoters have been used to construct vectors for RNA interference (RNAi). By embedding the shRNA into a micro RNA-context (miRNA) the endogenous miRNA processing machinery is exploited to achieve the mature synthetic miRNA (smiRNA), thereby expanding the possible promoter choices and eventually allowing cell type specific down-regulation of target genes. In the present study, we constructed lentiviral vectors expressing smiRNAs under the control of pol II promoters to knockdown gene expression in cell culture and in the brain. We demonstrate robust knockdown of green fluorescent protein using lentiviral vectors driving RNAi from the ubiquitously-expressing promoter of the cytomegalovirus (CMV) and, in addition, we show for the first time neuron-specific knockdown in the brain using a neuron-specific promoter. Furthermore, we show that the expression pattern of the presumed ubiquitously-expressing CMV promoter changes over time from being expressed initially in neurons and glial cells to being expressed almost exclusively in neurons in later stages. In the present study, we developed vectors for cell-specific RNAi for use in the brain. This offers the possibility of specifically targeting RNAi to a subset of cells in a complex tissue and may prove to be of great importance in the design of future gene therapeutic paradigms.

Alterations in miRNA processing and expression in pleomorphic adenomas of the salivary gland

Genome-wide microRNA (miRNA) expression profiling of salivary gland pleomorphic adenomas revealed a distinct expression signature consisting largely of upregulated miRNAs compared with matched normal tissue. Microarray data were confirmed by quantitative real time RT-PCR (q-RTPCR). Five miRNA genes upregulated in the tumours were found in close proximity to fragile sites and/or cancer associated genomic regions. Interestingly, q-RTPCR revealed an increase in the expression of components of the miRNA processing machinery (Dicer, Drosha, DGCR8 and p68) in tumours suggesting that the deregulation of miRNA expression may result from increased miRNA biogenesis. Target gene prediction analysis of the altered miRNAs indicated that genes in a number of signalling pathways important in tumourigenesis including WNT, MAPK and JAK-STAT were overrepresented. Significantly, the oncogene PLAG1 was overexpressed in our cohort and may be potentially regulated by these miRNAs. This is the first study to examine changes in the miRNA milieu in pleomorphic adenoma, the most common salivary gland tumour. This study has demonstrated an upregulation of both miRNAs genes and an upregulation of the miRNA processing machinery. These changes may be potential underlying mechanisms for the development of these benign tumours.

A distinct class of small RNAs arises from pre-miRNA–proximal regions in a simple chordate

MicroRNAs (miRNAs) have been implicated in various cellular processes. They are thought to function primarily as inhibitors of gene activity by attenuating translation or promoting mRNA degradation. A typical miRNA gene produces a predominant approximately 21-nucleotide (nt) RNA (the miRNA) along with a less abundant miRNA(*) product. We sought to identify miRNAs from the simple chordate Ciona intestinalis through comprehensive sequencing of small RNA libraries created from different developmental stages. Unexpectedly, half of the identified miRNA loci encode up to four distinct, stable small RNAs. The additional RNAs, miRNA-offset RNAs (moRs), are generated from sequences immediately adjacent to the predicted approximately 60-nt pre-miRNA. moRs seem to be produced by RNAse III-like processing, are approximately 20 nt long and, like miRNAs, are observed at specific developmental stages. We present evidence suggesting that the biogenesis of moRs results from an intrinsic property of the miRNA processing machinery in C. intestinalis.

Expression patterns of microRNAs in the chorioamniotic membranes: a role for microRNAs in human pregnancy and parturition

MicroRNAs (miRNAs) are involved in the post-transcriptional regulation of gene expression during development. This study was performed to determine gestational age-dependent changes in miRNA expression in the chorioamniotic membranes and to assess the significance of miRNAs in human pregnancy and parturition. The expression profile of 455 miRNAs was compared between patients at term without labour (TNL: n = 10), in labour (TL: n = 10), and preterm labour (PTL: n = 10) using microarrays. A total of 39 miRNAs were differentially expressed between term and preterm cases, of which 31 (79.5%) were down-regulated at term. Expression of ten miRNAs, including miR-338, differentially expressed between PTL and TL groups was decreased at term. Computational analyses using miRBase Targets have identified PLA2G4B, a phospholipase implicated in parturition, as a putative target of miR-338. Inhibition of endogenous miR-338 with anti-miR-338 increased the mRNA and protein expression of PLA2G4B in decidual cells. Luciferase assay with reporter constructs confirmed that the suppression of PLA2G4B occurs through binding of miR-338 to the 3UTR of PLA2G4B. Interestingly, the expression of Dicer, a key miRNA-processing enzyme, was markedly decreased at term, particularly with labour in the chorioamniotic membranes. Collectively, the novel findings reported herein strongly suggest that post-transcriptional regulation of genes by miRNAs, coupled with the changes of miRNA processing machinery in the chorioamniotic membranes, plays a role in pregnancy and parturition. Furthermore, the expression level of Dicer in the chorioamniotic membranes dichotomizes pathological preterm labour and physiological spontaneous labour at term.

Stimuli-dependent cleavage of Dicer during apoptosis

miRNAs (microRNAs) play important roles in diverse physiological processes, including stress response, apoptosis and carcinogenesis. Even though the role of individual miRNAs has been demonstrated, expression of proteins involved in miRNA production in response to acute stress or harmful agents has not been extensively investigated. Here, we have studied the role of Dicer, one of the central proteins of the miRNA processing machinery during apoptosis, and show that down-regulation of Dicer results in accelerated apoptosis of HeLa cells, triggered by TNFalpha (tumour necrosis factor alpha). We have also investigated the integrity of Dicer, and provide evidence that Dicer is a target for caspases during apoptosis. The cleavage of Dicer is stimulidependent and more pronounced when apoptosis is induced by PKC (protein kinase C) inhibitors, and can also be observed in HIV-1-infected cells at late stages of infection. Thus the apoptotic machinery may regulate the miRNA pathway by affecting individual proteins, such as Dicer.

Potential role of miR-9 and miR-223 in recurrent ovarian cancer

BackgroundMicroRNAs (miRNAs) are small, noncoding RNAs that negatively regulate gene expression by binding to target mRNAs. miRNAs have not been comprehensively studied in recurrent ovarian cancer, yet an incurable disease.ResultsUsing real-time RT-PCR, we obtained distinct miRNA expression profiles between primary and recurrent serous papillary ovarian adenocarcinomas (n = 6) in a subset of samples previously used in a transcriptome approach. Expression levels of top dysregulated miRNA genes, miR-223 and miR-9, were examined using TaqMan PCR in independent cohorts of fresh frozen (n = 18) and FFPE serous ovarian tumours (n = 22). Concordance was observed on TaqMan analysis for miR-223 and miR-9 between the training cohort and the independent test cohorts. Target prediction analysis for the above miRNA "recurrent metastatic signature" identified genes previously validated in our transcriptome study. Common biological pathways well characterised in ovarian cancer were shared by miR-9 and miR-223 lists of predicted target genes. We provide strong evidence that miR-9 acts as a putative tumour suppressor gene in recurrent ovarian cancer. Components of the miRNA processing machinery, such as Dicer and Drosha are not responsible for miRNA deregulation in recurrent ovarian cancer, as deluded by TaqMan and immunohistochemistry.ConclusionWe propose a miRNA model for the molecular pathogenesis of recurrent ovarian cancer. Some of the differentially deregulated miRNAs identified correlate with our previous transcriptome findings. Based on integrated transcriptome and miRNA analysis, miR-9 and miR-223 can be of potential importance as biomarkers in recurrent ovarian cancer.

Impaired microRNA processing enhances cellular transformation and tumorigenesis

MicroRNAs (miRNAs) are a new class of small noncoding RNAs that post-transcriptionally regulate the expression of target mRNA transcripts. Many of these target mRNA transcripts are involved in proliferation, differentiation and apoptosis, processes commonly altered during tumorigenesis. Recent work has shown a global decrease of mature miRNA expression in human cancers. However, it is unclear whether this global repression of miRNAs reflects the undifferentiated state of tumors or causally contributes to the transformed phenotype. Here we show that global repression of miRNA maturation promotes cellular transformation and tumorigenesis. Cancer cells expressing short hairpin RNAs (shRNAs) targeting three different components of the miRNA processing machinery showed a substantial decrease in steady-state miRNA levels and a more pronounced transformed phenotype. In animals, miRNA processing-impaired cells formed tumors with accelerated kinetics. These tumors were more invasive than control tumors, suggesting that global miRNA loss enhances tumorigenesis. Furthermore, conditional deletion of Dicer1 enhanced tumor development in a K-Ras-induced mouse model of lung cancer. Overall, these studies indicate that abrogation of global miRNA processing promotes tumorigenesis.

MicroRNA signatures in human cancers

MicroRNA (miRNA) alterations are involved in the initiation and progression of human cancer. The causes of the widespread differential expression of miRNA genes in malignant compared with normal cells can be explained by the location of these genes in cancer-associated genomic regions, by epigenetic mechanisms and by alterations in the miRNA processing machinery. MiRNA-expression profiling of human tumours has identified signatures associated with diagnosis, staging, progression, prognosis and response to treatment. In addition, profiling has been exploited to identify miRNA genes that might represent downstream targets of activated oncogenic pathways, or that target protein-coding genes involved in cancer.

MicroRNA Pathway, Synaptic Function, and Disease

Fragile X-related protein (FMRP) is an RNA-binding protein whose loss in fragile X syndrome results in mental retardation. When incorporated into a messenger ribonucleoprotein, it is thought to associate with ribosomes and to regulate protein synthesis locally in neurons. The Drosophila ortholog of FMRP associates with an RNA-induced silencing complex (RISC) that includes a protein called Argonaute 2 to control the processing of microRNAs (miRNAs). These miRNAs are short, noncoding RNAs that target mRNA for destruction. However, a link between the effect of FMRP on miRNAs and fragile X syndrome has not been clear. Jin et al. report that mammalian FMRP also interacts with the miRNA processing machinery in normal human cells. MiRNAs and possible precursor transcripts were detected in immunoprecipitates of FMRP from normal human cells but not from cells of fragile X patients. Activity of Dicer, an RNAse that processes miRNA precursors, and the mammalian Argonaute protein eIF2C2 were also present in FMRP immunoprecipitates from normal human cells only. Overexpression of FMRP in the Drosophila eye results in apoptosis. However, additional reduction of Argonaute 1 suppressed this phenotype, indicating a genetic interaction. The authors propose that FMRP may exert translational suppression through associated miRNAs and RISC complexes on target mRNAs. Disruption of this mechanism in neurons could affect the local protein synthesis response to synaptic stimulation and affect neural development and maintenance. P. Jin, D. C. Zarnescu, S. Ceman, M. Nakamoto, J. Mowrey, T. A. Jongens, D. L. Nelson, K. Moses, S. T. Warren, Biochemical and genetic interaction between the fragile X mental retardation protein and the microRNA pathway. Nature Neurosci. 7 , 113-117 (2004). [Online Journal]