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1.  microRNA-122 as a regulator of mitochondrial metabolic gene network in hepatocellular carcinoma 
A moderate loss of miR-122 function correlates with up-regulation of seed-matched genes and down-regulation of mitochondrially localized genes in both human hepatocellular carcinoma and in normal mice treated with anti-miR-122 antagomir.Putative direct targets up-regulated with loss of miR-122 and secondary targets down-regulated with loss of miR-122 are conserved between human beings and mice and are rapidly regulated in vitro in response to miR-122 over- and under-expression.Loss of miR-122 secondary target expression in either tumorous or adjacent non-tumorous tissue predicts poor survival of heptatocellular carcinoma patients.
Hepatocellular carcinoma (HCC) is one of the most aggressive human malignancies, common in Asia, Africa, and in areas with endemic infections of hepatitis-B or -C viruses (HBV or HCV) (But et al, 2008). Globally, the 5-year survival rate of HCC is <5% and about 600 000 HCC patients die each year. The high mortality associated with this disease is mainly attributed to the failure to diagnose HCC patients at an early stage and a lack of effective therapies for patients with advanced stage HCC. Understanding the relationships between phenotypic and molecular changes in HCC is, therefore, of paramount importance for the development of improved HCC diagnosis and treatment methods.
In this study, we examined mRNA and microRNA (miRNA)-expression profiles of tumor and adjacent non-tumor liver tissue from HCC patients. The patient population was selected from a region of endemic HBV infection, and HBV infection appears to contribute to the etiology of HCC in these patients. A total of 96 HCC patients were included in the study, of which about 88% tested positive for HBV antigen; patients testing positive for HCV antigen were excluded. Among the 220 miRNAs profiled, miR-122 was the most highly expressed miRNA in liver, and its expression was decreased almost two-fold in HCC tissue relative to adjacent non-tumor tissue, confirming earlier observations (Lagos-Quintana et al, 2002; Kutay et al, 2006; Budhu et al, 2008).
Over 1000 transcripts were correlated and over 1000 transcripts were anti-correlated with miR-122 expression. Consistent with the idea that transcripts anti-correlated with miR-122 are potential miR-122 targets, the most highly anti-correlated transcripts were highly enriched for the presence of the miR-122 central seed hexamer, CACTCC, in the 3′UTR. Although the complete set of negatively correlated genes was enriched for cell-cycle genes, the subset of seed-matched genes had no significant KEGG Pathway annotation, suggesting that miR-122 is unlikely to directly regulate the cell cycle in these patients. In contrast, transcripts positively correlated with miR-122 were not enriched for 3′UTR seed matches to miR-122. Interestingly, these 1042 transcripts were enriched for genes coding for mitochondrially localized proteins and for metabolic functions.
To analyze the impact of loss of miR-122 in vivo, silencing of miR-122 was performed by antisense inhibition (anti-miR-122) in wild-type mice (Figure 3). As with the genes negatively correlated with miR-122 in HCC patients, no significant biological annotation was associated with the seed-matched genes up-regulated by anti-miR-122 in mouse livers. The most significantly enriched biological annotation for anti-miR-122 down-regulated genes, as for positively correlated genes in HCC, was mitochondrial localization; the down-regulated mitochondrial genes were enriched for metabolic functions. Putative direct and downstream targets with orthologs on both the human and mouse microarrays showed significant overlap for regulations in the same direction. These overlaps defined sets of putative miR-122 primary and secondary targets. The results were further extended in the analysis of a separate dataset from 180 HCC, 40 cirrhotic, and 6 normal liver tissue samples (Figure 4), showing anti-correlation of proposed primary and secondary targets in non-healthy tissues.
To validate the direct correlation between miR-122 and some of the primary and secondary targets, we determined the expression of putative targets after transfection of miR-122 mimetic into PLC/PRF/5 HCC cells, including the putative direct targets SMARCD1 and MAP3K3 (MEKK3), a target described in the literature, CAT-1 (SLC7A1), and three putative secondary targets, PPARGC1A (PGC-1α) and succinate dehydrogenase subunits A and B. As expected, the putative direct targets showed reduced expression, whereas the putative secondary target genes showed increased expression in cells over-expressing miR-122 (Figure 4).
Functional classification of genes using the total ancestry method (Yu et al, 2007) identified PPARGC1A (PGC-1α) as the most connected secondary target. PPARGC1A has been proposed to function as a master regulator of mitochondrial biogenesis (Ventura-Clapier et al, 2008), suggesting that loss of PPARGC1A expression may contribute to the loss of mitochondrial gene expression correlated with loss of miR-122 expression. To further validate the link of miR-122 and PGC-1α protein, we transfected PLC/PRF/5 cells with miR-122-expression vector, and observed an increase in PGC-1α protein levels. Importantly, transfection of both miR-122 mimetic and miR-122-expression vector significantly reduced the lactate content of PLC/PRF/5 cells, whereas anti-miR-122 treatment increased lactate production. Together, the data support the function of miR-122 in mitochondrial metabolic functions.
Patient survival was not directly associated with miR-122-expression levels. However, miR-122 secondary targets were expressed at significantly higher levels in both tumor and adjacent non-tumor tissues among survivors as compared with deceased patients, providing supporting evidence for the potential relevance of loss of miR-122 function in HCC patient morbidity and mortality.
Overall, our findings reveal potentially new biological functions for miR-122 in liver physiology. We observed decreased expression of miR-122, a liver-specific miRNA, in HBV-associated HCC, and loss of miR-122 seemed to correlate with the decrease of mitochondrion-related metabolic pathway gene expression in HCC and in non-tumor liver tissues, a result that is consistent with the outcome of treatment of mice with anti-miR-122 and is of prognostic significance for HCC patients. Further investigation will be conducted to dissect the regulatory function of miR-122 on mitochondrial metabolism in HCC and to test whether increasing miR-122 expression can improve mitochondrial function in liver and perhaps in liver tumor tissues. Moreover, these results support the idea that primary targets of a given miRNA may be distributed over a variety of functional categories while resulting in a coordinated secondary response, potentially through synergistic action (Linsley et al, 2007).
Tumorigenesis involves multistep genetic alterations. To elucidate the microRNA (miRNA)–gene interaction network in carcinogenesis, we examined their genome-wide expression profiles in 96 pairs of tumor/non-tumor tissues from hepatocellular carcinoma (HCC). Comprehensive analysis of the coordinate expression of miRNAs and mRNAs reveals that miR-122 is under-expressed in HCC and that increased expression of miR-122 seed-matched genes leads to a loss of mitochondrial metabolic function. Furthermore, the miR-122 secondary targets, which decrease in expression, are good prognostic markers for HCC. Transcriptome profiling data from additional 180 HCC and 40 liver cirrhotic patients in the same cohort were used to confirm the anti-correlation of miR-122 primary and secondary target gene sets. The HCC findings can be recapitulated in mouse liver by silencing miR-122 with antagomir treatment followed by gene-expression microarray analysis. In vitro miR-122 data further provided a direct link between induction of miR-122-controlled genes and impairment of mitochondrial metabolism. In conclusion, miR-122 regulates mitochondrial metabolism and its loss may be detrimental to sustaining critical liver function and contribute to morbidity and mortality of liver cancer patients.
doi:10.1038/msb.2010.58
PMCID: PMC2950084  PMID: 20739924
hepatocellular carcinoma; microarray; miR-122; mitochondrial; survival
2.  MiR-10 Represses HoxB1a and HoxB3a in Zebrafish 
PLoS ONE  2008;3(1):e1396.
Background
The Hox genes are involved in patterning the anterior-posterior axis. In addition to the protein coding Hox genes, the miR-10, miR-196 and miR-615 families of microRNA genes are conserved within the vertebrate Hox clusters. The members of the miR-10 family are located at positions associated with Hox-4 paralogues. No function is yet known for this microRNA family but the genomic positions of its members suggest a role in anterior-posterior patterning.
Methodology/Principal Findings
Using sensor constructs, overexpression and morpholino knockdown, we show in Zebrafish that miR-10 targets HoxB1a and HoxB3a and synergizes with HoxB4 in the repression of these target genes. Overexpression of miR-10 also induces specific phenotypes related to the loss of function of these targets. HoxB1a and HoxB3a have a dominant hindbrain expression domain anterior to that of miR-10 but overlap in a weaker expression domain in the spinal cord. In this latter domain, miR-10 knockdown results in upregulation of the target genes. In the case of a HoxB3a splice variant that includes miR-10c within its primary transcript, we show that the microRNA acts in an autoregulatory fashion.
Conclusions/Significance
We find that miR-10 acts to repress HoxB1a and HoxB3a within the spinal cord and show that this repression works cooperatively with HoxB4. As with the previously described interactions between miR-196 and HoxA7 and Hox-8 paralogues, the target genes are located in close proximity to the microRNA. We present a model in which we postulate a link between the clustering of Hox genes and post-transcriptional gene regulation. We speculate that the high density of transcription units and enhancers within the Hox clusters places constraints on the precision of the transcriptional control that can be achieved within these clusters and requires the involvement of post-transcriptional gene silencing to define functional domains of genes appropriately.
doi:10.1371/journal.pone.0001396
PMCID: PMC2148072  PMID: 18167555
3.  MicroRNAs Located in the Hox Gene Clusters Are Implicated in Huntington's Disease Pathogenesis 
PLoS Genetics  2014;10(2):e1004188.
Transcriptional dysregulation has long been recognized as central to the pathogenesis of Huntington's disease (HD). MicroRNAs (miRNAs) represent a major system of post-transcriptional regulation, by either preventing translational initiation or by targeting transcripts for storage or for degradation. Using next-generation miRNA sequencing in prefrontal cortex (Brodmann Area 9) of twelve HD and nine controls, we identified five miRNAs (miR-10b-5p, miR-196a-5p, miR-196b-5p, miR-615-3p and miR-1247-5p) up-regulated in HD at genome-wide significance (FDR q-value<0.05). Three of these, miR-196a-5p, miR-196b-5p and miR-615-3p, were expressed at near zero levels in control brains. Expression was verified for all five miRNAs using reverse transcription quantitative PCR and all but miR-1247-5p were replicated in an independent sample (8HD/8C). Ectopic miR-10b-5p expression in PC12 HTT-Q73 cells increased survival by MTT assay and cell viability staining suggesting increased expression may be a protective response. All of the miRNAs but miR-1247-5p are located in intergenic regions of Hox clusters. Total mRNA sequencing in the same samples identified fifteen of 55 genes within the Hox cluster gene regions as differentially expressed in HD, and the Hox genes immediately adjacent to the four Hox cluster miRNAs as up-regulated. Pathway analysis of mRNA targets of these miRNAs implicated functions for neuronal differentiation, neurite outgrowth, cell death and survival. In regression models among the HD brains, huntingtin CAG repeat size, onset age and age at death were independently found to be inversely related to miR-10b-5p levels. CAG repeat size and onset age were independently inversely related to miR-196a-5p, onset age was inversely related to miR-196b-5p and age at death was inversely related to miR-615-3p expression. These results suggest these Hox-related miRNAs may be involved in neuroprotective response in HD. Recently, miRNAs have shown promise as biomarkers for human diseases and given their relationship to disease expression, these miRNAs are biomarker candidates in HD.
Author Summary
Huntington's disease (HD) is an inherited fatal neurological disorder that commonly affects people in midlife. Past studies have implicated abnormal patterns of gene expression as a candidate for causing the death of the brain cells affected in HD. MicroRNAs (miRNAs) are small molecules that regulate and target transcripts for either storage or destruction. We measured the levels of miRNAs, as well as the levels of gene expression (mRNAs) in twelve HD and nine control brain samples. We found five miRNAs that had greatly increased expression in the HD brains, including three that were not expressed in the normal samples. Four of these were related to important characteristics of the disease expression, including the age at disease onset, and the age at death of the individual. The genes that these miRNAs target for regulation were also altered in their expression with most being increased, suggesting they may have been targeted for storage. One of the miRNAs, miR-196a-5p was previously implicated in enhancing the survival of brain cells in HD. When we overexpressed miR-10b-5p in an HD cell model, the cells survived longer than untreated cells, suggesting these miRNAs may promote neuron survival and may hold new clues for treatments in HD.
doi:10.1371/journal.pgen.1004188
PMCID: PMC3937267  PMID: 24586208
4.  Conserved Regulation of p53 Network Dosage by MicroRNA–125b Occurs through Evolving miRNA–Target Gene Pairs 
PLoS Genetics  2011;7(9):e1002242.
MicroRNAs regulate networks of genes to orchestrate cellular functions. MiR-125b, the vertebrate homologue of the Caenorhabditis elegans microRNA lin-4, has been implicated in the regulation of neural and hematopoietic stem cell homeostasis, analogous to how lin-4 regulates stem cells in C. elegans. Depending on the cell context, miR-125b has been proposed to regulate both apoptosis and proliferation. Because the p53 network is a central regulator of both apoptosis and proliferation, the dual roles of miR-125b raise the question of what genes in the p53 network might be regulated by miR-125b. By using a gain- and loss-of-function screen for miR-125b targets in humans, mice, and zebrafish and by validating these targets with the luciferase assay and a novel miRNA pull-down assay, we demonstrate that miR-125b directly represses 20 novel targets in the p53 network. These targets include both apoptosis regulators like Bak1, Igfbp3, Itch, Puma, Prkra, Tp53inp1, Tp53, Zac1, and also cell-cycle regulators like cyclin C, Cdc25c, Cdkn2c, Edn1, Ppp1ca, Sel1l, in the p53 network. We found that, although each miRNA–target pair was seldom conserved, miR-125b regulation of the p53 pathway is conserved at the network level. Our results lead us to propose that miR-125b buffers and fine-tunes p53 network activity by regulating the dose of both proliferative and apoptotic regulators, with implications for tissue stem cell homeostasis and oncogenesis.
Author Summary
MicroRNAs (miRNAs) are tiny endogenous RNAs that can regulate the expression of hundreds of genes simultaneously, thus orchestrating changes in gene networks and mediating cellular functions in both plants and animals. Although the identification of individual targets of miRNAs is of major importance, to date few studies have sought to uncover miRNA targets at the gene network level and general principles of miRNA regulation at the network level. Here we describe how miR-125b targets 20 apoptosis and proliferation genes in the p53 network. We found that, although each miRNA-target pair evolves rapidly across vertebrates, regulation of the p53 pathway by miR-125b is conserved at the network level. The structure of the miR-125b regulatory network suggests that miR-125b buffers and fine-tunes p53 network activity. This buffering feature of miR-125b has implications for our understanding of how miR-125b regulates oncogenesis and tissue stem cell homeostasis. We believe these findings on miR-125b support a new fundamental principle for how miRNAs regulate gene networks in general.
doi:10.1371/journal.pgen.1002242
PMCID: PMC3174204  PMID: 21935352
5.  A microRNA network functioning in the regulation of radiobiological effects 
Journal of Radiation Research  2014;55(Suppl 1):i57-i58.
MicroRNA (miRNA), a small non-coding RNA molecule, is vital in genetic regulation, and miRNA pathway, which regulates gene expression through degradation or translational suppression of their target transcripts, is highly conservative in evolution.
Although profiles of miRNAs are different in various cell types and tissues, miRNAs have been considered as a crucial class of regulators in cellular response to ionizing radiation (IR). By carrying out a series of experiments, we have found that altered transcriptional regulation network composed of radiation-mediated miRNAs regulates the expression of their downstream target genes in most biological processes to control cell growth, cell cycle and apoptosis. For example, the newly identified miR-3928 negatively regulates the expression of Dicer, which has been validated by the luciferase assay and western blotting. Dicer is not only a key participant in responding to radiation, but also a critical factor for the maturation of miRNAs, suggesting that miR-3928 affects on the expression of other miRNAs through regulating Dicer. Among the miRNAs controlled by the Dicer, we reveal that miR-185 and miR-663 can efficiently suppress ATR and TGF-β1 expression, which are both important responders in the process of radiobiological effects. Further experiments reveal that the expression of Dicer is suppressed by miR-3928 induced by IR and consequently, the maturation of other miRNAs including miR-185 and miR-663 is inhibited, resulting in the abundantly enhanced expression of ATR and TGF-β1 respectively. This mechanism to hammer at fixing DNA damage or promote cells to apoptosis caused by IR has important implications in the decision of cell fates.
Moreover, it has been shown that the expression of BTG1 is characterized in response to factors that induce growth arrest and subsequent differentiation both in vivo and in vitro, affecting cellular physiological progresses of angiogenesis, follicular development and myoblast and B cell differentiation, through regulating cell growth, migration, cell cycle, apoptosis and differentiation. BTG1 gene is phylogenetically highly conserved in its coding and 3′-untranslated region (UTR), which is considered as a decisive element involved in regulation of BTG1 expression. We present evidence that BTG1 can be induced by IR and confirm that miR-454-3p, whose gene locates in the intron of Ska2 gene, can regulate BTG1 expression through directly binding to the 3′-UTR of BTG1 mRNA. These results point out that increased expression of BTG1 caused by the down-regulation of miR-454-3p in case that IR modulates endogenous activity of PRMT1, a BTG1-binding partner, which can methylate endogenous transcription factors to change gene expression pattern and reply radiostilumation. An inverse relationship between the levels of expression of BTG1 and miR-454-3p reveals that there exists a new pathway in response to IR stimulation. Furthermore, cell growth will be transiently increased by the knockdown of BTG1 by transfecting BTG1 siRNA or miR-454-3p mimic. However, the apoptotic state of cells can be tested after 2 days. Down-regulation of BTG1 by miR-454-3p increases the sensitivity of human renal cell carcinoma 786-O cells to IR-induced apoptosis, suggesting that BTG1 could serve as a terget for sensitizing renal carcinoma to standard radiotherapy.
Taken together, all these data indicate that alteration of miRNA expression is evident in the cellular response to IR. MiR-3928, miR-185, miR-663 and miR-454-3p may constitute a complex network contributing to the regulation of radiobiological effects. It is apparent that the study of radiation-related miRNAs is beneficial to qualitatively and quantitatively modulating radiobiological effects, and also in favor of the advanced research of miRNA functions.
doi:10.1093/jrr/rrt157
PMCID: PMC3941529
microRNA; network; Dicer; BTG1; ionizing radiation
6.  Evolution of coding and non-coding genes in HOX clusters of a marsupial 
BMC Genomics  2012;13:251.
Background
The HOX gene clusters are thought to be highly conserved amongst mammals and other vertebrates, but the long non-coding RNAs have only been studied in detail in human and mouse. The sequencing of the kangaroo genome provides an opportunity to use comparative analyses to compare the HOX clusters of a mammal with a distinct body plan to those of other mammals.
Results
Here we report a comparative analysis of HOX gene clusters between an Australian marsupial of the kangaroo family and the eutherians. There was a strikingly high level of conservation of HOX gene sequence and structure and non-protein coding genes including the microRNAs miR-196a, miR-196b, miR-10a and miR-10b and the long non-coding RNAs HOTAIR, HOTAIRM1 and HOXA11AS that play critical roles in regulating gene expression and controlling development. By microRNA deep sequencing and comparative genomic analyses, two conserved microRNAs (miR-10a and miR-10b) were identified and one new candidate microRNA with typical hairpin precursor structure that is expressed in both fibroblasts and testes was found. The prediction of microRNA target analysis showed that several known microRNA targets, such as miR-10, miR-414 and miR-464, were found in the tammar HOX clusters. In addition, several novel and putative miRNAs were identified that originated from elsewhere in the tammar genome and that target the tammar HOXB and HOXD clusters.
Conclusions
This study confirms that the emergence of known long non-coding RNAs in the HOX clusters clearly predate the marsupial-eutherian divergence 160 Ma ago. It also identified a new potentially functional microRNA as well as conserved miRNAs. These non-coding RNAs may participate in the regulation of HOX genes to influence the body plan of this marsupial.
doi:10.1186/1471-2164-13-251
PMCID: PMC3541083  PMID: 22708672
Marsupial; HOX cluster; MicroRNAs; Long non-coding RNAs
7.  Functional Specialization of the Plant miR396 Regulatory Network through Distinct MicroRNA–Target Interactions 
PLoS Genetics  2012;8(1):e1002419.
MicroRNAs (miRNAs) are ∼21 nt small RNAs that regulate gene expression in animals and plants. They can be grouped into families comprising different genes encoding similar or identical mature miRNAs. Several miRNA families are deeply conserved in plant lineages and regulate key aspects of plant development, hormone signaling, and stress response. The ancient miRNA miR396 regulates conserved targets belonging to the GROWTH-REGULATING FACTOR (GRF) family of transcription factors, which are known to control cell proliferation in Arabidopsis leaves. In this work, we characterized the regulation of an additional target for miR396, the transcription factor bHLH74, that is necessary for Arabidopsis normal development. bHLH74 homologs with a miR396 target site could only be detected in the sister families Brassicaceae and Cleomaceae. Still, bHLH74 repression by miR396 is required for margin and vein pattern formation of Arabidopsis leaves. MiR396 contributes to the spatio-temporal regulation of GRF and bHLH74 expression during leaf development. Furthermore, a survey of miR396 sequences in different species showed variations in the 5′ portion of the miRNA, a region known to be important for miRNA activity. Analysis of different miR396 variants in Arabidopsis thaliana revealed that they have an enhanced activity toward GRF transcription factors. The interaction between the GRF target site and miR396 has a bulge between positions 7 and 8 of the miRNA. Our data indicate that such bulge modulates the strength of the miR396-mediated repression and that this modulation is essential to shape the precise spatio-temporal pattern of GRF2 expression. The results show that ancient miRNAs can regulate conserved targets with varied efficiency in different species, and we further propose that they could acquire new targets whose control might also be biologically relevant.
Author Summary
Plants and other multicellular organisms need precise spatio-temporal control of gene expression, and this regulatory capacity depends, in part, on small RNAs. MicroRNAs (miRNAs) are one class of ∼21 nt small RNAs that originate from endogenous fold-back precursors found in plants and animals. They recognize complementary target sites in target mRNAs and guide them to cleavage or translational arrest. Studies of conserved miRNA networks in Arabidopsis and other plants have revealed that they fulfill essential regulatory roles. Most of the ancient miRNAs regulate transcription factors involved in plant development and hormone signaling. Here, we characterize the miR396 regulatory network. While miR396 regulates GRF transcription factors, at least in angiosperms and gymnosperms, this miRNA additionally regulates another transcription factor of the bHLH class but only in Arabidopsis thaliana and closely related species. Most conspicuously, the regulation of both conserved and new targets is important for leaf development in Arabidopsis. We also show that miRNA variants can exist in certain species and that they can display an enhanced activity towards their targets. In summary, we propose that conserved miRNA regulatory networks might expand their functions by the recruitment of additional targets as well as by slight variations in the small RNA sequences.
doi:10.1371/journal.pgen.1002419
PMCID: PMC3252272  PMID: 22242012
8.  Evolution of MIR159/319 microRNA genes and their post-transcriptional regulatory link to siRNA pathways 
Background
MicroRNAs (miRNAs) are prevalent and important endogenous gene regulators in eukaryotes. MiR159 and miR319 are highly conserved miRNAs essential for plant development and fertility. Despite high similarity in conservation pattern and mature miRNA sequences, miR159 and miR319 have distinct expression patterns, targets and functions. In addition, both MIR319 and MIR159 precursors produce multiple miRNAs in a phased loop-to-base manner. Thus, MIR159 and MIR319 appear to be related in origin and considerably diverged. However the phylogeny of MIR159 and MIR319 genes and why such unusual style of miRNA production has been conserved during evolution is not well understood.
Results
We reconstructed the phylogeny of MIR159/319 genes and analyzed their mature miRNA expression. The inferred phylogeny suggests that the MIR159/319 genes may have formed at least ten extant early-branching clades through gene duplication and loss. A series of duplications occurred in the common ancestor of seed plants leading to the original split of flowering plant MIR159 and MIR319. The results also indicate that the expression of MIR159/319 is regulated at post-transcriptional level to switch on the expression of alternative miRNAs during development in a highly spatio-temporal specific manner, and to selectively respond to the disruption of defensive siRNA pathways. Such intra-stem-loop regulation appears diverged across the early-branching clades of MIR159/319 genes.
Conclusions
Our results support that the MIR159 and MIR319 genes evolve from a common ancestor, which is likely to be a phased stem-loop small RNA. Through duplication and loss of genes this miRNA gene family formed clades specific to moss, lycopods, gymnosperms and angiosperms including the two major clades of flowering plants containing the founding members of MIR319 and MIR159 genes in A.thaliana. Our analyses also suggest that some MIR159/319 have evolved into unusual miRNA genes that are regulated at post-transcriptional level to express multiple mature products with variable proportions under different circumstances. Moreover, our analyses reveal conserved regulatory link of MIR159/319 genes to siRNA pathway through post-transcriptional regulation.
doi:10.1186/1471-2148-11-122
PMCID: PMC3118147  PMID: 21569383
9.  Over-expression of miR172 causes loss of spikelet determinacy and floral organ abnormalities in rice (Oryza sativa) 
BMC Plant Biology  2009;9:149.
Background
Regulation of gene expression by microRNAs (miRNAs) plays a crucial role in many developmental and physiological processes in plants. miRNAs act to repress expression of their target genes via mRNA cleavage or translational repression. Dozens of miRNA families have been identified in rice, 21 of which are conserved between rice and Arabidopsis. miR172 is a conserved miRNA family which has been shown to regulate expression of APETALA2 (AP2)-like transcription factors in Arabidopsis and maize. The rice genome encodes five AP2-like genes predicted to be targets of miR172. To determine whether these rice AP2-like genes are regulated by miR172 and investigate the function of the target genes, we studied the effect of over-expressing two members of the miR172 family on rice plant development.
Results
Analysis of miR172 expression showed that it is most highly expressed in late vegetative stages and developing panicles. Analyses of expression of three miR172 targets showed that SUPERNUMERARY BRACT (SNB) and Os03g60430 have high expression in developing panicles. Expression of miR172 was not inversely correlated with expression of its targets although miR172-mediated cleavage of SNB was detected by 5' rapid amplification of cDNA ends (RACE). Over-expression of miR172b in rice delayed the transition from spikelet meristem to floral meristem, and resulted in floral and seed developmental defects, including changes to the number and identity of floral organs, lower fertility and reduced seed weight. Plants over-expressing miR172b not only phenocopied the T-DNA insertion mutant of SNB but showed additional defects in floret development not seen in the snb mutant. However SNB expression was not reduced in the miR172b over-expression plants.
Conclusions
The phenotypes resulting from over-expression of miR172b suggests it represses SNB and at least one of the other miR172 targets, most likely Os03g60430, indicating roles for other AP2-like genes in rice floret development. miR172 and the AP2-like genes had overlapping expression patterns in rice and their expression did not show an obvious negative correlation. There was not a uniform decrease in the expression of the AP2-like miR172 target mRNAs in the miR172b over-expression plants. These observations are consistent with miR172 functioning via translational repression or with expression of the AP2-like genes being regulated by a negative feedback loop.
doi:10.1186/1471-2229-9-149
PMCID: PMC2803185  PMID: 20017947
10.  The mir-51 Family of microRNAs Functions in Diverse Regulatory Pathways in Caenorhabditis elegans 
PLoS ONE  2012;7(5):e37185.
The mir-51 family of microRNAs (miRNAs) in C. elegans are part of the deeply conserved miR-99/100 family. While loss of all six family members (mir-51-56) in C. elegans results in embryonic lethality, loss of individual mir-51 family members results in a suppression of retarded developmental timing defects associated with the loss of alg-1. The mechanism of this suppression of developmental timing defects is unknown. To address this, we characterized the function of the mir-51 family in the developmental timing pathway. We performed genetic analysis and determined that mir-51 family members regulate the developmental timing pathway in the L2 stage upstream of hbl-1. Loss of the mir-51 family member, mir-52, suppressed retarded developmental timing defects associated with the loss of let-7 family members and lin-46. Enhancement of precocious defects was observed for mutations in lin-14, hbl-1, and mir-48(ve33), but not later acting developmental timing genes. Interestingly, mir-51 family members showed genetic interactions with additional miRNA-regulated pathways, which are regulated by the let-7 and mir-35 family miRNAs, lsy-6, miR-240/786, and miR-1. Loss of mir-52 likely does not suppress miRNA-regulated pathways through an increase in miRNA biogenesis or miRNA activity. We found no increase in the levels of four mature miRNAs, let-7, miR-58, miR-62 or miR-244, in mir-52 or mir-52/53/54/55/56 mutant worms. In addition, we observed no increase in the activity of ectopic lsy-6 in the repression of a downstream target in uterine cells in worms that lack mir-52. We propose that the mir-51 family functions broadly through the regulation of multiple targets, which have not yet been identified, in diverse regulatory pathways in C. elegans.
doi:10.1371/journal.pone.0037185
PMCID: PMC3353893  PMID: 22615936
11.  Cloning, characterization, and expression of microRNAs from the Asian malaria mosquito, Anopheles stephensi 
BMC Genomics  2008;9:244.
Background
microRNAs (miRNAs) are non-coding RNAs that are now recognized as a major class of gene-regulating molecules widely distributed in metozoans and plants. miRNAs have been found to play important roles in apoptosis, cancer, development, differentiation, inflammation, longevity, and viral infection. There are a few reports describing miRNAs in the African malaria mosquito, Anopheles gambiae, on the basis of similarity to known miRNAs from other species. An. stephensi is the most important malaria vector in Asia and it is becoming a model Anopheline species for physiological and genetics studies.
Results
We report the cloning and characterization of 27 distinct miRNAs from 17-day old An. stephensi female mosquitoes. Seventeen of the 27 miRNAs matched previously predicted An. gambiae miRNAs, offering the first experimental verification of miRNAs from mosquito species. Ten of the 27 are miRNAs previously unknown to mosquitoes, four of which did not match any known miRNAs in any organism. Twenty-five of the 27 Anopheles miRNAs had conserved sequences in the genome of a divergent relative, the yellow fever mosquito Aedes aegypti. Two clusters of miRNAs were found within introns of orthologous genes in An. gambiae, Ae. aegypti, and Drosophila melanogaster. Mature miRNAs were detected in An. stephensi for all of the nine selected miRNAs, including the four novel miRNAs (miR-x1- miR-x4), either by northern blot or by Ribonuclease Protection Assay. Expression profile analysis of eight of these miRNAs revealed distinct expression patterns from early embryo to adult stages in An. stephensi. In both An. stephensi and Ae. aegypti, the expression of miR-x2 was restricted to adult females and predominantly in the ovaries. A significant reduction of miR-x2 level was observed 72 hrs after a blood meal. Thus miR-x2 is likely involved in female reproduction and its function may be conserved among divergent mosquitoes. A mosquito homolog of miR-14, a regulator of longevity and apoptosis in D. melanogaster, represented 25% of all sequenced miRNA clones from 17-day old An. stephensi female mosquitoes. An. stephensi miR-14 displayed a relatively strong signal from late embryonic to adult stages. miR-14 expression is consistent during the adult lifespan regardless of age, sex, and blood feeding status. Thus miR-14 is likely important across all mosquito life stages.
Conclusion
This study provides experimental evidence for 23 conserved and four new microRNAs in An. stephensi mosquitoes. Comparisons between miRNA gene clusters in Anopheles and Aedes mosquitoes, and in D. melanogaster suggest the loss or significant change of two miRNA genes in Ae. aegypti. Expression profile analysis of eight miRNAs, including the four new miRNAs, revealed distinct patterns from early embryo to adult stages in An. stephensi. Further analysis showed that miR-x2 is likely involved in female reproduction and its function may be conserved among divergent mosquitoes. Consistent expression of miR-14 suggests that it is likely important across all mosquito life stages from embryos to aged adults. Understanding the functions of mosquito miRNAs will undoubtedly contribute to a better understanding of mosquito biology including longevity, reproduction, and mosquito-pathogen interactions, which are important to disease transmission.
doi:10.1186/1471-2164-9-244
PMCID: PMC2430712  PMID: 18500992
12.  Building a Robust A-P Axis 
Current Genomics  2012;13(4):278-288.
Since the last common ancestor of Metazoa, animals have evolved complex body plans with specialized cells and spatial organization of tissues and organs. Arguably, one of the most significant innovations during animal evolutionary history was the establishment of a bilateral plane of symmetry on which morphological features (e.g. tissues, organs, appendages, skeleton) could be given specific coordinates within the animal along the anterior-posterior (A-P) and dorsal-ventral (D-V) axes. Hox genes are a known group of eumetazoan transcription factors central to regulating A-P patterning, but less well known and under current investigation is the broader regulatory landscape incorporating these genes, including microRNA (miRNA) regulation. The degree to which evolutionarily conserved targeting of Hox genes by Hox-embedded miRNAs contributes directly to A-P patterning is under investigation, yielding contrasting information dependent on the organism and miRNA of interest. The widespread A-P patterning defects observed in recent miR-196 loss-of-function studies solidifies the importance of miRNA regulation in Hox genetic hierarchies, and elucidating the developmental and evolutionary importance of all Hox-embedded miRNAs remains a challenge for the future.
doi:10.2174/138920212800793348
PMCID: PMC3394115  PMID: 23204917
Hox gene; microRNA; A-P patterning; miR-10; miR-196.
13.  Downregulation of the Host Gene jigr1 by miR-92 Is Essential for Neuroblast Self-Renewal in Drosophila 
PLoS Genetics  2015;11(5):e1005264.
Intragenic microRNAs (miRNAs), located mostly in the introns of protein-coding genes, are often co-expressed with their host mRNAs. However, their functional interaction in development is largely unknown. Here we show that in Drosophila, miR-92a and miR-92b are embedded in the intron and 3’UTR of jigr1, respectively, and co-expressed with some jigr1 isoforms. miR-92a and miR-92b are highly expressed in neuroblasts of larval brain where Jigr1 expression is low. Genetic deletion of both miR-92a and miR-92b demonstrates an essential cell-autonomous role for these miRNAs in maintaining neuroblast self-renewal through inhibiting premature differentiation. We also show that miR-92a and miR-92b directly target jigr1 in vivo and that some phenotypes due to the absence of these miRNAs are partially rescued by reducing the level of jigr1. These results reveal a novel function of the miR-92 family in Drosophila neuroblasts and provide another example that local negative feedback regulation of host genes by intragenic miRNAs is essential for animal development.
Author Summary
Animal development is regulated by many genes including a class of small RNAs called microRNAs (miRNAs). Nearly half of the miRNAs are located in the protein coding genes but functional importance of this genomic organization is unclear. Here we use Drosophila stem cells in the brain as a model system to investigate the interactions between miR-92a and miR-92b and their host gene jing interacting regulatory protein 1 (jigr1). Our studies reveal that these miRNAs prevent premature differentiation of neural stem cells and they do so in part through directly targeting and suppressing their host gene, jigr1. Our results reveal a novel function of the miR-92 family and identify another negative feedback loop as an essential regulator in neural stem cell development.
doi:10.1371/journal.pgen.1005264
PMCID: PMC4441384  PMID: 26000445
14.  The miR-35-41 Family of MicroRNAs Regulates RNAi Sensitivity in Caenorhabditis elegans 
PLoS Genetics  2012;8(3):e1002536.
RNA interference (RNAi) utilizes small interfering RNAs (siRNAs) to direct silencing of specific genes through transcriptional and post-transcriptional mechanisms. The siRNA guides can originate from exogenous (exo–RNAi) or natural endogenous (endo–RNAi) sources of double-stranded RNA (dsRNA). In Caenorhabditis elegans, inactivation of genes that function in the endo–RNAi pathway can result in enhanced silencing of genes targeted by siRNAs from exogenous sources, indicating cross-regulation between the pathways. Here we show that members of another small RNA pathway, the mir-35-41 cluster of microRNAs (miRNAs) can regulate RNAi. In worms lacking miR-35-41, there is reduced expression of lin-35/Rb, the C. elegans homolog of the tumor suppressor Retinoblastoma gene, previously shown to regulate RNAi responsiveness. Genome-wide microarray analyses show that targets of endo–siRNAs are up-regulated in mir-35-41 mutants, a phenotype also displayed by lin-35/Rb mutants. Furthermore, overexpression of lin-35/Rb specifically rescues the RNAi hypersensitivity of mir-35-41 mutants. Although the mir-35-41 miRNAs appear to be exclusively expressed in germline and embryos, their effect on RNAi sensitivity is transmitted to multiple tissues and stages of development. Additionally, we demonstrate that maternal contribution of miR-35-41 or lin-35/Rb is sufficient to reduce RNAi effectiveness in progeny worms. Our results reveal that miRNAs can broadly regulate other small RNA pathways and, thus, have far reaching effects on gene expression beyond directly targeting specific mRNAs.
Author Summary
RNA interference (RNAi) has become a widely used approach for silencing genes of interest. This tool is possible because endogenous RNA silencing pathways exist broadly across organisms, including humans, worms, and plants. The general RNAi pathway utilizes small ∼21-nucleotide RNAs to target specific protein-coding genes through base-pairing interactions. Since RNAs from exogenous sources require some of the same factors as endogenous small RNAs to silence gene expression, there can be competition between the pathways. Thus, perturbations in the endogenous RNAi pathway can result in enhanced silencing efficiency by exogenous small RNAs. MicroRNAs (miRNAs) comprise another endogenous small RNA pathway, but their biogenesis and mechanism of gene silencing are distinct in many ways from RNAi pathways. Here we show that a family of miRNAs regulates the effectiveness of RNAi in Caenorhabditis elegans. Loss of mir-35-41 results in enhanced RNAi by exogenous RNAs and reduced silencing of endogenous RNAi targets. The embryonic miR-35-41 miRNAs regulate the sensitivity to RNAi through lin-35/Rb, a homolog of the human Retinoblastoma tumor suppressor gene previously shown to regulate RNAi effectiveness in C. elegans. Additionally, we show that this sensitivity can be passed on to the next generation of worms, demonstrating a far-reaching effect of the miR-35-41 miRNAs on gene regulation by other small RNA pathways.
doi:10.1371/journal.pgen.1002536
PMCID: PMC3297572  PMID: 22412382
15.  Targeted Inhibition of miRNA Maturation with Morpholinos Reveals a Role for miR-375 in Pancreatic Islet Development 
PLoS Biology  2007;5(8):e203.
Several vertebrate microRNAs (miRNAs) have been implicated in cellular processes such as muscle differentiation, synapse function, and insulin secretion. In addition, analysis of Dicer null mutants has shown that miRNAs play a role in tissue morphogenesis. Nonetheless, only a few loss-of-function phenotypes for individual miRNAs have been described to date. Here, we introduce a quick and versatile method to interfere with miRNA function during zebrafish embryonic development. Morpholino oligonucleotides targeting the mature miRNA or the miRNA precursor specifically and temporally knock down miRNAs. Morpholinos can block processing of the primary miRNA (pri-miRNA) or the pre-miRNA, and they can inhibit the activity of the mature miRNA. We used this strategy to knock down 13 miRNAs conserved between zebrafish and mammals. For most miRNAs, this does not result in visible defects, but knockdown of miR-375 causes defects in the morphology of the pancreatic islet. Although the islet is still intact at 24 hours postfertilization, in later stages the islet cells become scattered. This phenotype can be recapitulated by independent control morpholinos targeting other sequences in the miR-375 precursor, excluding off-target effects as cause of the phenotype. The aberrant formation of the endocrine pancreas, caused by miR-375 knockdown, is one of the first loss-of-function phenotypes for an individual miRNA in vertebrate development. The miRNA knockdown strategy presented here will be widely used to unravel miRNA function in zebrafish.
Author Summary
The striking tissue-specific expression patterns of microRNAs (miRNAs) suggest that they play a role in tissue development. These small RNA molecules (∼22 bases in length) are processed from long primary transcripts (pri-miRNA) and regulate gene expression at the posttranscriptional level. There are hundreds of different miRNAs, many of which are strongly conserved. Vertebrate embryonic development is most easily studied in zebrafish, but genetically disrupting miRNA genes to see which miRNA does what is technically challenging. In this study, we interfere with miRNA function during the first few days of zebrafish embryonic development by introducing specific antisense morpholino oligonucleotides (morpholinos have been used previously to interfere with the synthesis of the much larger mRNAs). We show that morpholinos targeting the miRNA precursor can block processing of the pri-miRNA or directly inhibit the activity of the mature miRNA. We also used morpholinos to study the developmental effects of miRNA knockdown. Although we did not observe gross phenotypic defects for many miRNAs, we found that zebrafish miR-375 is essential for formation of the insulin-secreting pancreatic islet. Loss of miR-375 results in dispersed islet cells by 36 hours postfertilization, representing one of the first vertebrate miRNA loss-of-function phenotypes.
The authors show that morpholinos can be used to knock down zebrafish miRNAs, revealing that miR-375 is important for vertebrate pancreas development.
doi:10.1371/journal.pbio.0050203
PMCID: PMC1925136  PMID: 17676975
16.  Computational and in vitro Investigation of miRNA-Gene Regulations in Retinoblastoma Pathogenesis: miRNA Mimics Strategy 
PURPOSE
Retinoblastoma (RB), a primary pediatric intraocular tumor, arises from primitive retinal layers. Several novel molecular strategies are being developed for the clinical management of RB. miRNAs are known to regulate cancer-relevant biological processes. Here, the role of selected miRNAs, namely, miR-532-5p and miR-486-3p, has been analyzed for potential therapeutic targeting in RB.
METHODS
A comprehensive bioinformatic analysis was performed to predict the posttranscriptional regulators (miRNAs) of the select panel of genes [Group 1: oncogenes (HMGA2, MYCN, SYK, FASN); Group 2: cancer stem cell markers (TACSTD, ABCG2, CD133, CD44, CD24) and Group 3: cell cycle regulatory proteins (p53, MDM2)] using Microcosm, DIANALAB, miRBase v 18, and REFSEQ database, and RNA hybrid. The expressions of five miRNAs, namely, miR-146b-5p, miR-532-5p, miR-142-5p, miR-328, and miR-486-3p, were analyzed by qRT–PCR on primary RB tumor samples (n = 30; including 17 invasive RB tumors and 13 noninvasive RB tumors). Detailed complementary alignment between 5’ seed sequence of differentially expressed miRNAs and the sequence of target genes was determined. Based on minimum energy level and piCTAR scores, the gene targets were selected. Functional roles of these miRNA clusters were studied by using mimics in cultured RB (Y79, Weri Rb-1) cells in vitro. The gene targets (SYK and FASN) of the studied miRNAs were confirmed by qRT-PCR and western blot analysis. Cell proliferation and apoptotic studies were performed.
RESULTS
Nearly 1948 miRNAs were identified in the in silico analysis, From this list, only 9 upregulated miRNAs (miR-146b-5p, miR-305, miR-663b, miR-299, miR-532-5p, miR-892b, miR-501, miR-142-5p, and miR-513b) and 10 downregulated miRNAs (miR-1254, miR-328, miR-133a, miR-1287, miR-1299, miR-375, miR-486-3p, miR-720, miR-98, and miR-122*) were found to be common with the RB serum miRNA profile. Downregulation of five miRNAs (miR-146b-5p, miR-532-5p, miR-142-5p, miR-328, and miR-486-3p) was confirmed experimentally. Predicted common oncogene targets (SYK and FASN) of miR-486-3p and miR-532-5p were evaluated for their mRNA and protein expression in these miRNA mimic-treated RB cells. Experimental overexpression of these miRNAs mediated apoptotic cell death without significantly altering the cell cycle in RB cells.
CONCLUSION
Key miRNAs in RB pathogenesis were identified by an in silico approach. Downregulation of miR-486-3p and miR-532-5p in primary retinoblastoma tissues implicates their role in tumorigenesis. Prognostic and therapeutic potential of these miRNA was established by the miRNA mimic strategy.
doi:10.4137/BBI.S21742
PMCID: PMC4429751  PMID: 25983556
bio-informatics analysis; miRNA-mRNA; mimics; retinoblastoma
17.  Analysis of the mRNA Targetome of MicroRNAs Expressed by Marek’s Disease Virus 
mBio  2014;5(1):e01060-13.
ABSTRACT
Marek’s disease virus 1 (MDV-1), an oncogenic α-herpesvirus that induces T-cell lymphomas in chickens, serves as model system to study transformation by lymphotropic herpesviruses. Like the oncogenic human γ-herpesviruses Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), MDV-1 encodes several viral microRNAs (miRNAs). One MDV-1 miRNA, miR-M4, shares the same “seed” targeting sequence with both a KSHV miRNA, miR-K11, and cellular miR-155. Importantly, miR-M4 plays a critical role in T-cell transformation by MDV-1, while miR-K11 and cellular miR-155 are thought to play key roles in B-cell transformation by KSHV and EBV, respectively. Here, we present an analysis of the mRNAs targeted by viral miRNAs expressed in the chicken T-cell line MSB1, which is naturally coinfected with MDV-1 and the related nonpathogenic virus MDV-2. Our analysis identified >1,000 endogenous mRNAs targeted by miRNAs encoded by each virus, many of which are targeted by both MDV-1 and MDV-2 miRNAs. We present a functional analysis of an MDV-1 gene, RLORF8, targeted by four MDV-1 miRNAs and a cellular gene, encoding interleukin-18 (IL-18) and targeted by both MDV-1 and MDV-2 miRNAs, and show that ectopic expression of either protein in a form resistant to miRNA inhibition results in inhibition of cell proliferation. Finally, we present a restricted list of 9 genes targeted by not only MDV-1 miR-M4 but also KSHV miR-K11 and human miR-155. Given the critical role played by miR-155 seed family members in lymphomagenesis in humans and chickens, these mRNA targets may contain genes whose inhibition plays a conserved role in herpesvirus transformation.
IMPORTANCE
Herpesviruses cause lymphomas in both humans and chickens, and in both cases, evidence indicates that virally encoded miRNAs, or virally subverted cellular miRNAs, belonging to the miR-155 seed family, play a critical role in this process. However, because each miRNA regulates numerous cellular mRNAs species, it has been difficult to elucidate which miRNA targets are important. Given the evolutionary distance between chickens and humans and the observation that miR-155 is nevertheless highly conserved in both species, we reasoned that the identification of shared miR-155 targets might shed light on this process. Here, we present an analysis of the mRNAs targeted by miRNAs encoded by the oncogenic avian herpesvirus MDV-1 in transformed chicken T cells, including a short list of mRNAs that are also targeted by miR-155 seed family miRNAs in EBV- or KSHV-transformed human B cells, and present an initial functional analysis of some of these miRNA targets.
doi:10.1128/mBio.01060-13
PMCID: PMC3903288  PMID: 24449754
18.  MicroRNA-146 function in the innate immune transcriptome response of zebrafish embryos to Salmonella typhimurium infection 
BMC Genomics  2013;14:696.
Background
MicroRNAs (miRNAs) have recently been shown to play important roles in development of the immune system and in fine-tuning of immune responses. Human miR-146 family members are known as inflammation-inducible miRNAs involved in negative feedback regulation of Toll-like receptor (TLR) signalling. Dysregulation of the miR-146 family has often been linked to inflammatory diseases and malignancies. This study reports on miR-146a and miR-146b as infection-inducible miRNAs in zebrafish, which has emerged as a model species for human disease.
Results
Using a custom-designed microarray platform for miRNA expression we found that both members of the zebrafish miR-146 family, miR-146a and miR-146b, were commonly induced by infection of zebrafish embryos with Salmonella typhimurium and by infection of adult fish with Mycobacterium marinum. The induction of these miRNAs was confirmed by Taqman miRNA assays. Subsequently, we used zebrafish embryos, in which adaptive immunity is not yet active, as an in vivo system to investigate the role of miR-146 in the innate immune response to S. typhimurium infection. Knockdown of traf6 and use of myd88 mutants demonstrated that the induction of miR-146a and miR-146b by S. typhimurium infection was affected by disruption of the MyD88-Traf6 pathway that mediates transduction of TLR signals and cytokine responses. In turn, knockdown of miR-146 itself had no major effects on the expression of known targets of MyD88-Traf6 signalling. Instead, RNA sequencing analysis showed that miR-146 knockdown led to an increased induction of six members of the apolipoprotein gene family in S. typhimurium-infected embryos.
Conclusion
Based on microarray analysis and Taqman miRNA assays we conclude that members of the miR-146 family, which is highly conserved between fish and human, are induced by bacterial infection in zebrafish in a MyD88 and Traf6 dependent manner. The combined knockdown of miR-146a and miR-146b in zebrafish embryos infected with S. typhimurium had no major effect on the expression of pro-inflammatory genes and transcription factors known to be downstream of the MyD88-Traf6 pathway. In contrast, apolipoprotein-mediated lipid transport emerged as an infection-inducible pathway under miR-146 knockdown conditions, suggesting a possible function of miR-146 in regulating lipid metabolism during inflammation.
doi:10.1186/1471-2164-14-696
PMCID: PMC3852110  PMID: 24112639
MicroRNA; miR-146; Innate immunity; Infection; Salmonella typhimurium; Mycobacterium marinum; MyD88; Traf6; Apolipoproteins; Zebrafish
19.  microRNA-1 regulates sarcomere formation and suppresses smooth muscle gene expression in the mammalian heart 
eLife  2013;2:e01323.
microRNA-1 (miR-1) is an evolutionarily conserved, striated muscle-enriched miRNA. Most mammalian genomes contain two copies of miR-1, and in mice, deletion of a single locus, miR-1-2, causes incompletely penetrant lethality and subtle cardiac defects. Here, we report that deletion of miR-1-1 resulted in a phenotype similar to that of the miR-1-2 mutant. Compound miR-1 knockout mice died uniformly before weaning due to severe cardiac dysfunction. miR-1-null cardiomyocytes had abnormal sarcomere organization and decreased phosphorylation of the regulatory myosin light chain-2 (MLC2), a critical cytoskeletal regulator. The smooth muscle-restricted inhibitor of MLC2 phosphorylation, Telokin, was ectopically expressed in the myocardium, along with other smooth muscle genes. miR-1 repressed Telokin expression through direct targeting and by repressing its transcriptional regulator, Myocardin. Our results reveal that miR-1 is required for postnatal cardiac function and reinforces the striated muscle phenotype by regulating both transcriptional and effector nodes of the smooth muscle gene expression network.
DOI: http://dx.doi.org/10.7554/eLife.01323.001
eLife digest
MicroRNAs are tiny RNAs that do not encode proteins. Instead, they regulate the expression of genes by preventing protein-encoding messenger RNAs from being translated into protein. MicroRNAs are expressed throughout the body, including the heart, where the most abundant microRNA is called miR-1. This is encoded by two nearly identical genes: miR-1-1 and miR-1-2.
Mice that lack the miR-1-2 gene have various heart abnormalities, but generally survive because they still produce some miR-1 from their remaining miR-1-1 gene. Now, Heidersbach et al. have generated the first mice that specifically lack both miR-1 genes, and shown that these animals die before weaning.
When viewed under the electron microscope, heart muscle from miR-1 double knockout mice lacks the characteristic ‘striped’, or striated, appearance of normal heart muscle. Additionally, miR-1 double knockout hearts have some gene expression characteristics more similar to the smooth muscle found in the gut and in the walls of blood vessels. Smooth muscle differs from striated muscle in that it lacks sarcomeres: these are bands of fibrous proteins, such as myosin, that are essential for muscle contraction.
In normal mice, an enzyme called MLCK contributes to the formation and function of sarcomeres by adding phosphate groups to myosin molecules. By contrast, in smooth muscle an enzyme called Telokin promotes phosphate group removal, and thus affects the function of sarcomeres. Heidersbach et al. showed that miR-1 interacts directly with Telokin mRNA to prevent its expression in the heart, and simultaneously represses a protein called Myocardin, which directly activates transcription of Telokin. However, when miR-1 is absent, as in the miR-1 double knockout mice, Telokin is expressed in heart muscle, along with many other genes characteristic of smooth muscle.
As well as improving our understanding of the development and functioning of the heart, these findings should shed new light on the role of microRNAs in maintaining the patterns of gene expression that characterize unique cell fates.
DOI: http://dx.doi.org/10.7554/eLife.01323.002
doi:10.7554/eLife.01323
PMCID: PMC3833424  PMID: 24252873
microRNA-1; cardiac; sarcomere; Telokin; Myocardin; smooth muscle gene expression; Mouse
20.  Identification of nuclear-enriched miRNAs during mouse granulopoiesis 
Background
MicroRNAs (miRNAs) are coordinators of cellular differentiation, including granulopoiesis. Although differential expression of many miRNAs is associated with the maturation of granulocytes, analysis of differentially expressed miRNAs and their cellular localization across all stages of granulopoiesis, starting from hemopoietic stems cells, is not well characterized.
Methods
We analyzed whole cell miRNA and mRNA expression during granulopoiesis using Taqman low-density and Affymetrix arrays respectively. We also performed nuclear and cytoplasmic fractionation followed by Taqman low-density array and/or quantitative PCR to identify nuclear-enriched miRNAs in hemopoietic stem/progenitor cells, promyelocytes, myelocytes, granulocytes and several hemopoietic cell lines. Anti-correlation between the expression of miRNA and target pairs was used to determine putative miRNA targets.
Results
Analyses of our array data revealed distinct clusters of differentially expressed miRNAs that are specific to promyelocytes and granulocytes. While the roles of many of these miRNAs in granulopoiesis are not currently known, anti-correlation of the expression of miRNA/mRNA target pairs identified a suite of novel target genes. Clusters of miRNAs (including members of the let-7 and miR-17-92 families) are downregulated in hemopoietic stem/progenitor cells, potentially allowing the expression of target genes known to facilitate stem cell proliferation and homeostasis. Additionally, four miRNAs (miR-709, miR-706, miR-690 and miR-467a*) were found to be enriched in the nucleus of myeloid cells and multiple hemopoietic cell lines compared to other miRNAs, which are predominantly cytoplasmic-enriched. Both miR-709 and miR-706 are nuclear-enriched throughout granulopoiesis and have putative binding sites of extensive complementarity downstream of pri-miRNAs. Nuclear enrichment of miR-467a* is specific to hemopoietic stem/progenitors and promyelocytes. These miRNAs are also nuclear-enriched in other hemopoietic cell lines, where nuclear sequestering may fine-tune the expression of cytoplasmic mRNA targets.
Conclusions
Overall, we have demonstrated differentially expressed miRNAs that have not previously been associated with hemopoietic differentiation and provided further evidence of regulated nuclear-enrichment of miRNAs. Further studies into miRNA function in granulocyte development may shed light on fundamental aspects of regulatory RNA biology and the role of nuclear miRNAs.
doi:10.1186/1756-8722-7-42
PMCID: PMC4046156  PMID: 24886830
miRNAs; mRNA targets; Nuclear; Granulopoiesis; Gene expression; Stem cell
21.  Regulation of microRNA during cardiomyocyte maturation in sheep 
BMC Genomics  2015;16(1):541.
Background
There is a limited capacity to repair damage in the mammalian heart after birth, which is primarily due to the inability of cardiomyocytes to proliferate after birth. This is in contrast to zebrafish and salamander, in which cardiomyocytes retain the ability to proliferate throughout life and can regenerate their heart after significant damage. Recent studies in zebrafish and rodents implicate microRNA (miRNA) in the regulation of genes responsible for cardiac cell cycle progression and regeneration, in particular, miR-133a, the miR-15 family, miR-199a and miR-590. However, the significance of these miRNA and miRNA in general in the regulation of cardiomyocyte proliferation in large mammals, including humans, where the timing of heart development relative to birth is very different than in rodents, is unclear. To determine the involvement of miRNA in the down-regulation of cardiomyocyte proliferation occurring before birth in large mammals, we investigated miRNA and target gene expression in sheep hearts before and after birth. The experimental approach included targeted transcriptional profiling of miRNA and target mRNA previously identified in rodent studies as well as genome-wide miRNA profiling using microarrays.
Results
The cardiac expression of miR-133a increased and its target gene IGF1R decreased with increasing age, reaching their respective maximum and minimum abundance when the majority of ovine cardiomyocytes were quiescent. The expression of the miR-15 family members was variable with age, however, four of their target genes decreased with age. These latter profiles are inconsistent with the direct involvement of this family of miRNA in cardiomyocyte quiescence in late gestation sheep. The expression patterns of ‘pro-proliferative’ miR-199a and miR-590 were also inconsistent with their involvement in cardiomyocyte quiescence. Consequently, miRNA microarray analysis was undertaken, which identified six discrete clusters of miRNA with characteristic developmental profiles. The functions of predicted target genes for the miRNA in four of the six clusters were enriched for aspects of cell division and regulation of cell proliferation suggesting a potential role of these miRNA in regulating cardiomyocyte proliferation.
Conclusion
The results of this study show that the expression of miR-133a and one of its target genes is consistent with it being involved in the suppression of cardiomyocyte proliferation, which occurs across the last third of gestation in sheep. The expression patterns of the miR-15 family, miR-199a and miR-590 were inconsistent with direct involvement in the regulation cardiomyocyte proliferation in sheep, despite studies in rodents demonstrating that their manipulation can influence the degree of cardiomyocyte proliferation. miRNA microarray analysis suggests a coordinated and potentially more complex role of multiple miRNA in the regulation of cardiomyocyte quiescence and highlights significant differences between species that may reflect their substantial differences in the timing of this developmental process.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1693-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s12864-015-1693-z
PMCID: PMC4509559  PMID: 26198574
Microarray; miR-133; miR-15 family; miR-590; miR-199a; Cardiomyocyte; Proliferation
22.  Estrogen Mediated-Activation of miR-191/425 Cluster Modulates Tumorigenicity of Breast Cancer Cells Depending on Estrogen Receptor Status 
PLoS Genetics  2013;9(3):e1003311.
MicroRNAs (miRNAs), single-stranded non-coding RNAs, influence myriad biological processes that can contribute to cancer. Although tumor-suppressive and oncogenic functions have been characterized for some miRNAs, the majority of microRNAs have not been investigated for their ability to promote and modulate tumorigenesis. Here, we established that the miR-191/425 cluster is transcriptionally dependent on the host gene, DALRD3, and that the hormone 17β-estradiol (estrogen or E2) controls expression of both miR-191/425 and DALRD3. MiR-191/425 locus characterization revealed that the recruitment of estrogen receptor α (ERα) to the regulatory region of the miR-191/425-DALRD3 unit resulted in the accumulation of miR-191 and miR-425 and subsequent decrease in DALRD3 expression levels. We demonstrated that miR-191 protects ERα positive breast cancer cells from hormone starvation-induced apoptosis through the suppression of tumor-suppressor EGR1. Furthermore, enforced expression of the miR-191/425 cluster in aggressive breast cancer cells altered global gene expression profiles and enabled us to identify important tumor promoting genes, including SATB1, CCND2, and FSCN1, as targets of miR-191 and miR-425. Finally, in vitro and in vivo experiments demonstrated that miR-191 and miR-425 reduced proliferation, impaired tumorigenesis and metastasis, and increased expression of epithelial markers in aggressive breast cancer cells. Our data provide compelling evidence for the transcriptional regulation of the miR-191/425 cluster and for its context-specific biological determinants in breast cancers. Importantly, we demonstrated that the miR-191/425 cluster, by reducing the expression of an extensive network of genes, has a fundamental impact on cancer initiation and progression of breast cancer cells.
Author Summary
MicroRNAs are small noncoding RNAs that act as posttranscriptional repressors of gene expression. A pivotal role for miRNAs in all the molecular processes driving initiation and progression of various malignancies, including breast cancer, has been described. Divergent miRNA expression between normal and neoplastic breast tissues has been demonstrated, as well as differential miRNA expression among the molecular subtypes of breast cancer. Over half of all breast cancers overexpress ERα, and several studies have shown that miRNA expression is controlled by ERα. We assessed the global change in microRNA expression after estrogen starvation and stimulation in breast cancer cells and identified that miR-191/425 and the host gene DALRD3 are positively associated to ERα-positive tumors. We demonstrated that ERα regulates the miR-191/425 cluster and verified the existence of a transcriptional network that allows a dual effect of estrogen on miR-191/425 and their host gene. We show that estrogen induction of miR-191/425 supports in vitro and in vivo the estrogen-dependent proliferation of ERα positive breast cancer cells. On the contrary, miR-191/425 cluster reprograms gene expression to impair tumorigenicity and metastatic potential of highly aggressive ERα negative breast cancer cells.
doi:10.1371/journal.pgen.1003311
PMCID: PMC3591271  PMID: 23505378
23.  Evidence for Positive Selection on a Number of MicroRNA Regulatory Interactions during Recent Human Evolution 
PLoS Genetics  2012;8(3):e1002578.
MicroRNA (miRNA)–mediated gene regulation is of critical functional importance in animals and is thought to be largely constrained during evolution. However, little is known regarding evolutionary changes of the miRNA network and their role in human evolution. Here we show that a number of miRNA binding sites display high levels of population differentiation in humans and thus are likely targets of local adaptation. In a subset we demonstrate that allelic differences modulate miRNA regulation in mammalian cells, including an interaction between miR-155 and TYRP1, an important melanosomal enzyme associated with human pigmentary differences. We identify alternate alleles of TYRP1 that induce or disrupt miR-155 regulation and demonstrate that these alleles are selected with different modes among human populations, causing a strong negative correlation between the frequency of miR-155 regulation of TYRP1 in human populations and their latitude of residence. We propose that local adaptation of microRNA regulation acts as a rheostat to optimize TYRP1 expression in response to differential UV radiation. Our findings illustrate the evolutionary plasticity of the microRNA regulatory network in recent human evolution.
Author Summary
MicroRNAs (miRNAs) are endogenous small RNAs that bind to their target mRNAs to post-transcriptionally repress protein production. miRNA–mediated gene regulation is usually considered to be strongly conserved among and within species, and thus alteration of such regulations is usually considered as detrimental. However, it is likely that evolutionary divergence of miRNA regulation may actually be selectively advantageous and could even serve as a genetic reservoir for innovation and adaptation. Towards this goal, we identified a number of polymorphic miRNA binding sites that display extreme population differentiation and show evidence of positive selection. We experimentally validated 3 regulations, including a regulation by miR-155 on TYRP1, a melanosomal enzyme associated with human pigmentation. We found that the two alternate alleles on the 3′ UTR of TYRP1, either inducing or disrupting repression by miR-155, are under opposite selections among human populations. This results in a strong negative correlation between the degree of fixation of miR-155–mediated repression of TYRP1 in a population and the population's latitude of residence. These observations collectively suggest miR-155 acts a rheostat to optimize TYRP1 expression for local adaptation to differential UV radiation along the latitudes. Our findings demonstrate the plasticity of miRNA regulation in recent human evolution.
doi:10.1371/journal.pgen.1002578
PMCID: PMC3310733  PMID: 22457636
24.  An Intronic microRNA Links Rb/E2F and EGFR Signaling 
PLoS Genetics  2014;10(7):e1004493.
The importance of microRNAs in the regulation of various aspects of biology and disease is well recognized. However, what remains largely unappreciated is that a significant number of miRNAs are embedded within and are often co-expressed with protein-coding host genes. Such a configuration raises the possibility of a functional interaction between a miRNA and the gene it resides in. This is exemplified by the Drosophila melanogaster dE2f1 gene that harbors two miRNAs, mir-11 and mir-998, within its last intron. miR-11 was demonstrated to limit the proapoptotic function of dE2F1 by repressing cell death genes that are directly regulated by dE2F1, however the biological role of miR-998 was unknown. Here we show that one of the functions of miR-998 is to suppress dE2F1-dependent cell death specifically in rbf mutants by elevating EGFR signaling. Mechanistically, miR-998 operates by repressing dCbl, a negative regulator of EGFR signaling. Significantly, dCbl is a critical target of miR-998 since dCbl phenocopies the effects of miR-998 on dE2f1-dependent apoptosis in rbf mutants. Importantly, this regulation is conserved, as the miR-998 seed family member miR-29 repressed c-Cbl, and enhanced MAPK activity and wound healing in mammalian cells. Therefore, the two intronic miRNAs embedded in the dE2f1 gene limit the apoptotic function of dE2f1, but operate in different contexts and act through distinct mechanisms. These results also illustrate that examining an intronic miRNA in the context of its host's function can be valuable in elucidating the biological function of the miRNA, and provide new information about the regulation of the host gene itself.
Author Summary
Animal genomes encode hundreds of microRNA genes that impact all areas of biology by limiting the expression of their targets. What remains largely unappreciated is that a significant proportion of microRNA genes are embedded within protein-coding genes, and are often co-expressed with their hosts, which raises the possibility of a functional interaction between them. The mir-998 gene is located within an intron of the gene encoding Drosophila E2F1 transcription factor. E2F1 can induce the expression of cell death genes, and its activity is negatively regulated by the pRB tumour suppressor protein. In certain settings, unrestrained E2F1 activity is sufficient to induce cell death in cells lacking functional pRB. Here, we show that miR-998 limits cell death in Rb-deficient cells by repressing dCbl, a negative regulator of Epidermal Growth Factor Receptor signaling (EGFR). miR-998 also augments EGFR signaling in differentiating photoreceptor cells. Furthermore, we show that the interaction between miR-998 and Cbl is conserved: in human cells, miR-29, a mir-29/998 seed family member, enhances EGFR signaling by targeting c-Cbl. Therefore, by examining the role of an intronic microRNA in the context of its host's function, we identified an important microRNA target and uncovered a biological function of the microRNA.
doi:10.1371/journal.pgen.1004493
PMCID: PMC4109884  PMID: 25058496
25.  Molecular insights into the origin of the Hox-TALE patterning system 
eLife  2014;3:e01939.
Despite tremendous body form diversity in nature, bilaterian animals share common sets of developmental genes that display conserved expression patterns in the embryo. Among them are the Hox genes, which define different identities along the anterior–posterior axis. Hox proteins exert their function by interaction with TALE transcription factors. Hox and TALE members are also present in some but not all non-bilaterian phyla, raising the question of how Hox–TALE interactions evolved to provide positional information. By using proteins from unicellular and multicellular lineages, we showed that these networks emerged from an ancestral generic motif present in Hox and other related protein families. Interestingly, Hox-TALE networks experienced additional and extensive molecular innovations that were likely crucial for differentiating Hox functions along body plans. Together our results highlight how homeobox gene families evolved during eukaryote evolution to eventually constitute a major patterning system in Eumetazoans.
DOI: http://dx.doi.org/10.7554/eLife.01939.001
eLife digest
Any animal with a body that is symmetric about an imaginary line that runs from its head to its tail is known as a bilaterian. Humans and most animals are bilateral, whereas jellyfish and starfish are not. Bilateral symmetry can take many forms—as demonstrated by the differences between flies, frogs and humans—but all bilaterians express many of the same genes during development.
One of these groups of genes is known as the Hox family. The expression of specific Hox genes at specific times instructs cells in the developing embryo to adopt different fates according to their position along the anterior–posterior (head to tail) axis. The patterning function of Hox genes relies on the presence of two additional cofactors that belong to the so-called TALE family. Although both Hox and TALE proteins were present early on during animal evolution, it is unclear how and when the interactions between them first began to generate symmetrical body plans.
Now, Hudry et al. have provided insights into the origin of the Hox-TALE network by analysing the expression and molecular properties of Hox and TALE proteins from various multicellular and unicellular organisms. These experiments revealed that Hox and TALE proteins of the sea anemone Nematostella, which belongs to a group of animals called cnidarians that have radial rather than bilateral symmetry, interact with one another in a similar manner to the interactions seen in bilaterians.
Hudry et al. then showed that two Nematostella Hox genes were able to substitute for their bilaterian equivalents in fruit flies, and that a Nematostella TALE gene was able to take over neuronal functions of its equivalent in Xenopus frogs. This striking conservation of function between species suggests that Hox and TALE genes were already working together in the common ancestor of all bilaterian and cnidarian animals.
By contrast, TALE members from a unicellular amoeba were unable to interact with Hox proteins, suggesting that Hox–TALE interactions first emerged in multicellular animals. In addition to increasing our knowledge of highly conserved Hox signalling, these data provide insight into the molecular mechanisms that gave rise to the symmetrical body plan that has been adopted, and adapted, by the majority of animals since.
DOI: http://dx.doi.org/10.7554/eLife.01939.002
doi:10.7554/eLife.01939
PMCID: PMC3957477  PMID: 24642410
Hox; TALE; evolution; network; transcription factors; Nematostella vectensis; D. melanogaster; other

Results 1-25 (1470149)