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1.  Epigenetic Regulation of miR-17∼92 Contributes to the Pathogenesis of Pulmonary Fibrosis 
Rationale: Idiopathic pulmonary fibrosis (IPF) is a disease of progressive lung fibrosis with a high mortality rate. In organ repair and remodeling, epigenetic events are important. MicroRNAs (miRNAs) regulate gene expression post-transcriptionally and can target epigenetic molecules important in DNA methylation. The miR-17∼92 miRNA cluster is critical for lung development and lung epithelial cell homeostasis and is predicted to target fibrotic genes and DNA methyltransferase (DNMT)-1 expression.
Objectives: We investigated the miR-17∼92 cluster expression and its role in regulating DNA methylation events in IPF lung tissue.
Methods: Expression and DNA methylation patterns of miR-17∼92 were determined in human IPF lung tissue and fibroblasts and fibrotic mouse lung tissue. The relationship between the miR-17∼92 cluster and DNMT-1 expression was examined in vitro. Using a murine model of pulmonary fibrosis, we examined the therapeutic potential of the demethylating agent, 5′-aza-2′-deoxycytidine.
Measurements and Main Results: Compared with control samples, miR-17∼92 expression was reduced in lung biopsies and lung fibroblasts from patients with IPF, whereas DNMT-1 expression and methylation of the miR-17∼92 promoter was increased. Several miRNAs from the miR-17∼92 cluster targeted DNMT-1 expression resulting in a negative feedback loop. Similarly, miR-17∼92 expression was reduced in the lungs of bleomycin-treated mice. Treatment with 5′-aza-2′-deoxycytidine in a murine bleomycin-induced pulmonary fibrosis model reduced fibrotic gene and DNMT-1 expression, enhanced miR-17∼92 cluster expression, and attenuated pulmonary fibrosis.
Conclusions: This study provides insight into the pathobiology of IPF and identifies a novel epigenetic feedback loop between miR-17∼92 and DNMT-1 in lung fibrosis.
doi:10.1164/rccm.201205-0888OC
PMCID: PMC3603596  PMID: 23306545
microRNA; miR-17∼92; pulmonary fibrosis; DNA methylation; DNMT-1
2.  miR-21 mediates fibrogenic activation of pulmonary fibroblasts and lung fibrosis 
The Journal of Experimental Medicine  2010;207(8):1589-1597.
Uncontrolled extracellular matrix production by fibroblasts in response to tissue injury contributes to fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), a progressive and ultimately fatal process that currently has no cure. Although dysregulation of miRNAs is known to be involved in a variety of pathophysiologic processes, the role of miRNAs in fibrotic lung diseases is unclear. In this study, we found up-regulation of miR-21 in the lungs of mice with bleomycin-induced fibrosis and also in the lungs of patients with IPF. Increased miR-21 expression was primarily localized to myofibroblasts. Administration of miR-21 antisense probes diminished the severity of experimental lung fibrosis in mice, even when treatment was started 5–7 d after initiation of pulmonary injury. TGF-β1, a central pathological mediator of fibrotic diseases, enhanced miR-21 expression in primary pulmonary fibroblasts. Increasing miR-21 levels promoted, whereas knocking down miR-21 attenuated, the pro-fibrogenic activity of TGF-β1 in fibroblasts. A potential mechanism for the role of miR-21 in fibrosis is through regulating the expression of an inhibitory Smad, Smad7. These experiments demonstrate an important role for miR-21 in fibrotic lung diseases and also suggest a novel approach using miRNA therapeutics in treating clinically refractory fibrotic diseases, such as IPF.
doi:10.1084/jem.20100035
PMCID: PMC2916139  PMID: 20643828
3.  Role of MicroRNAs in Fibrosis 
Fibrosis is the leading cause of organ dysfunction in diseases such as systemic sclerosis, liver cirrhosis, cardiac fibrosis, progressive kidney disease, and idiopathic pulmonary fibrosis. The hallmark of fibrosis is tissue remodeling with excess deposition of extracellular matrix components, predominantly collagens. Different cell types, cytokines, growth factors, and enzymes interact in complex pathogenic networks with myofibroblasts playing a pivotal role. MicroRNAs are small non-coding RNAs acting as negative regulators of gene expression at the post-transcriptional level. MicroRNAs have been associated with many basic cellular processes as well as with a wide spectrum of diseases, most notably cancer. This review provides a comprehensive overview of microRNAs regulating profibrotic pathways and extracellular matrix synthesis. The potential of miRNA for targeted therapeutic approaches in fibrotic disorders is also discussed.
doi:10.2174/1874312901206010130
PMCID: PMC3396185  PMID: 22802911
Fibrosis; fibroblasts; microRNA (miRNA)-mediated gene regulation regulation; transforming growth factor-beta (TGF-β); connective tissue growth factor (CTGF); extracellular matrix (ECM); epithelial-to-mesenchymal transition (EMT); signaling pathways; antagomirs.
4.  MicroRNA profiling implicates the insulin-like growth factor pathway in bleomycin-induced pulmonary fibrosis in mice 
Background
Idiopathic pulmonary fibrosis is a disease characterized by alveolar epithelial cell injury, inflammatory cell infiltration and deposition of extracellular matrix in lung tissue. As mouse models of bleomycin-induced pulmonary fibrosis display many of the same phenotypes observed in patients with idiopathic pulmonary fibrosis, they have been used to study various aspects of the disease, including altered expression of microRNAs.
Results
In this work, microRNA expression profiling of the lungs from treated C57BL/6J mice, relative to that of untreated controls, was undertaken to determine which alterations in microRNAs could in part regulate the fibrosis phenotype induced by bleomycin delivered through mini-osmotic pumps. We identified 11 microRNAs, including miR-21 and miR-34a, to be significantly differentially expressed (P < 0.01) in lungs of bleomycin treated mice and confirmed these data with real time PCR measurements. In situ hybridization of both miR-21 and miR-34a indicated that they were expressed in alveolar macrophages. Using a previously reported gene expression profile, we identified 195 genes to be both predicted targets of the 11 microRNAs and of altered expression in bleomycin-induced lung disease of C57BL/6J mice. Pathway analysis with these 195 genes indicated that altered microRNA expression may be associated with hepatocyte growth factor signaling, cholecystokinin/gastrin-mediated signaling, and insulin-like growth factor (IGF-1) signaling, among others, in fibrotic lung disease. The relevance of the IGF-1 pathway in this model was then demonstrated by showing lung tissue of bleomycin treated C57BL/6J mice had increased expression of Igf1 and that increased numbers of Igf-1 positive cells, predominantly in macrophages, were detected in the lungs.
Conclusions
We conclude that altered microRNA expression in macrophages is a feature which putatively influences the insulin-like growth factor signaling component of bleomycin-induced pulmonary fibrosis.
doi:10.1186/1755-1536-6-16
PMCID: PMC3766165  PMID: 23987664
Pulmonary fibrosis; microRNA; Bleomycin; Insulin-like growth factor; Pathway analysis; Mouse model
5.  Profibrotic Role of miR-154 in Pulmonary Fibrosis 
In this study, we explored the regulation and the role of up-regulated microRNAs in idiopathic pulmonary fibrosis (IPF), a progressive interstitial lung disease of unknown origin. We analyzed the expression of microRNAs in IPF lungs and identified 43 significantly up-regulated microRNAs. Twenty-four of the 43 increased microRNAs were localized to the chromosome 14q32 microRNA cluster. We validated the increased expression of miR-154, miR-134, miR-299–5p, miR-410, miR-382, miR-409–3p, miR-487b, miR-31, and miR-127 by quantitative RT-PCR and determined that they were similarly expressed in embryonic lungs. We did not find evidence for differential methylation in this region, but analysis of transcription factor binding sites identified multiple SMAD3-binding elements in the 14q32 microRNA cluster. TGF-β1 stimulation of normal human lung fibroblasts (NHLF) caused up-regulation of microRNAs on chr14q32 that were also increased in IPF lungs. Chromatin immunoprecipitation confirmed binding of SMAD3 to the putative promoter of miR-154. Mir-154 was increased in IPF fibroblasts, and transfection of NHLF with miR-154 caused significant increases in cell proliferation and migration. The increase in proliferation induced by TGF-β was not observed when NHLF or IPF fibroblasts were transfected with a mir-154 inhibitor. Transfection with miR-154 caused activation of the WNT pathway in NHLF. ICG-001 and XAV939, inhibitors of the WNT/β-catenin pathway, reduced the proliferative effect of miR-154. The potential role of miR-154, one of multiple chr14q32 microRNA cluster members up-regulated in IPF and a regulator of fibroblast migration and proliferation, should be further explored in IPF.
doi:10.1165/rcmb.2011-0377OC
PMCID: PMC3547095  PMID: 23043088
idiopathic pulmonary fibrosis; microRNA; TGF-β; WNT/β-catenin; developmental pathways
6.  miR-93, miR-98 and miR-197 regulate expression of tumor suppressor gene FUS1 
Molecular cancer research : MCR  2009;7(8):1234-1243.
FUS1 is a tumor suppressor gene located on human chromosome 3p21, and expression of Fus1 protein is highly regulated at various levels, leading to lost or greatly diminished tumor suppressor function in many lung cancers. Here we show that selected microRNAs (miRNAs) interact with the 3’ untranslated region (3’ UTR) of FUS1, leading to down-regulation of protein expression. Using computational methods, we first predicted that FUS1 is a target of three miRNAs, miR-93, miR-98 and miR-197, and then showed that exogenous over-expression of these miRNAs inhibited Fus1 protein expression. We then confirmed that the three miRNAs target the 3’UTR region of the FUS1 transcript, and that individual deletion of the three miRNA target sites in the FUS1 3’UTR restores the expression level of Fus1 protein. We further found that miR-93 and miR-98 are expressed at higher levels in small cell lung cancer cell lines (SCLC) than in non-small cell lung cancer cell lines (NSCLC) and immortalized human bronchial epithelial cells (HBECs), and that miR-197 is expressed at higher levels in both SCLC and NSCLC than in HBECs. Finally, we found that elevated miR-93 and miR-197 expression is correlated with reduced Fus1 expression in NSCLC tumor specimens. These results suggest that the three miRNAs are negative regulators of Fus1 expression in lung cancers.
doi:10.1158/1541-7786.MCR-08-0507
PMCID: PMC2741087  PMID: 19671678
microRNA; lung cancer; tumor suppressor gene
7.  MiR-101 and miR-144 Regulate the Expression of the CFTR Chloride Channel in the Lung 
PLoS ONE  2012;7(11):e50837.
The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a chloride channel that plays a critical role in the lung by maintaining fluid homeostasis. Absence or malfunction of CFTR leads to Cystic Fibrosis, a disease characterized by chronic infection and inflammation. We recently reported that air pollutants such as cigarette smoke and cadmium negatively regulate the expression of CFTR by affecting several steps in the biogenesis of CFTR protein. MicroRNAs (miRNAs) have recently received a great deal of attention as both biomarkers and therapeutics due to their ability to regulate multiple genes. Here, we show that cigarette smoke and cadmium up-regulate the expression of two miRNAs (miR-101 and miR-144) that are predicted to target CFTR in human bronchial epithelial cells. When premature miR-101 and miR-144 were transfected in human airway epithelial cells, they directly targeted the CFTR 3′UTR and suppressed the expression of the CFTR protein. Since miR-101 was highly up-regulated by cigarette smoke in vitro, we investigated whether such increase also occurred in vivo. Mice exposed to cigarette smoke for 4 weeks demonstrated an up-regulation of miR-101 and suppression of CFTR protein in their lungs. Finally, we show that miR-101 is highly expressed in lung samples from patients with severe chronic obstructive pulmonary disease (COPD) when compared to control patients. Taken together, these results suggest that chronic cigarette smoking up-regulates miR-101 and that this miRNA could contribute to suppression of CFTR in the lungs of COPD patients.
doi:10.1371/journal.pone.0050837
PMCID: PMC3511328  PMID: 23226399
8.  Increased Circulating miR-21 Levels Are Associated with Kidney Fibrosis 
PLoS ONE  2013;8(2):e58014.
MicroRNAs (miRNAs) are a class of noncoding RNA acting at a post-transcriptional level to control the expression of large sets of target mRNAs. While there is evidence that miRNAs deregulation plays a causative role in various complex disorders, their role in fibrotic kidney diseases is largely unexplored. Here, we found a strong up-regulation of miR-21 in the kidneys of mice with unilateral ureteral obstruction and also in the kidneys of patients with severe kidney fibrosis. In addition, mouse primary fibroblasts derived from fibrotic kidneys exhibited higher miR-21 expression level compared to those derived from normal kidneys. Expression of miR-21 in normal primary kidney fibroblasts was induced upon TGFβ exposure, a key growth factor involved in fibrogenesis. Finally, ectopic expression of miR-21 in primary kidney fibroblasts was sufficient to promote myofibroblast differentiation. As circulating miRNAs have been suggested as promising non-invasive biomarkers, we further assess whether circulating miR-21 levels are associated with renal fibrosis using sera from 42 renal transplant recipients, categorized according to their renal fibrosis severity, evaluated on allograft biopsies (Interstitial Fibrosis/Tubular Atrophy (IF/TA). Circulating miR-21 levels are significantly increased in patients with severe IF/TA grade (IF/TA grade 3: 3.0±1.0 vs lower grade of fibrosis: 1.5±1.2; p = 0.001). By contrast, circulating miR-21 levels were not correlated with other renal histological lesions. In a multivariate linear regression model including IF/TA grade and estimated GFR, independent associations were found between circulating miR-21 levels and IF/TA score (ß = 0.307, p = 0.03), and between miR-21 levels and aMDRD (ß = −0.398, p = 0.006). Altogether, these data suggest miR-21 has a key pathogenic role in kidney fibrosis and may represent a novel, predictive and reliable blood marker of kidney fibrosis.
doi:10.1371/journal.pone.0058014
PMCID: PMC3585177  PMID: 23469132
9.  Hepatic miR-29ab1 expression modulates chronic hepatic injury 
microRNAs (miRNAs) are small, regulatory noncoding RNAs that have potent effects on gene expression. Several miRNA are deregulated in cellular processes involved in human liver diseases and regulation of cellular processes. Recent studies have identified the involvement of miR-29 in hepatic fibrosis and carcinogenesis. Although several targets of miR-29 have been identified, there is limited information regarding the cell-type specific roles of miR-29 in the liver, and we sought to evaluate the role of this miRNA in hepatic pathobiology. We report the generation of a tissue–specific knock-out mouse to evaluate the role of miR-29 in hepatic fibrosis and carcinogenesis in response to injury. We hypothesized that miR-29 contributes to the hepatocyte driven response to chronic cellular injury that results in fibrosis. In support of this hypothesis, fibrosis and mortality were enhanced in miR29 knockout mice in response to carbon tetrachloride. Genome-wide gene expression analysis identified an over-representation of genes associated with fibrosis. The oncofetal RNA H19 was modulated in a miR-29 dependent manner following exposure to carbon tetrachloride in vivo. The impact of a hepatocyte specific miR-29 knock-out on survival following chronic hepatic injury in vivo implicates this miRNA as a potential target for intervention. These results provide evidence of the involvement of miR-29 in chronic hepatic injury, and suggest a role for deregulated hepatocyte expression of miR-29 in the response to hepatic injury, fibrosis and carcinogenesis.
doi:10.1111/j.1582-4934.2012.01578.x
PMCID: PMC3923513  PMID: 22469499
fibrosis; liver cancer; transgenic
10.  Identification of Dynamically Regulated microRNA and mRNA Networks in Developing Oligodendrocytes 
MicroRNAs (miRNAs) play important roles in modulating gene expression at the post-transcriptional level. In postnatal oligodendrocyte lineage cells, the miRNA expression profile -“microRNAome” - contains 43 miRNAs whose expression dynamically changes during the transition from A2B5+ oligodendrocyte progenitor cells to premyelinating GalC+ cells. The combination of microRNAome profiling with analyses of the oligodendrocyte transcriptome reveals a target bias for a class of miRNAs which includes miR-9. We show that miR-9 is down-regulated during oligodendrocyte differentiation. In addition, miR-9 expression level inversely correlates with the expression of its predicted targets, among which is the peripheral myelin protein PMP22. We found that PMP22 mRNA but not protein is detectable in oligodendrocytes, while Schwann cells producing PMP22 protein lack miR-9. We demonstrate that miR-9 interacts with the 3’ untranslated region of PMP22 and down-regulates its expression. Our results support models in which miRNAs can act as guardians of the transcriptome.
doi:10.1523/JNEUROSCI.1932-08.2008
PMCID: PMC2646797  PMID: 18987208
microRNA; post-transcriptional regulation; oligodendrocyte; PMP22; myelin; glia
11.  miRNA-127 Inhibits Lung Inflammation by Targeting IgG Fcγ Receptor I 
The molecular mechanisms of acute lung injury are incompletely understood. MicroRNAs (miRNAs) are crucial biological regulators that act by suppressing their target genes and are involved in a variety of pathophysiologic processes. miR-127 appears to be down-regulated during lung injury. We set out to investigate the role of miR-127 in lung injury and inflammation. Expression of miR-127 significantly reduced cytokine release by macrophages. Looking into the mechanisms of regulation of inflammation by miR-127, we found that IgG Fcγ Receptor I (FcγRI/CD64) was a target of miR-127, as evidenced by reduced CD64 protein expression in macrophages over-expressing miR-127. Furthermore, miR-127 significantly reduced the luciferase activity with a reporter construct containing the native 3′-UTR of CD64. Importantly, we demonstrated that miR-127 attenuated lung inflammation in an IgG immune complex (IgG IC) model in vivo. Collectively, these data show that miR-127 targets macrophage CD64 expression and promotes the reduction of lung inflammation. Understanding how miRNAs regulate lung inflammation may represent an attractive way to control inflammation induced by infectious or non-infectious lung injury.
doi:10.4049/jimmunol.1101070
PMCID: PMC3288289  PMID: 22287715
12.  miR-199a-5p Is Upregulated during Fibrogenic Response to Tissue Injury and Mediates TGFbeta-Induced Lung Fibroblast Activation by Targeting Caveolin-1 
PLoS Genetics  2013;9(2):e1003291.
As miRNAs are associated with normal cellular processes, deregulation of miRNAs is thought to play a causative role in many complex diseases. Nevertheless, the precise contribution of miRNAs in fibrotic lung diseases, especially the idiopathic form (IPF), remains poorly understood. Given the poor response rate of IPF patients to current therapy, new insights into the pathogenic mechanisms controlling lung fibroblasts activation, the key cell type driving the fibrogenic process, are essential to develop new therapeutic strategies for this devastating disease. To identify miRNAs with potential roles in lung fibrogenesis, we performed a genome-wide assessment of miRNA expression in lungs from two different mouse strains known for their distinct susceptibility to develop lung fibrosis after bleomycin exposure. This led to the identification of miR-199a-5p as the best miRNA candidate associated with bleomycin response. Importantly, miR-199a-5p pulmonary expression was also significantly increased in IPF patients (94 IPF versus 83 controls). In particular, levels of miR-199a-5p were selectively increased in myofibroblasts from injured mouse lungs and fibroblastic foci, a histologic feature associated with IPF. Therefore, miR-199a-5p profibrotic effects were further investigated in cultured lung fibroblasts: miR-199a-5p expression was induced upon TGFβ exposure, and ectopic expression of miR-199a-5p was sufficient to promote the pathogenic activation of pulmonary fibroblasts including proliferation, migration, invasion, and differentiation into myofibroblasts. In addition, we demonstrated that miR-199a-5p is a key effector of TGFβ signaling in lung fibroblasts by regulating CAV1, a critical mediator of pulmonary fibrosis. Remarkably, aberrant expression of miR-199a-5p was also found in unilateral ureteral obstruction mouse model of kidney fibrosis, as well as in both bile duct ligation and CCl4-induced mouse models of liver fibrosis, suggesting that dysregulation of miR-199a-5p represents a general mechanism contributing to the fibrotic process. MiR-199a-5p thus behaves as a major regulator of tissue fibrosis with therapeutic potency to treat fibroproliferative diseases.
Author Summary
Fibrosis is the final common pathway in virtually all forms of chronic organ failure, including lung, liver, and kidney, and is a leading cause of morbidity and mortality worldwide. Fibrosis results from the excessive activity of fibroblasts, in particular a differentiated form known as myofibroblast that is responsible for the excessive and persistent accumulation of scar tissue and ultimately organ failure. Idiopathic Lung Fibrosis (IPF) is a chronic and often rapidly fatal pulmonary disorder of unknown origin characterized by fibrosis of the supporting framework (interstitium) of the lungs. Given the poor prognosis of IPF patients, new insights into the biology of (myo)fibroblasts is of major interest to develop new therapeutics aimed at reducing (myo)fibroblast activity to slow or even reverse disease progression, thereby preserving organ function and prolonging life. MicroRNAs (miRNAs), a class of non-coding RNA recently identified, are associated with normal cellular processes; and deregulation of miRNAs plays a causative role in a vast array of complex diseases. In this study, we identified a particular miRNA: miR-199a-5p that governs lung fibroblast activation and ultimately lung fibrosis. Overall we showed that miR-199a-5p is a major regulator of fibrosis with strong therapeutic potency to treat fibroproliferative diseases such as IPF.
doi:10.1371/journal.pgen.1003291
PMCID: PMC3573122  PMID: 23459460
13.  Inhibition of hypoxia-induced miR-155 radiosensitizes hypoxic lung cancer cells 
Cancer Biology & Therapy  2011;12(10):908-914.
miR-155 is a prominent microRNA (miRNA) that regulates genes involved in immunity and cancer-related pathways. miR-155 is overexpressed in lung cancer, which correlates with poor patient prognosis. It is unclear how miR-155 becomes increased in lung cancers and how this increase contributes to reduced patient survival. Here, we show that hypoxic conditions induce miR-155 expression in lung cancer cells and trigger a corresponding decrease in a validated target, FOXO3A. Furthermore, we find that increased levels of miR-155 radioprotects lung cancer cells, while inhibition of miR-155 radiosensitizes these cells. Moreover, we reveal a therapeutically important link between miR-155 expression, hypoxia, and irradiation by demonstrating that anti-miR-155 molecules also sensitize hypoxic lung cancer cells to irradiation. Our study helps explain how miR-155 becomes elevated in lung cancers, which contain extensive hypoxic microenvironments, and demonstrates that inhibition of miR-155 may have important therapeutic potential as a means to radiosensitize hypoxic lung cancer cells.
doi:10.4161/cbt.12.10.17681
PMCID: PMC3280906  PMID: 22027557
microRNAs; miR-155; hypoxia; radiosensitizer; lung cancer
14.  MicroRNAs and fibrosis 
Purpose of review
MicroRNAs (miRNAs) are short noncoding RNAs that inhibit gene expression in plants and animals. miRNAs have emerged as key players in virtually all aspects of mammalian biology. Aberrant miRNA expression is observed in numerous human diseases such as diabetes, hypercholesterolemia, cancer, and tissue fibrosis. Therefore, approaches to correct miRNA expression represent the novel therapeutic strategies for these diseases.
Recent findings
miRNAs are essential for kidney development and homeostasis. Aberrant miRNA expression is observed in the mouse models of kidney fibrosis. Three TGF-β-regulated miRNA families, miR-21, miR-200, and miR-29 have been shown to modulate renal fibrosis. miR-21, through a feed-forward loop, amplifies TGF-β signaling and promotes fibrosis. Conversely, miR-200 and miR-29 reduce fibrosis by inhibiting epithelial-tomesenchymal transition and preventing the deposition of extracellular matrix, respectively. Inhibition of miR-21 expression or augmenting miR-29 expression prevents kidney fibrosis in mice.
Summary
Aberrant miRNA expression perturbs signaling pathways that lead to progression of kidney fibrosis. Thus, miRNAs represent novel biomarkers and therapeutic targets in the treatment of kidney fibrosis.
doi:10.1097/MNH.0b013e328354e559
PMCID: PMC3399722  PMID: 22622653
kidney fibrosis; microRNA; miR-200; miR-21; miR-29
15.  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
16.  Decreased microRNA-214 levels in breast cancer cells coincides with increased cell proliferation, invasion and accumulation of the Polycomb Ezh2 methyltransferase 
Carcinogenesis  2011;32(11):1607-1614.
MicroRNAs (miRNAs) are small non-coding RNAs, which regulate gene expression by inhibiting translation or promoting degradation of specific target messenger RNAs (mRNAs). Alteration of the levels of a number of miRNAs is common in solid and hematological tumors. We have shown previously that miR-214 regulates Ezh2 in skeletal muscle and embryonic stem cells. The current study was aimed at examining the role of miR-214 in breast cancer where miR-214 levels are reduced but whether this phenomenon bears a functional relevance is unknown. MiR-214 expression was inversely correlated with Ezh2 mRNA and protein levels in breast cancer cell lines and at least one copy of the miR-214 alleles was found to be deleted in 24% (6/25) of primary breast tumors. Experimental increase of miR-214 in breast cancer cell lines correlated with reduction of Ezh2 protein levels, a known marker of invasion and aggressive breast cancer behavior. Supporting a direct targeting mechanism, miR-214 decreased luciferase activity from a construct containing the Ezh2 3′ untranslated region. Expression of miR-214 specifically reduced cell proliferation of breast cancer cells and inhibited the invasive potential of a highly metastatic breast cancer cell line. These findings indicate that reduced miR-214 levels may contribute to breast tumorigenesis by allowing abnormally elevated Ezh2 accumulation and subsequent unchecked cell proliferation and invasion.
doi:10.1093/carcin/bgr184
PMCID: PMC3204346  PMID: 21828058
17.  Involvement of microRNA-Mediated Gene Expression Regulation in the Pathological Development of Stem Canker Disease in Populus trichocarpa 
PLoS ONE  2012;7(9):e44968.
MicroRNAs (miRNAs), a type of short (21–23 nucleotides), non-coding RNA molecule, mediate repressive gene regulation through RNA silencing at the post-transcriptional level, and play an important role in defense and response to abiotic and biotic stresses. In the present study, Affymetrix® miRNA Array, real-time quantitative PCR (qPCR) for miRNAs and their targets, and miRNA promoter analysis were used to validate the gene expression patterns of miRNAs in Populus trichocarpa plantlets induced with the poplar stem canker pathogen, Botryosphaeria dothidea. Twelve miRNAs (miR156, miR159, miR160, miR164, miR166, miR168, miR172, miR319, miR398, miR408, miR1448, and miR1450) were upregulated in the stem bark of P. trichocarpa, but no downregulated miRNAs were found. Based on analysis of the miRNAs and their targets, a potential co-regulatory network was developed to describe post-transcriptional regulation in the pathological development of poplar stem canker. There was highly complex cross-talk between diverse miRNA pathway responses to biotic and abiotic stresses. The results suggest that miR156 is probably an integral component of the miRNA response to all environmental stresses in plants. Cis-regulatory elements were binding sites for the transcription factors (TFs) on DNA. Promoter analysis revealed that TC-rich repeats and a W1-box motif were both tightly related disease response motifs in Populus. Promoter analysis and target analysis of miRNAs also revealed that some TFs regulate their activation/repression. Furthermore, a feedback regulatory network in the pathological development of poplar stem canker is provided. The results confirm that miRNA pathways regulate gene expression during the pathological development of plant disease, and provide new insights into understanding the onset and development of poplar stem canker.
doi:10.1371/journal.pone.0044968
PMCID: PMC3445618  PMID: 23028709
18.  MiR-21 plays an Important Role in Radiation Induced Carcinogenesis in BALB/c Mice by Directly Targeting the Tumor Suppressor Gene Big-h3 
Dysregulation of certain microRNAs (miRNAs) in cancer can promote tumorigenesis, metastasis and invasion. However, the functions and targets of only a few mammalian miRNAs are known. In particular, the miRNAs that participates in radiation induced carcinogenesis and the miRNAs that target the tumor suppressor gene Big-h3 remain undefined. Here in this study, using a radiation induced thymic lymphoma model in BALB/c mice, we found that the tumor suppressor gene Big-h3 is down-regulated and miR-21 is up-regulated in radiation induced thymic lymphoma tissue samples. We also found inverse correlations between Big-h3 protein and miR-21 expression level among different tissue samples. Furthermore, our data indicated that miR-21 could directly target Big-h3 in a 3′UTR dependent manner. Finally, we found that miR-21 could be induced by TGFβ, and miR-21 has both positive and negative effects in regulating TGFβ signaling. We conclude that miR-21 participates in radiation induced carcinogenesis and it regulates TGFβ signaling.
PMCID: PMC3076505  PMID: 21494432
Radiation-induced carcinogenesis; Radiation induced thymic lymphoma; MicroRNA; miR-21; Big-h3; TGFβ.
19.  Role of MicroRNA-26b in Glioma Development and Its Mediated Regulation on EphA2 
PLoS ONE  2011;6(1):e16264.
Background
MicroRNAs (miRNAs) are short, non-coding RNAs that regulate the expression of multiple target genes. Deregulation of miRNAs is common in human tumorigenesis. Low level expression of miR-26b has been found in glioma cells. However, its underlying mechanism of action has not been determined.
Methodology/Principal Findings
Real-time PCR was employed to measure the expression level of miR-26b in glioma patients and cells. The level of miR-26b was inversely correlated with the grade of glioma. Ectopic expression of miR-26b inhibited the proliferation, migration and invasion of human glioma cells. A binding site for miR-26b was identified in the 3′UTR of EphA2. Over-expression of miR-26b in glioma cells repressed the endogenous level of EphA2 protein. Vasculogenic mimicry (VM) experiments were performed to further confirm the effects of miR-26b on the regulation of EphA2, and the results showed that miR-26b inhibited the VM processes which regulated by EphA2.
Significance
This study demonstrated that miR-26b may act as a tumor suppressor in glioma and it directly regulates EphA2 expression. EphA2 is a direct target of miR-26b, and the down-regulation of EphA2 mediated by miR-26b is dependent on the binding of miR-26b to a specific response element of microRNA in the 3′UTR region of EphA2 mRNA.
doi:10.1371/journal.pone.0016264
PMCID: PMC3021542  PMID: 21264258
20.  Clinical relevance of microRNA miR-21, miR-31, miR-92a, miR-101, miR-106a and miR-145 in colorectal cancer 
BMC Cancer  2012;12:505.
Background
MicroRNAs (miRNAs) regulate gene expression by binding to mRNA, and can function as oncogenes or tumor suppressors depending on the target. In this study, using qRT-PCR, we examined the expression of six miRNAs (miR-21, miR-31, miR-92a, miR-101, miR-106a and miR-145) in tumors from 193 prospectively recruited patients with colorectal cancer, and associations with clinicopathological parameters and patient outcome were analyzed. The miRNAs were chosen based on previous studies for their biomarker potential and suggested biological relevance in colorectal cancer.
Methods
The miRNA expression was examined by qRT-PCR. Associations between miRNA expression and clinicopathological variables were explored using Mann–Whitney U and Kruskal-Wallis test while survival was estimated using the Kaplan-Meier method and compared using the log-rank test.
Results
MiR-101 was hardly expressed in the tumor samples, while for the other miRNAs, variable expression levels and expression ranges were observed, with miR-21 being most abundantly expressed relative to the reference (RNU44). In our study cohort, major clinical significance was demonstrated only for miR-31, as high expression was associated with advanced tumor stage and poor differentiation. No significant associations were found between expression of the investigated miRNAs and metastasis-free or overall survival.
Conclusions
Investigating the expression of six miRNAs previously identified as candidate biomarkers in colorectal cancer, few clinically relevant associations were detected in our patient cohort. Our results emphasize the importance of validating potential tumor markers in independent patient cohorts, and indicate that the role of miRNAs as colorectal cancer biomarkers is still undetermined.
doi:10.1186/1471-2407-12-505
PMCID: PMC3519622  PMID: 23121918
MiRNA; Colorectal cancer; Prognostic biomarker
21.  HBx Down-Regulated Gld2 Plays a Critical Role in HBV-Related Dysregulation of miR-122 
PLoS ONE  2014;9(3):e92998.
miR-122 is a liver-rich-specific microRNA that plays an important role in hepatic gene expression via post-transcription regulation, and it is potentially associated with the development of hepatocellular carcinoma. It has been confirmed that miR-122 is down-regulated during HBV infection; however, how HBV affects miR-122 is still debated. One research provided evidence that HBx could reduce the miR-122 transcription level, but the other insisted that HBV had no significant effect on miR-122 transcription level but reduce miR-122 level via binding and sequestering endogenous miR-122. It is determinate that Gld2 could increase the specific miRNA stabilization by monoadenylation which was a post-transcription regulation. In this study, we aimed to investigate the mechanism of HBV-induced reduction of miR-122 and examine whether Gld2 is involved in it. According to the results of a microRNA microarray, we found miR-122 was the most down-regulated microRNA in HepG2.2.15 compared to HepG2. As revealed by qRT-PCR and western blotting analyses, both miR-122 and Gld2 levels were reduced in hepatic cell lines with expression of HBV or HBx but not other proteins of HBV, and over-expression of Gld2 could abolish the effect of HBV and HBx on the miR-122 level. What's more, both HBV and HBx have no significant effect on pre-miR-122 levels. And the dual-luciferase assay implicated that HBx could reduce the Gld2 promoter activity but had no significant effect on miR-122 promoter activity. In conclusion, HBx is a critical protein derived from HBV, which regulates miR-122 via down-regulating Gld2.
doi:10.1371/journal.pone.0092998
PMCID: PMC3965513  PMID: 24667324
22.  Negative Regulation of the Tumor Suppressor p53 Gene by MicroRNAs 
Oncogene  2010;30(7):843-853.
The tumor suppressor p53, encoded by the TP53 gene, is recognized as the guardian of the human genome because it regulates many downstream genes to exercise its function in cell cycle and cell death. Recent reports have revealed that several microRNAs (miRNAs) are important components of the p53 tumor suppressor network with miR-125b and miR-504 directly targeting TP53. In this report, we use a screening method to identify that two miRNAs (miR-25 and miR-30d) directly target the 3'UTR of TP53 to down-regulate p53 protein levels and reduce the expression of genes that are transcriptionally activated by p53. Correspondingly, both miR-25 and miR-30d adversely affect apoptotic cell death, cell cycle arrest, and cellular senescence. Inhibition of either miR-25 or miR-30d expression increases endogenous p53 expression and elevates cellular apoptosis in several cell lines, including one from multiple myeloma that has little TP53 mutations. Thus, beyond miR-125b and miR-504, the human TP53 gene is negatively regulated by two more miRNAs: miR-25 and miR-30d.
doi:10.1038/onc.2010.457
PMCID: PMC3021102  PMID: 20935678
microRNA; miR-25; miR-30d; p53; senescence; apoptosis; cell cycle arrest; multiple myeloma
23.  Selective microRNA Suppression in Human Thoracic Aneurysms: Relationship of miR-29a to Aortic Size and Proteolytic Induction 
Background
Increasing evidence points to a direct role for altered microRNA (miRNA or miR) expression levels in cardiovascular remodeling and disease progression. While alterations in miR expression levels have been directly linked to cardiac hypertrophy, fibrosis, and remodeling, their role in regulating gene expression during thoracic aortic aneurysm (TAA) development has yet to be explored.
Methods and Results
The present study examined miR expression levels in aortic tissue specimens collected from patients with ascending TAAs by quantitative real-time PCR, and observed decreased miR expression (miRs -1, -21, -29a, -133a, and -486) as compared to normal aortic specimens. A significant relationship between miR expression levels (miRs -1, -21, -29a, and -133a) and aortic diameter was identified; as aortic diameter increased, miR expression decreased. Using a bioinformatics approach, members of the matrix metalloproteinase (MMP) family, proteins involved in TAA development, were examined for putative miR binding sites. MMP-2 and MMP-9 were identified as potential targets for miR-29a and miR-133a respectively, and MMP-2 was subsequently verified as a miR-29a target in vitro. A significant inverse relationship between miR-29a and total MMP-2 was then identified in the clinical TAA specimens.
Conclusions
These findings demonstrate altered miR expression patterns in clinical TAA specimens, suggesting that the loss of specific miR expression may allow for the elaboration of specific MMPs capable of driving aortic remodeling during TAA development. Importantly, these data suggest that these miRs have biological and clinical relevance to the behavior of TAAs, and may provide significant targets for therapeutic and diagnostic applications.
doi:10.1161/CIRCGENETICS.111.960419
PMCID: PMC3246193  PMID: 22010139
aneurysm; thoracic aorta; microRNA; MMP; remodeling
24.  Angiotensin II upregulated the expression of microRNA-224 but not microRNA-21 in adult rat cardiac fibroblasts 
Biomedical Reports  2013;1(5):776-780.
The role of microRNA-21 (miRNA-21, miR-21) in cardiac fibrosis remains controversial, while the role of microRNA-224 (miRNA-224, miR-224) in cardiac fibroblasts has not been reported. Angiotensin II (Ang II) is known to play a pivotal role in the pathogenesis of cardiac fibrosis. The aim of this study was to confirm whether the expression of miR-21 and miR-224 is regulated by Ang II in adult rat cardiac fibroblasts. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and quantitative PCR (qPCR) were performed to measure the levels of miR-21 and miR-224 in Ang II-treated or untreated adult rat cardiac fibroblasts. The RT-PCR, qPCR and previous miRNA array results demonstrated that treatment with Ang II (100 nM) for 24 h did not induce the increase of miR-21 in cardiac fibroblasts, although the level of miR-21 in cardiac fibroblasts was not considered as low. The results of the present study also demonstrated that Ang II significantly upregulated the expression of miR-224 in adult rat cardiac fibroblasts. Bioinformatic analysis revealed that the potential target genes of miR-224 included SMAD4, SMAD5, cyclin-dependent kinase 9 and early growth response 1/2. In previous studies, it was reported that miR-224 was upregulated in tumors by promoting cell proliferation and targeting SMAD4. Those results indicated the potential roles of miR-224 in cardiac fibroblasts and cardiac fibrosis. In conclusion, results of the present study demonstrated that miR-21 was not induced by Ang II, whereas Ang II upregulated miR-224 expression in adult rat cardiac fibroblasts, a finding that may provide a starting point for the investigation of the potential role of miR-224 in cardiac fibrosis.
doi:10.3892/br.2013.144
PMCID: PMC3916944  PMID: 24649028
angiotensin II; cardiac fibroblasts; microRNA-224; microRNA-21
25.  microRNA-101 inhibits lung cancer invasion through the regulation of enhancer of zeste homolog 2 
microRNAs (miRNAs) may function as oncogenes or tumor-suppressor genes depending on the targets that are regulated. Enhancer of zeste homolog 2 (EZH2) is the target of miR-101 and a member of the polycomb repressive complex 2, which is involved in the methylation of histone H3 at lysine 27 (H3K27). Therefore, we aimed to ascertain whether or not the overexpression of miR-101 inhibits invasion of lung cancer through regulation of EZH2. In this study, the expression of miR-101 was down-regulated and the expression of EZH2 was up-regulated in lung cancer. Global methylation of H3K27 was higher in metastatic lung cancer than in early lung cancer lesions. Overexpression of miR-101 induced a marked reduction in EZH2 mRNA levels in several lung cancer cell lines. A reduction in the trimethyl H3K27 histone mark was detected at the CDH1 promoter in miR-101 precursor-transfected cells. Moreover, the expression of CDH1 and MMP-2 was reversed by miR-101 transfection. Therefore, the overexpression of miR-101 inhibits the invasiveness of lung cancer. miR-101 may be a potent tumor suppressor by altering chromatin structure through repression of EZH2 and may be a potential therapeutic tool for patients with lung cancer.
doi:10.3892/etm.2011.284
PMCID: PMC3440726  PMID: 22977606
microRNA; miR-101; enhancer of zeste homolog 2; invasion; lung cancer

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