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Fibrosis is defined by the overgrowth, hardening, and/or scarring of various tissues and is attributed to excess deposition of extracellular matrix components including collagen. Fibrosis is the end result of chronic inflammatory reactions induced by a variety of stimuli including persistent infections, autoimmune reactions, allergic responses, chemical insults, radiation, and tissue injury. Although current treatments for fibrotic diseases such as idiopathic pulmonary fibrosis, liver cirrhosis, systemic sclerosis, progressive kidney disease, and cardiovascular fibrosis typically target the inflammatory response, there is accumulating evidence that the mechanisms driving fibrogenesis are distinct from those regulating inflammation. In fact, some studies have suggested that ongoing inflammation is needed to reverse established and progressive fibrosis. The key cellular mediator of fibrosis is the myofibroblast, which when activated serves as the primary collagen-producing cell. Myofibroblasts are generated from a variety of sources including resident mesenchymal cells, epithelial and endothelial cells in processes termed epithelial/endothelial-mesenchymal (EMT/EndMT) transition, as well as from circulating fibroblast-like cells called fibrocytes that are derived from bone-marrow stem cells. Myofibroblasts are activated by a variety of mechanisms, including paracrine signals derived from lymphocytes and macrophages, autocrine factors secreted by myofibroblasts, and pathogen-associated molecular patterns (PAMPS) produced by pathogenic organisms that interact with pattern recognition receptors (i.e. TLRs) on fibroblasts. Cytokines (IL-13, IL-21, TGF-β1), chemokines (MCP-1, MIP-1β), angiogenic factors (VEGF), growth factors (PDGF), peroxisome proliferator-activated receptors (PPARs), acute phase proteins (SAP), caspases, and components of the renin–angiotensin–aldosterone system (ANG II) have been identified as important regulators of fibrosis and are being investigated as potential targets of antifibrotic drugs. This review explores our current understanding of the cellular and molecular mechanisms of fibrogenesis.

MicroRNA (miRNA) are small non-coding RNA molecules that posttranscriptionally effect mRNA stability and translation by targeting the 3′-untranslated region (3′-UTR) of various transcripts. Thus, dysregulation of miRNA affects a wide range of cellular processes such as cell proliferation and differentiation involved in organ remodeling processes. Divergent miRNA patterns were observed during chronic liver diseases of various etiologies. Chronic liver diseases result in uncontrolled scar formation ending up in liver fibrosis or even cirrhosis. Since it has been shown that miR-29 dysregulation is involved in synthesis of extracellular matrix proteins, miR-29 is of special interest. The importance of miR-29 in hepatic collagen homeostasis is underlined by in vivo data showing that experimental severe fibrosis is associated with a prominent miR-29 decrease. The loss of miR-29 is due to the response of hepatic stellate cells to exposure to the profibrogenic mediators TGF-β and PDGF-BB. Several putative binding sites for the Smad proteins and the Ap1 complex are located in the miR-29 promoter, which are suggested to mediate miR-29 decrease in fibrosis. Other miRNA are highly increased after profibrogenic stimulation, such as miR-21. miR-21 is transcriptionally upregulated in response to Smad-3 rather than Smad-2 activation after TGF-β stimulation. In addition, TGF-β promotes miR-21 expression by formation of a microprocessor complex containing Smad proteins. Elevated miR-21 may then act as a profibrogenic miRNA by its repression of the TGF-β inhibitory Smad-7 protein.

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.

MicroRNAs (miRNA) are small regulatory RNAs that control gene expression by translational suppression and destabilization of target mRNAs. There is increasing evidence that miRNAs regulate genes associated with fibrosis in organs, such as the heart, kidney, liver, and the lung. In a large-scale screening for miRNAs potentially involved in bleomycin-induced fibrosis, we found expression of miR-29 family members significantly reduced in fibrotic lungs. Analysis of normal lungs showed the presence of miR-29 in subsets of interstitial cells of the alveolar wall, pleura, and at the entrance of the alveolar duct, known sites of pulmonary fibrosis. miR-29 levels inversely correlated with the expression levels of profibrotic target genes and the severity of the fibrosis. To study the impact of miR-29 down-regulation in the lung interstitium, we characterized gene expression profiles of human fetal lung fibroblast IMR-90 cells in which endogenous miR-29 was knocked down. This confirmed the derepression of reported miR-29 targets, including several collagens, but also revealed up-regulation of a large number of previously unrecognized extracellular matrix–associated and remodeling genes. Moreover, we found that miR-29 is suppressed by transforming growth factor (TGF)–β1 in these cells, and that many fibrosis-associated genes up-regulated by TGF-β1 are derepressed by miR-29 knockdown. Interestingly, a comparison of TGF-β1 and miR-29 targets revealed that miR-29 controls an additional subset of fibrosis-related genes, including laminins and integrins, independent of TGF-β1. Together, these strongly suggest a role of miR-29 in the pathogenesis of pulmonary fibrosis. miR-29 may be a potential new therapeutic target for this disease.

MicroRNAs (miRNAs) are small non-coding RNAs of 18–25 nucleotides that are generally believed to either block the translation or induce the degradation of target mRNA. miRNAs have been shown to play fundamental roles in diverse biological and pathological processes including cell proliferation, differentiation, apoptosis and carcinogenesis. Fibrosis results from an imbalance in the turnover of extracellular matrix molecules and is a highly debilitating process that can eventually lead to organ dysfunction. A growing body of evidence suggests that miRNAs participate in the fibrotic process in a number of organs including the heart, kidney, liver and lung. In this review, we summarize our current understanding of the role of miRNAs in the development of tissue fibrosis and their potential as novel drug targets.

Macrophages are found in close proximity with collagen-producing myofibroblasts and indisputably play a key role in fibrosis. They produce profibrotic mediators that directly activate fibroblasts, including transforming growth factor-β1 and platelet-derived growth factor, and control extracellular matrix turnover by regulating the balance of various matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases. Macrophages also regulate fibrogenesis by secreting chemokines that recruit fibroblasts and other inflammatory cells. With their potential to act in both a pro- and antifibrotic capacity, as well as their ability to regulate the activation of resident and recruited myofibroblasts, macrophages and the factors they express are integrated into all stages of the fibrotic process. These various, and sometimes opposing, functions may be performed by distinct macrophage subpopulations, the identification of which is a growing focus of fibrosis research. Although collagen-secreting myofibroblasts once were thought of as the master “producers” of fibrosis, this review will illustrate how macrophages function as the master “regulators” of fibrosis.

Fibrosis is the end result of pathologic wound healing and is characterized by inflammation, excessive proliferation of fibroblasts, and abnormal deposition of extracellular matrix (ECM) proteins. Despite the advanced treatments for the fibrotic diseases, as well as the researches on the fibrosis, pathologic fibrotic diseases remain to be hard cured and the molecular mechanism of fibrosis is still unclear. In our previous studies we found ITGB4BP was involved in the myofibroblast differentiation. However there were no studies about the roles of ITGB4BP in fibrosis. On this background this review explores the basic features and the biological function of ITGB4BP which might imply the underlying cellular and molecular mechanism in the regulation of fibrotic diseases.

Background

MicroRNAs are small endogenously expressed non-coding RNA molecules that regulate target gene expression through translation repression or messenger RNA degradation. MicroRNA regulation is performed through pairing of the microRNA to sites in the messenger RNA of protein coding genes. Since experimental identification of miRNA target genes poses difficulties, computational microRNA target prediction is one of the key means in deciphering the role of microRNAs in development and disease.

Results

DIANA-microT 3.0 is an algorithm for microRNA target prediction which is based on several parameters calculated individually for each microRNA and combines conserved and non-conserved microRNA recognition elements into a final prediction score, which correlates with protein production fold change. Specifically, for each predicted interaction the program reports a signal to noise ratio and a precision score which can be used as an indication of the false positive rate of the prediction.

Conclusion

Recently, several computational target prediction programs were benchmarked based on a set of microRNA target genes identified by the pSILAC method. In this assessment DIANA-microT 3.0 was found to achieve the highest precision among the most widely used microRNA target prediction programs reaching approximately 66%. The DIANA-microT 3.0 prediction results are available online in a user friendly web server at

OBJECTIVE

Progressive fibrosis in the diabetic kidney is driven and sustained by a diverse range of profibrotic factors. This study examines the critical role of microRNAs (miRNAs) in the regulation of the key fibrotic mediators, TGF-β1 and TGF-β2.

RESEARCH DESIGN AND METHODS

Rat proximal-tubular epithelial cells (NRK52E) were treated with TGF-β1 and TGF-β2 for 3 days, and expression of markers of epithelial-to-mesenchymal transition (EMT) and fibrogenesis were assessed by RT-PCR and Western blotting. The expression of miR-141 and miR-200a was also assessed, as was their role as translational repressors of TGF-β signaling. Finally, these pathways were explored in two different mouse models, representing early and advanced diabetic nephropathy.

RESULTS

Both TGF-β1 and TGF-β2 induced EMT and fibrogenesis in NRK52E cells. TGF-β1 and TGF-β2 also downregulated expression of miR-200a. The importance of these changes was demonstrated by the finding that ectopic expression miR-200a downregulated smad-3 activity and the expression of matrix proteins and prevented TGF-β–dependent EMT. miR-200a also downregulated the expression of TGF-β2, via direct interaction with the 3′ untranslated region of TGF-β2. The renal expression of miR-141 and miR-200a was also reduced in mouse models representing early and advanced kidney disease.

CONCLUSIONS

miR-200a and miR-141 significantly impact on the development and progression of TGF-β–dependent EMT and fibrosis in vitro and in vivo. These miRNAs appear to be intricately involved in fibrogenesis, both as downstream mediators of TGF-β signaling and as components of feedback regulation, and as such represent important new targets for the prevention of progressive kidney disease in the context of diabetes.

MicroRNAs are small noncoding RNAs that play an important role in the regulation of various biological processes through their interaction with cellular messenger RNAs. They are frequently dysregulated in cancer and have shown great potential as tissue-based markers for cancer classification and prognostication. microRNAs are also present in extracellular human body fluids such as serum, plasma, saliva, and urine. Most of circulating microRNAs are present in human plasma and serum cofractionate with the Argonaute2 (Ago2) protein. However, circulating microRNAs have been also found in membrane-bound vesicles such as exosomes. Since microRNAs circulate in the bloodstream in a highly stable, extracellular form, they may be used as blood-based biomarkers for cancer and other diseases. A knowledge base of extracellular circulating miRNAs is a fundamental tool for biomedical research. In this work, we present miRandola, a comprehensive manually curated classification of extracellular circulating miRNAs. miRandola is connected to miRò, the miRNA knowledge base, allowing users to infer the potential biological functions of circulating miRNAs and their connections with phenotypes. The miRandola database contains 2132 entries, with 581 unique mature miRNAs and 21 types of samples. miRNAs are classified into four categories, based on their extracellular form: miRNA-Ago2 (173 entries), miRNA-exosome (856 entries), miRNA-HDL (20 entries) and miRNA-circulating (1083 entries). miRandola is available online at: http://atlas.dmi.unict.it/mirandola/index.html.

Organ fibrosis is a pathological condition associated with chronic inflammatory diseases. In fibrosis, excessive deposition of extracellular matrix (ECM) severely impairs tissue architecture and function, eventually resulting in organ failure. This process is mediated primarily by the induction of myofibroblasts, which produce large amounts of collagen I, the main component of the ECM. Accordingly, the origin, developmental pathways, and mechanisms of myofibroblast regulation are attracting increasing attention as potential therapeutic targets. The fibrotic cascade, from initial epithelial damage to eventual myofibroblast induction, is mediated by complex biological processes such as macrophage infiltration, a shift from Th1 to Th2 phenotype, and by inflammatory mediators such as transforming growth factor-β. Here, we review the current understanding of the cellular and molecular mechanisms underlying organ fibrosis.

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.

We present an overview of selected computational methods for microRNA prediction. It is especially aimed at viral miRNA detection. As the number of microRNAs increases and the range of genomes encoding miRNAs expands, it seems that these small regulators have a more important role than has been previously thought. Most microRNAs have been detected by cloning and Northern blotting, but experimental methods are biased towards abundant microRNAs as well as being time-consuming. Computational detection methods must therefore be refined to serve as a faster, better, and more affordable method for microRNA detection. We also present data from a small study investigating the problems of computational miRNA prediction. Our findings suggest that the prediction of microRNA precursor candidates is fairly easy, while excluding false positives as well as exact prediction of the mature microRNA is hard. Finally, we discuss possible improvements to computational microRNA detection.

Hyaluronan synthase 2 and CD44 are required for severe lung fibrosis in response to bleomycin.

Tissue fibrosis is a major cause of morbidity, and idiopathic pulmonary fibrosis (IPF) is a terminal illness characterized by unremitting matrix deposition in the lung. The mechanisms that control progressive fibrosis are unknown. Myofibroblasts accumulate at sites of tissue remodeling and produce extracellular matrix components such as collagen and hyaluronan (HA) that ultimately compromise organ function. We found that targeted overexpression of HAS2 (HA synthase 2) by myofibroblasts produced an aggressive phenotype leading to severe lung fibrosis and death after bleomycin-induced injury. Fibroblasts isolated from transgenic mice overexpressing HAS2 showed a greater capacity to invade matrix. Conditional deletion of HAS2 in mesenchymal cells abrogated the invasive fibroblast phenotype, impeded myofibroblast accumulation, and inhibited the development of lung fibrosis. Both the invasive phenotype and the progressive fibrosis were inhibited in the absence of CD44. Treatment with a blocking antibody to CD44 reduced lung fibrosis in mice in vivo. Finally, fibroblasts isolated from patients with IPF exhibited an invasive phenotype that was also dependent on HAS2 and CD44. Understanding the mechanisms leading to an invasive fibroblast phenotype could lead to novel approaches to the treatment of disorders characterized by severe tissue fibrosis.

MicroRNAs (miRNAs) have emerged as a class of regulatory RNAs with immense significance in numerous biological processes. When aberrantly expressed miRNAs have been shown to play a role in the pathogenesis of several disease states. Extensive research has explored miRNA involvement in the development and fate of immune cells and in both the innate and adaptive immune responses whereby strong evidence links miRNA expression to signalling pathways and receptors with critical roles in the inflammatory response such as NF-κB and the toll-like receptors, respectively. Recent studies have revealed that unique miRNA expression profiles exist in inflammatory lung diseases such as cystic fibrosis, chronic obstructive pulmonary disease, asthma, idiopathic pulmonary fibrosis and lung cancer. Evaluation of the global expression of miRNAs provides a unique opportunity to identify important target gene sets regulating susceptibility and response to infection and treatment, and control of inflammation in chronic airway disorders. Over 800 human miRNAs have been discovered to date, however the biological function of the majority remains to be uncovered. Understanding the role that miRNAs play in the modulation of gene expression leading to sustained chronic pulmonary inflammation is important for the development of new therapies which focus on prevention of disease progression rather than symptom relief. Here we discuss the current understanding of miRNA involvement in innate immunity, specifically in LPS/TLR4 signalling and in the progression of the chronic inflammatory lung diseases cystic fibrosis, COPD and asthma. miRNA in lung cancer and IPF are also reviewed.

MicroRNAs (miRNAs) regulate post-transcriptional gene expression during development and disease. We have determined the miRNA expression levels of early- and end-stage hypertrophic cardiomyopathy (HCM) in a severe, transgenic mouse model of the disease. Five miRNAs were differentially expressed at an early stage of HCM development. Time-course analysis revealed that decreased expression of miR-1 and miR-133a commences at a pre-disease stage, and precedes upregulation of target genes causal of cardiac hypertrophy and extracellular matrix remodelling, suggesting a role for miR-1 and miR-133a in early disease development. At end-stage HCM, 16 miRNA are dysregulated to form an expression profile resembling that of other forms of cardiac hypertrophy, suggesting common responses. Analysis of the mRNA transcriptome revealed that miRNAs potentially target 15.7% upregulated and 4.8% downregulated mRNAs at end-stage HCM, and regulate mRNAs associated with cardiac hypertrophy and electrophysiology, calcium signalling, fibrosis, and the TGF-β signalling pathway. Collectively, these results define the miRNA expression signatures during development and progression of severe HCM and highlight critical miRNA regulated gene networks that are involved in disease pathogenesis.

Liver fibrosis is the excessive accumulation of extracellular matrix proteins including collagen that occurs in most types of chronic liver diseases. Advanced liver fibrosis results in cirrhosis, liver failure, and portal hypertension and often requires liver transplantation. Our knowledge of the cellular and molecular mechanisms of liver fibrosis has greatly advanced. Activated hepatic stellate cells, portal fibroblasts, and myofibroblasts of bone marrow origin have been identified as major collagen-producing cells in the injured liver. These cells are activated by fibrogenic cytokines such as TGF-β1, angiotensin II, and leptin. Reversibility of advanced liver fibrosis in patients has been recently documented, which has stimulated researchers to develop antifibrotic drugs. Emerging antifibrotic therapies are aimed at inhibiting the accumulation of fibrogenic cells and/or preventing the deposition of extracellular matrix proteins. Although many therapeutic interventions are effective in experimental models of liver fibrosis, their efficacy and safety in humans is unknown. This review summarizes recent progress in the study of the pathogenesis and diagnosis of liver fibrosis and discusses current antifibrotic strategies.

Alcoholic liver disease (ALD) is a major global health problem. Chronic alcohol use results in inflammation and fatty liver, and in some cases, it leads to fibrosis and cirrhosis or hepatocellular carcinoma. Increased proinflammatory cytokines, particularly TNF alpha, play a central role in the pathogenesis of ALD. TNF alpha is tightly regulated at transcriptional and posttranscriptional levels. Recently, microRNAs (miRNAs) have been shown to modulate gene functions. The role of miRNAs in ALD is getting attention, and recent studies suggest that alcohol modulates miRNAs. Recently, we showed that alcohol induces miR-155 expression both in vitro (RAW 264.7 macrophage) and in vivo (Kupffer cells, KCs of alcohol-fed mice). Induction of miR-155 contributed to increased TNF alpha production and to the sensitization of KCs to produce more TNF alpha in response to LPS. In this paper, we summarize the current knowledge of miRNAs in ALD and also report increased expression of miR-155 and miR-132 in the total liver as well as in isolated hepatocytes and KCs of alcohol-fed mice. Our novel finding of the alcohol-induced increase of miRNAs in hepatocytes and KCs after alcohol feeding provides further insight into the evolving knowledge regarding the role of miRNAs in ALD.

The polycystic liver and kidney diseases are a family of disorders with heterogeneous etiologies. Proposed mechanisms of disease include ciliary dysfunction, excess cell proliferation, and altered cell-cell or cell-matrix interactions. In this issue of the JCI, Lee and colleagues provide data to support a novel mechanism for cystogenesis involving microRNA (miRNA) (see the related article beginning on page 3714). They demonstrate that levels of the miRNA miR15a are decreased in livers of patients with autosomal recessive and autosomal dominant polycystic kidney disease (ARPKD and ADPKD, respectively) and congenital hepatic fibrosis as well as in the PKC rat model of ARPKD. This results in increased expression of the cell-cycle regulator Cdc25A, which is a direct target of miR15a, and increased cellular proliferation and cystogenesis in vitro. These findings suggest that other miRNAs may also participate in the molecular pathogenesis of cystic liver and kidney diseases.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and usually lethal fibrotic lung disease characterized by profound changes in epithelial cell phenotype and fibroblast proliferation.

Objectives: To determine changes in expression and role of microRNAs in IPF.

Methods: RNA from 10 control and 10 IPF tissues was hybridized on Agilent microRNA microarrays and results were confirmed by quantitative real-time polymerase chain reaction and in situ hybridization. SMAD3 binding to the let-7d promoter was confirmed by chromatin immunoprecipitation, electrophoretic mobility shift assay, luciferase assays, and reduced expression of let-7d in response to transforming growth factor-β. HMGA2, a let-7d target, was localized by immunohistochemistry. In mice, let-7d was inhibited by intratracheal administration of a let-7d antagomir and its effects were determined by immunohistochemistry, immunofluorescence, quantitative real-time polymerase chain reaction, and morphometry.

Measurements and Main Results: Eighteen microRNAs including let-7d were significantly decreased in IPF. Transforming growth factor-β down-regulated let-7d expression, and SMAD3 binding to the let-7d promoter was demonstrated. Inhibition of let-7d caused increases in mesenchymal markers N-cadherin-2, vimentin, and α-smooth muscle actin (ACTA2) as well as HMGA2 in multiple epithelial cell lines. let-7d was significantly reduced in IPF lungs and the number of epithelial cells expressing let-7d correlated with pulmonary functions. HMGA2 was increased in alveolar epithelial cells of IPF lungs. let-7d inhibition in vivo caused alveolar septal thickening and increases in collagen, ACTA2, and S100A4 expression in SFTPC (pulmonary-associated surfactant protein C) expressing alveolar epithelial cells.

Conclusions: Our results indicate a role for microRNAs in IPF. The down-regulation of let-7d in IPF and the profibrotic effects of this down-regulation in vitro and in vivo suggest a key regulatory role for this microRNA in preventing lung fibrosis.

Clinical trial registered with www.clinicaltrials.gov (NCT 00258544).

MicroRNAs are evolutionarily conserved, small (~20–25 nucleotides), single-stranded molecules that suppress the expression of protein-coding genes by translational repression, messenger RNA degradation, or both. More than 700 microRNAs have been identified in the human genome. Amazingly, a single miRNA can regulate the expression of hundreds of mRNAs/proteins within a cell. The small RNAs are fast emerging as master regulators of innate and adaptive immunity, and very likely to play a pivotal role in transplantation. The clinical application of RNA sequencing (“next – generation sequencing”) should facilitate transcriptome profiling at an unprecedented resolution. We provide an overview of microRNA biology and their hypothesized roles in transplantation.

Fibrosis is a common end-point of a number of different diseases such as hypertension, diabetes, liver cirrhosis, and those associated with chronic inflammation. Fibrosis is characterized by excessive deposition of extracellular matrix that interferes with normal tissue architecture and function. Increased expression of secreted protein acidic and rich in cysteine (SPARC) in fibrotic tissues has been reported in numerous studies. SPARC is a 43 kDa collagen-binding protein secreted from several different cell types into the extracellular matrix and has been shown to be anti-proliferative and counter-adhesive in vitro. SPARC is a matricellular protein; meaning SPARC is secreted into the extracellular space but does not serve a structural function. Instead, SPARC modulates interactions between cells and the surrounding extracellular matrix. In animal models of fibrotic disease and in human fibrotic tissues, elevated expression of SPARC has been reported in many tissues including heart, lungs, kidneys, liver, dermis, intestine, and eyes. In this review, we will summarize current studies that have examined the expression and functional importance of SPARC in various animal models of fibrosis and in human tissues. Although cellular mechanisms of SPARC in fibrosis remain to be fully elucidated, the studies summarized here provide impetus to further explore the efficacy of SPARC as a potential target for reducing fibrosis.

Multiple etiologies of liver disease lead to liver fibrosis through integrated signaling networks that regulate the deposition of extracellular matrix. This cascade of responses drives the activation of hepatic stellate cell (HSC) into a myofibroblast like phenotype that is contractile, proliferative and fibrogenic. Collagen and other extracellular matrix (ECM) components are deposited as the liver generates a wound healing response to encapsulate injury. Sustained fibrogenesis leads to cirrhosis, characterized by a distortion of the liver parenchyma and vascular architecture. Uncovering the intricate mechanisms that underlie liver fibrogenesis forms the basis for efforts to develop targeted therapies to reverse the fibrotic response and improve the outcomes of patients with chronic liver disease.

MicroRNAs (miRNAs) are a recently discovered class of small, non-coding RNAs that regulate protein levels post-transcriptionally. miRNAs play important regulatory roles in many cellular processes, including differentiation, neoplastic transformation, and cell replication and regeneration. Because of these regulatory roles, it is not surprising that aberrant miRNA expression has been implicated in several diseases. Recent studies have reported significant levels of miRNAs in serum and other body fluids, raising the possibility that circulating miRNAs could serve as useful clinical biomarkers. Here, we provide a brief overview of miRNA biogenesis and function, the identification and potential roles of circulating extracellular miRNAs, and the prospective uses of miRNAs as clinical biomarkers. Finally, we address several issues associated with the accurate measurement of miRNAs from biological samples.

Background

MicroRNAs are small non-coding RNA gene products that play diversified roles from species to species. The explosive growth of microRNA researches in recent years proves the importance of microRNAs in the biological system and it is believed that microRNAs have valuable therapeutic potentials in human diseases. Continual efforts are therefore required to locate and verify the unknown microRNAs in various genomes. As many miRNAs are found to be arranged in clusters, meaning that they are in close proximity with their neighboring miRNAs, we are interested in utilizing the concept of microRNA clustering and applying it in microRNA computational prediction.

Results

We first validate the microRNA clustering phenomenon in the human, mouse and rat genomes. There are 45.45%, 51.86% and 48.67% of the total miRNAs that are clustered in the three genomes, respectively. We then conduct sequence and secondary structure similarity analyses among clustered miRNAs, non-clustered miRNAs, neighboring sequences of clustered miRNAs and random sequences, and find that clustered miRNAs are structurally more similar to one another, and the RNAdistance score can be used to assess the structural similarity between two sequences. We therefore design a clustering-based approach which utilizes this observation to filter false positives from a list of candidates generated by a selected microRNA prediction program, and successfully raise the positive predictive value by a considerable amount ranging from 15.23% to 23.19% in the human, mouse and rat genomes, while keeping a reasonably high sensitivity.

Conclusion

Our clustering-based approach is able to increase the effectiveness of currently available microRNA prediction program by raising the positive predictive value while maintaining a high sensitivity, and hence can serve as a filtering step. We believe that it is worthwhile to carry out further experiments and tests with our approach using data from other genomes and other prediction software tools. Better results may be achieved with fine-tuning of parameters.