Differential pathogenesis of closely related 2018 Nigerian outbreak clade III Lassa virus isolates.

Nigeria continues to experience ever increasing annual outbreaks of Lassa fever (LF). The World Health Organization has recently declared Lassa virus (LASV) as a priority pathogen for accelerated research leading to a renewed international effort to develop relevant animal models of disease and effective countermeasures to reduce LF morbidity and mortality in endemic West African countries. A limiting factor in evaluating medical countermeasures against LF is a lack of well characterized animal models outside of those based on infection with LASV strain Josiah originating form Sierra Leone, circa 1976. Here we genetically characterize five recent LASV isolates collected from the 2018 outbreak in Nigeria. Three isolates were further evaluated in vivo and despite being closely related and from the same spatial / geographic region of Nigeria, only one of the three isolates proved lethal in strain 13 guinea pigs and non-human primates (NHP). Additionally, this isolate exhibited atypical pathogenesis characteristics in the NHP model, most notably respiratory failure, not commonly described in hemorrhagic cases of LF. These results suggest that there is considerable phenotypic heterogeneity in LASV infections in Nigeria, which leads to a multitude of pathogenesis characteristics that could account for differences between subclinical and lethal LF infections. Most importantly, the development of disease models using currently circulating LASV strains in West Africa are critical for the evaluation of potential vaccines and medical countermeasures.

Differential pathogenesis between Andes virus strains CHI-7913 and Chile-9717869in Syrian Hamsters.

Hantavirus cardiopulmonary syndrome (HCPS) is a severe respiratory disease caused by orthohantaviruses in the Americas with a fatality rate as high as 35%. In South America, Andes orthohantavirus (Hantaviridae, Orthohantavirus, ANDV) is a major cause of HCPS, particularly in Chile and Argentina, where thousands of cases have been reported since the virus was discovered. Two strains of ANDV that are classically used for experimental studies of the virus are Chile-9717869, isolated from the natural reservoir, the long-tailed pygmy rice rat, and CHI-7913, an isolate from a lethal human case of HCPS. An important animal model for studying pathogenesis of HCPS is the lethal Syrian golden hamster model of ANDV infection. In this model, ANDV strain Chile-9717869 is uniformly lethal and has been used extensively for pathogenesis, vaccination, and therapeutic studies. Here we show that the CHI-7913 strain, despite having high sequence similarity with Chile-9717869, does not cause lethal disease in Syrian hamsters. CHI-7913, while being able to infect hamsters and replicate to moderate levels, showed a reduced ability to replicate within the tissues compared with Chile-9717869. Hamsters infected with CHI-7913 had reduced expression of cytokines IL-4, IL-6, and IFN-γ compared with Chile-9717869 infected animals, suggesting potentially limited immune-mediated pathology. These results demonstrate that certain ANDV strains may not be lethal in the classical Syrian hamster model of infection, and further exploration into the differences between lethal and non-lethal strains provide important insights into molecular determinants of pathogenic hantavirus infection.Importance:Andes orthohantavirus (ANDV) is a New World hantavirus that is a major cause of hantavirus cardiopulmonary syndrome (HCPS, also referred to as hantavirus pulmonary syndrome) in South America, particularly in Chile and Argentina. ANDV is one of the few hantaviruses for which there is a reliable animal model, the Syrian hamster model, which recapitulates important aspects of human disease. Here we infected hamsters with a human isolate of ANDV, CHI-7913, to assess its pathogenicity compared with the classical lethal Chile-9717869 strain. CHI-7913 had 22 amino acid differences compared with Chile-9717869, did not cause lethal disease in hamsters, and showed reduced ability to replicate in vivo Our data indicate potentially important molecular signatures for pathogenesis of ANDV infection in hamsters and may lead to insights into what drives pathogenesis of certain hantaviruses in humans.

New insights into lung development and diseases: the role of microRNAs.

MicroRNAs (miRNAs) are short endogenous noncoding RNA molecules (∼ 22 nucleotides) that can regulate gene expression at the post-transcription level. Research interest in the role of miRNAs in lung biology is emerging. MiRNAs have been implicated in a range of processes such as development, homeostasis, and inflammatory diseases in lung tissues and are capable of inducing differentiation, morphogenesis, and apoptosis. In recent years, several studies have reported that miRNAs are differentially regulated in lung development and lung diseases in response to epigenetic changes, providing new insights for their versatile role in various physiological and pathological processes in the lung. In this review, we discuss the contribution of miRNAs to lung development and diseases and possible future implications in the field of lung biology.

High-throughput measurement of gap junctional intercellular communication.

Gap junctional intercellular communication (GJIC) is a critical part of cellular activities and is necessary for electrical propagation among contacting cells. Disorders of gap junctions are a major cause for cardiac arrhythmias. Dye transfer through microinjection is a conventional technique for measuring GJIC. To overcome the limitations of manual microinjection and perform high-throughput GJIC measurement, here we present a new robotic microinjection system that is capable of injecting a large number of cells at a high speed. The highly automated system enables large-scale cell injection (thousands of cells vs. a few cells) without major operator training. GJIC of three cell lines of differing gap junction density, i.e., HeLa, HEK293, and HL-1, was evaluated. The effect of a GJIC inhibitor (18-α-glycyrrhetinic acid) was also quantified in the three cell lines. System operation speed, success rate, and cell viability rate were quantitatively evaluated based on robotic microinjection of over 4,000 cells. Injection speed was 22.7 cells per min, with 95% success for cell injection and >90% survival. Dye transfer cell counts and dye transfer distance correlated with the expected connexin expression of each cell type, and inhibition of dye transfer correlated with the concentration of GJIC inhibitor. Additionally, real-time monitoring of dye transfer enables the calculation of coefficients of molecular diffusion through gap junctions. This robotic microinjection dye transfer technique permits rapid assessment of gap junction function in confluent cell cultures.

Seasonal human coronaviruses OC43, 229E, and NL63 induce cell surface modulation of entry receptors and display host cell-specific viral replication kinetics.

The emergence of the COVID-19 pandemic prompted an increased interest in seasonal human coronaviruses. OC43, 229E, NL63, and HKU1 are endemic seasonal coronaviruses that cause the common cold and are associated with generally mild respiratory symptoms. In this study, we identified cell lines that exhibited cytopathic effects (CPE) upon infection by three of these coronaviruses and characterized their viral replication kinetics and the effect of infection on host surface receptor expression. We found that NL63 produced CPE in LLC-MK2 cells, while OC43 produced CPE in MRC-5, HCT-8, and WI-38 cell lines, while 229E produced CPE in MRC-5 and WI-38 by day 3 post-infection. We observed a sharp increase in nucleocapsid and spike viral RNA (vRNA) from day 3 to day 5 post-infection for all viruses; however, the abundance and the proportion of vRNA copies measured in the supernatants and cell lysates of infected cells varied considerably depending on the virus-host cell pair. Importantly, we observed modulation of coronavirus entry and attachment receptors upon infection. Infection with 229E and OC43 led to a downregulation of CD13 and GD3, respectively. In contrast, infection with NL63 and OC43 leads to an increase in ACE2 expression. Attempts to block entry of NL63 using either soluble ACE2 or anti-ACE2 monoclonal antibodies demonstrated the potential of these strategies to greatly reduce infection. Overall, our results enable a better understanding of seasonal coronaviruses infection kinetics in permissive cell lines and reveal entry receptor modulation that may have implications in facilitating co-infections with multiple coronaviruses in humans.IMPORTANCESeasonal human coronavirus is an important cause of the common cold associated with generally mild upper respiratory tract infections that can result in respiratory complications for some individuals. There are no vaccines available for these viruses, with only limited antiviral therapeutic options to treat the most severe cases. A better understanding of how these viruses interact with host cells is essential to identify new strategies to prevent infection-related complications. By analyzing viral replication kinetics in different permissive cell lines, we find that cell-dependent host factors influence how viral genes are expressed and virus particles released. We also analyzed entry receptor expression on infected cells and found that these can be up- or down-modulated depending on the infecting coronavirus. Our findings raise concerns over the possibility of infection enhancement upon co-infection by some coronaviruses, which may facilitate genetic recombination and the emergence of new variants and strains.

Intravenous immunoglobulin ameliorates ITP via activating Fc gamma receptors on dendritic cells.

Despite a more than 20-year experience of therapeutic benefit, the relevant molecular and cellular targets of intravenous immunoglobulin (IVIg) in autoimmune disease remain unclear. Contrary to the prevailing theories of IVIg action in autoimmunity, we show that IVIg drives signaling through activating Fc gamma receptors (Fc gammaR) in the amelioration of mouse immune thrombocytopenic purpura (ITP). The actual administration of IVIg was unnecessary because as few as 10(5) IVIg-treated cells could, upon adoptive transfer, ameliorate ITP. IVIg did not interact with the inhibitory Fc gammaRIIB on the initiator cell, although Fc gammaRIIB does have a role in the late phase of IVIg action. Notably, only IVIg-treated CD11c+ dendritic cells could mediate these effects. We hypothesize that IVIg forms soluble immune complexes in vivo that prime dendritic-cell regulatory activity. In conclusion, the clinical effects of IVIg in ameliorating ITP seem to involve the acute interaction of IVIg with activating Fc gammaR on dendritic cells.

Atypical Ebola Virus Disease in a Nonhuman Primate following Monoclonal Antibody Treatment Is Associated with Glycoprotein Mutations within the Fusion Loop.

Ebola virus (EBOV) is responsible for numerous devastating outbreaks throughout Africa, including the 2013-2016 West African outbreak as well as the two recent outbreaks in the Democratic Republic of the Congo (DRC), one of which is ongoing. Although EBOV disease (EVD) has typically been considered a highly lethal acute infection, increasing evidence suggests that the virus can persist in certain immune-privileged sites and occasionally lead to EVD recrudescence. Little is understood about the processes that contribute to EBOV persistence and recrudescence, in part because of the rarity of these phenomena but also because of the absence of an animal model that recapitulates them. Here, we describe a case of EBOV persistence associated with atypical EVD in a nonhuman primate (NHP) following inoculation with EBOV and treatment with an experimental monoclonal antibody cocktail. Although this animal exhibited only mild signs of acute EVD, it developed severe disease 2 weeks later and succumbed shortly thereafter. Viremia was undetectable at the time of death, despite abundant levels of viral RNA in most tissues, each of which appeared to harbor a distinct viral quasispecies. Remarkably, sequence analysis identified a single mutation in glycoprotein (GP) that not only resisted antibody-mediated neutralization but also increased viral growth kinetics and virulence. Overall, this report represents the most thoroughly characterized case of atypical EVD in an NHP described thus far, and it provides valuable insight into factors that may contribute to EBOV persistence and recrudescent disease.IMPORTANCE Ebola virus remains a global threat to public health and biosecurity, yet we still know relatively little about its pathogenesis and the complications that arise following recovery. With nearly 20,000 survivors from the 2013-2016 West African outbreak, as well as over 1,000 survivors of the recent outbreak in the DRC, we must consider the consequences of virus persistence and recrudescent disease, even if they are rare. In this study, we describe a case of atypical Ebola virus disease in a nonhuman primate after treatment with a monoclonal antibody. Not only does this study underscore the potential for atypical disease presentations, but it also emphasizes the importance of considering how medical countermeasures might relate to these phenomena, especially as antibodies are incorporated into the standard of care. The results presented herein provide a foundation from which we can continue to investigate these facets of Ebola virus disease.

Relative Ratios of Human Seasonal Coronavirus Antibodies Predict the Efficiency of Cross-Neutralization of SARS-CoV-2 Spike Binding to ACE2.

BACKGROUND: Antibodies raised against human seasonal coronaviruses (sCoVs), which are responsible for the common cold, are known to cross-react with SARS-CoV-2 antigens. This prompts questions about their protective role against SARS-CoV-2 infections and COVID-19 severity. However, the relationship between sCoVs exposure and SARS-CoV-2 correlates of protection are not clearly identified. METHODS: We performed a cross-sectional analysis of cross-reactivity and cross-neutralization to SARS-CoV-2 antigens (S-RBD, S-trimer, N) using pre-pandemic sera from four different groups: pediatrics and adolescents, individuals 21 to 70 years of age, older than 70 years of age, and individuals living with HCV or HIV. Data was then further analysed using machine learning to identify predictive patterns of neutralization based on sCoVs serology. FINDINGS: Antibody cross-reactivity to SARS-CoV-2 antigens varied between 1.6% and 15.3% depending on the cohort and the isotype-antigen pair analyzed. We also show a range of neutralizing activity (0-45%) with median inhibition ranging from 17.6 % to 23.3 % in serum that interferes with SARS-CoV-2 spike attachment to ACE2 independently of age group. While the abundance of sCoV antibodies did not directly correlate with neutralization, we show that neutralizing activity is rather dependent on relative ratios of IgGs in sera directed to all four sCoV spike proteins. More specifically, we identified antibodies to NL63 and OC43 as being the most important predictors of neutralization. INTERPRETATION: Our data support the concept that exposure to sCoVs triggers antibody responses that influence the efficiency of SARS-CoV-2 spike binding to ACE2, which may potentially impact COVID-19 disease severity through other latent variables. FUNDING: This study was supported by a grant by the CIHR (VR2 -172722) and by a grant supplement by the CITF, and by a NRC Collaborative R&D Initiative Grant (PR031-1).

Transfusion of IgG-opsonized foreign red blood cells mediates reduction of antigen-specific B cell priming in a murine model.

Hemolytic disease of the fetus and newborn can be effectively prevented by administration of anti-D to the mother. The administered IgG results in the attenuation of RBC-specific Ab production, a process termed Ab-mediated immune suppression (AMIS). Because in animal models of AMIS no major effect on T cell priming occurs, we hypothesized that the effect of the IgG on the immune system under AMIS conditions may involve a deficiency in B cell priming. We therefore challenged mice with either untreated RBCs or IgG-opsonized RBCs (AMIS) and assessed B cell priming. B cells from mice transfused with untreated RBCs, but not from mice treated under AMIS conditions, were primed as assessed by their ability to function as Ag-specific APCs to appropriate T cells. To our knowledge, this is the first report demonstrating that AMIS inhibits the appearance of Ag-primed RBC-specific B cells.

Assessment of antiviral therapeutics in animal models of Lassa fever.

Lassa virus (LASV) is an emerging zoonotic virus endemic in West Africa that can cause severe haemorrhagic Lassa fever (LF) in humans. LF recently gained international attention as a prominent infectious disease, leading to increasingly severe outbreaks in Nigeria over the past three years. Morbidity and mortality associated with LF disease in Nigeria continue to rise with 106 deaths reported in 2016, 143 in 2017 and 562 in 2018. Despite the significant health impact LF imposes on West Africa there are currently no FDA-approved therapeutics or vaccines available for treatment and prevention. This review focuses on the assessment and current state of LF antiviral therapeutics in animal models and their potential role in reducing disease burden throughout West Africa.

The Cytokine Response Profile of Ebola Virus Disease in a Large Cohort of Rhesus Macaques Treated With Monoclonal Antibodies.

Ebola virus (EBOV) is a highly pathogenic filovirus that causes outbreaks of a severe hemorrhagic fever known as EBOV disease (EVD). Ebola virus disease is characterized in part by a dysregulated immune response and massive production of both pro- and anti-inflammatory cytokines. To better understand the immune response elicited by EVD in the context of treatment with experimental anti-EBOV antibody cocktails, we analyzed 29 cytokines in 42 EBOV-infected rhesus macaques. In comparison to the surviving treated animals, which exhibited minimal aberrations in only a few cytokine levels, nonsurviving animals exhibited a dramatically upregulated inflammatory response that was delayed by antibody treatment.

Immunoglobulin G-mediated regulation of the murine immune response to transfused red blood cells occurs in the absence of active immune suppression: implications for the mechanism of action of anti-D in the prevention of haemolytic disease of the fetus and newborn?

Anti-D has been widely and effectively used in Rhesus blood group D negative mothers for the prevention of haemolytic disease of the fetus and newborn; its mechanism of action however, often referred to as antibody-mediated immune suppression (AMIS), remains largely unresolved. We investigated, in a murine model, whether active immune suppression or clonal deletion mediated by anti-red blood cell (RBC) immunoglobulin G (IgG) could explain the phenomenon of AMIS. Transfusion of IgG-opsonized foreign RBCs (i.e. AMIS) strongly attenuated antibody responses compared to transfusion of untreated foreign RBCs. When the AMIS-mice were subsequently transfused with untreated RBCs, no immune suppression was observed at 5 and 35 days after AMIS induction; in fact, the mice responded to retransfusion with untreated RBCs in a manner that was characteristic of a secondary immune response. When IgG-opsonized RBCs were transfused concurrently with untreated RBCs, a dose-dependent reduction of the antibody response was observed. This work suggests that the attenuation of the antibody responsiveness by anti-RBC IgG is not associated with active immune suppression or clonal deletion at either the T-cell or B-cell level; rather, the effect appears more characteristic of B-cell unresponsiveness to IgG-opsonized RBCs. These results may have implications for the understanding of the mechanism of action of anti-D in haemolytic disease of the fetus and newborn.

Can antibodies with specificity for soluble antigens mimic the therapeutic effects of intravenous IgG in the treatment of autoimmune disease?

Intravenous Ig (IVIg) mediates protection from the effects of immune thrombocytopenic purpura (ITP) as well as numerous other autoimmune states; however, the active antibodies within IVIg are unknown. There is some evidence that antibodies specific for a cell-associated antigen on erythrocytes are responsible, at least in part, for the therapeutic effect of IVIg in ITP. Yet whether an IVIg directed to a soluble antigen can likewise be beneficial in ITP or other autoimmune diseases is also unknown. A murine model of ITP was used to determine the effectiveness of IgG specific to soluble antigens in treating immune thrombocytopenic purpura. Mice experimentally treated with soluble OVA + anti-OVA versus mice treated with OVA conjugated to rbcs (OVA-rbcs) + anti-OVA were compared. In both situations, mice were protected from ITP. Both these experimental therapeutic regimes acted in a complement-independent fashion and both also blocked reticuloendothelial function. In contrast to OVA-rbcs + anti-OVA, soluble OVA + anti-OVA (as well as IVIg) did not have any effect on thrombocytopenia in mice lacking the inhibitory receptor FcgammaRIIB (FcgammaRIIB(-/-) mice). Similarly, antibodies reactive with the endogenous soluble antigens albumin and transferrin also ameliorated ITP in an FcgammaRIIB-dependent manner. Finally, broadening the significance of these experiments was the finding that anti-albumin was protective in a K/BxN serum-induced arthritis model. We conclude that IgG antibodies directed to soluble antigens ameliorated 2 disparate IVIg-treatable autoimmune diseases.

Animal models for filovirus infections.

The family Filoviridae, which includes the genera Marburgvirus and Ebolavirus, contains some of the most pathogenic viruses in humans and non-human primates (NHPs), causing severe hemorrhagic fevers with high fatality rates. Small animal models against filoviruses using mice, guinea pigs, hamsters, and ferrets have been developed with the goal of screening candidate vaccines and antivirals, before testing in the gold standard NHP models. In this review, we summarize the different animal models used to understand filovirus pathogenesis, and discuss the advantages and disadvantages of each model with respect to filovirus disease research.

Antiviral activity of quercetin-3-ß-O-D-glucoside against Zika virus infection.

Q3G is a natural derivative of quercetin and is already widely used in various foods and drinks. Our results clearly demonstrated that Q3G exerts antiviral activity against ZIKV in both tissue culture and knockout mice, and that post-exposure in vivo treatment with Q3G could have a beneficial effect. In the future, Q3G should be tested in human cell lines (such as Huh-7, HeLa, or K048, a fetal brain neural stem cell line) to provide further data supporting its potential efficacy in humans; in addition, live viral loads or viremia should be tested in treated animals to supplement the survival results observed in this study. Although the treatment regimens will need to be further optimized (i.e., dosage, frequency of treatment, and administration routes), our results support the results of Q3G efficacy studies in nonhuman primates against ZIKV infection. Further studies will also be needed to investigate the mechanism of Q3G antiviral action, in order to obtain valuable insights into the design of novel targets for antiviral therapeutics in the future.

Robotic adherent cell injection for characterizing cell-cell communication.

Compared to robotic injection of suspended cells (e.g., embryos and oocytes), fewer attempts were made to automate the injection of adherent cells (e.g., cancer cells and cardiomyocytes) due to their smaller size, highly irregular morphology, small thickness (a few micrometers thick), and large variations in thickness across cells. This paper presents a robotic system for automated microinjection of adherent cells. The system is embedded with several new capabilities: automatically locating micropipette tips; robustly detecting the contact of micropipette tip with cell culturing surface and directly with cell membrane; and precisely compensating for accumulative positioning errors. These new capabilities make it practical to perform adherent cell microinjection truly via computer mouse clicking in front of a computer monitor, on hundreds and thousands of cells per experiment (versus a few to tens of cells as state of the art). System operation speed, success rate, and cell viability rate were quantitatively evaluated based on robotic microinjection of over 4000 cells. This paper also reports the use of the new robotic system to perform cell-cell communication studies using large sample sizes. The gap junction function in a cardiac muscle cell line (HL-1 cells), for the first time, was quantified with the system.

TMEM43 mutation p.S358L alters intercalated disc protein expression and reduces conduction velocity in arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a myocardial disease characterized by fibro-fatty replacement of myocardium in the right ventricular free wall and frequently results in life-threatening ventricular arrhythmias and sudden cardiac death. A heterozygous missense mutation in the transmembrane protein 43 (TMEM43) gene, p.S358L, has been genetically identified to cause autosomal dominant ARVC type 5 in a founder population from the island of Newfoundland, Canada. Little is known about the function of the TMEM43 protein or how it leads to the pathogenesis of ARVC. We sought to determine the distribution of TMEM43 and the effect of the p.S358L mutation on the expression and distribution of various intercalated (IC) disc proteins as well as functional effects on IC disc gap junction dye transfer and conduction velocity in cell culture. Through Western blot analysis, transmission electron microscopy (TEM), immunofluorescence (IF), and electrophysiological analysis, our results showed that the stable expression of p.S358L mutation in the HL-1 cardiac cell line resulted in decreased Zonula Occludens (ZO-1) expression and the loss of ZO-1 localization to cell-cell junctions. Junctional Plakoglobin (JUP) and α-catenin proteins were redistributed to the cytoplasm with decreased localization to cell-cell junctions. Connexin-43 (Cx43) phosphorylation was altered, and there was reduced gap junction dye transfer and conduction velocity in mutant TMEM43-transfected cells. These observations suggest that expression of the p.S358L mutant of TMEM43 found in ARVC type 5 may affect localization of proteins involved in conduction, alter gap junction function and reduce conduction velocity in cardiac tissue.

Congenital heart block maternal sera autoantibodies target an extracellular epitope on the α1G T-type calcium channel in human fetal hearts.

BACKGROUND: Congenital heart block (CHB) is a transplacentally acquired autoimmune disease associated with anti-Ro/SSA and anti-La/SSB maternal autoantibodies and is characterized primarily by atrioventricular (AV) block of the fetal heart. This study aims to investigate whether the T-type calcium channel subunit α1G may be a fetal target of maternal sera autoantibodies in CHB. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate differential mRNA expression of the T-type calcium channel CACNA1G (α1G gene) in the AV junction of human fetal hearts compared to the apex (18-22.6 weeks gestation). Using human fetal hearts (20-22 wks gestation), our immunoprecipitation (IP), Western blot analysis and immunofluorescence (IF) staining results, taken together, demonstrate accessibility of the α1G epitope on the surfaces of cardiomyocytes as well as reactivity of maternal serum from CHB affected pregnancies to the α1G protein. By ELISA we demonstrated maternal sera reactivity to α1G was significantly higher in CHB maternal sera compared to controls, and reactivity was epitope mapped to a peptide designated as p305 (corresponding to aa305-319 of the extracellular loop linking transmembrane segments S5-S6 in α1G repeat I). Maternal sera from CHB affected pregnancies also reacted more weakly to the homologous region (7/15 amino acids conserved) of the α1H channel. Electrophysiology experiments with single-cell patch-clamp also demonstrated effects of CHB maternal sera on T-type current in mouse sinoatrial node (SAN) cells. CONCLUSIONS/SIGNIFICANCE: Taken together, these results indicate that CHB maternal sera antibodies readily target an extracellular epitope of α1G T-type calcium channels in human fetal cardiomyocytes. CHB maternal sera also show reactivity for α1H suggesting that autoantibodies can target multiple fetal targets.

MicroRNA miR-98 inhibits tumor angiogenesis and invasion by targeting activin receptor-like kinase-4 and matrix metalloproteinase-11.

Angiogenesis and invasion are essential processes for solid tumor growth and dissemination. The tumor development process can be dependent on the activation of a series of signaling pathways, including growth factor-activated pathways. MicroRNAs have been shown to be critical for tumorigenesis, but their roles in cancer angiogenesis, invasion and other signaling pathways important for tumor development are still unclear in the context of tumor biology. We investigated the role of microRNA miR-98 in regulating tumor growth, invasion, and angiogenesis using a highly aggressive breast cancer model in vitro and in vitro. We found that the expression of miR-98 inhibited breast cancer cell proliferation, survival, tumor growth, invasion, and angiogenesis. Conversely, inhibition of endogenous miR-98 promoted cell proliferation, survival, tumor growth, invasion, and angiogenesis. It appeared that miR-98 inhibited angiogenesis by modulating endothelial cell activities including cell spreading, cell invasion and tubule formation. Interestingly, miR-98 reduced the expression of ALK4 and MMP11, both of which were potential targets of miR-98. Transfection of an anti-miR-98 construct increased the expression of both targets. We confirmed that mir-98 targeted the 3'-untranslated regions of ALK4 and MMP11. Finally, ALK4- and MMP11-specific siRNAs inhibited breast cancer cell proliferation, survival, and angiogenesis. Rescue experiments with ALK4 and MMP11 constructs reversed the anti-proliferative, anti-invasive and anti-angiogenic effects of miR-98. Our findings define a regulatory role of miR-98 in tumor angiogenesis and invasion through repressed ALK4 and MMP11 expression.

MicroRNA MiR-17 retards tissue growth and represses fibronectin expression.

MicroRNAs (miRNAs) are single-stranded regulatory RNAs, frequently expressed as clusters. Previous studies have demonstrated that the six-miRNA cluster miR-17~92 has important roles in tissue development and cancers. However, the precise role of each miRNA in the cluster is unknown. Here we show that overexpression of miR-17 results in decreased cell adhesion, migration and proliferation. Transgenic mice overexpressing miR-17 showed overall growth retardation, smaller organs and greatly reduced haematopoietic cell lineages. We found that fibronectin and the fibronectin type-III domain containing 3A (FNDC3A) are two targets that have their expression repressed by miR-17, both in vitro and in transgenic mice. Several lines of evidence support the notion that miR-17 causes cellular defects through its repression of fibronectin expression. Our single miRNA expression assay may be evolved to allow the manipulation of individual miRNA functions in vitro and in vivo. We anticipate that this could serve as a model for studying gene regulation by miRNAs in the development of gene therapy.