Dehydration mediated microRNA response in the African clawed frog Xenopus laevis

Exposure to various environmental stresses induces metabolic rate depression in many animal species, an adaptation that conserves energy until the environment is again conducive to normal life. The African clawed frog, Xenopus laevis, is periodically subjected to arid summers in South Africa, and utilizes entry into the hypometabolic state of estivation as a mechanism of long term survival. During estivation, frogs must typically deal with substantial dehydration as their ponds dry out and X. laevis can endure > 30% loss of its body water. We hypothesize that microRNAs play a vital role in establishing a reversible hypometabolic state and responding to dehydration stress that is associated with amphibian estivation. The present study analyzes the effects of whole body dehydration on microRNA expression in three tissues of X. laevis. Compared to controls, levels of miR-1, miR-125b, and miR-16-1 decreased to 37 ± 6, 64 ± 8, and 80 ± 4% of control levels during dehydration in liver. By contrast, miR-210, miR-34a and miR-21 were significantly elevated by 3.05 ± 0.45, 2.11 ± 0.08, and 1.36 ± 0.05-fold, respectively, in the liver. In kidney tissue, miR-29b, miR-21, and miR-203 were elevated by 1.40 ± 0.09, 1.31 ± 0.05, and 2.17 ± 0.31-fold, respectively, in response to dehydration whereas miR-203 and miR-34a were elevated in ventral skin by 1.35 ± 0.05 and 1.74 ± 0.12-fold, respectively. Bioinformatic analysis of the differentially expressed microRNAs suggests that these are mainly involved in two processes: (1) expression of solute carrier proteins, and (2) regulation of mitogen-activated protein kinase signaling. This study is the first report that shows a tissue specific mode of microRNA expression during amphibian dehydration, providing evidence for microRNAs as crucial regulators of metabolic depression.     

miRNAs may play a major role in the control of gene expression in key pathobiological processes in Chagas disease cardiomyopathy

Chronic Chagas disease cardiomyopathy (CCC), an especially aggressive inflammatory dilated cardiomyopathy caused by lifelong infection with the protozoan Trypanosoma cruzi, is a major cause of cardiomyopathy in Latin America. Although chronic myocarditis may play a major pathogenetic role, little is known about the molecular mechanisms responsible for its severity. The aim of this study is to study the genes and microRNAs expression in tissues and their connections in regards to the pathobiological processes. To do so, we integrated for the first time global microRNA and mRNA expression profiling from myocardial tissue of CCC patients employing pathways and network analyses. We observed an enrichment in biological processes and pathways associated with the immune response and metabolism. IFNγ, TNF and NFkB were the top upstream regulators. The intersections between differentially expressed microRNAs and differentially expressed target mRNAs showed an enrichment in biological processes such as Inflammation, inflammation, Th1/IFN-γ-inducible genes, fibrosis, hypertrophy, and mitochondrial/oxidative stress/antioxidant response. MicroRNAs also played a role in the regulation of gene expression involved in the key cardiomyopathy-related processes fibrosis, hypertrophy, myocarditis and arrhythmia. Significantly, a discrete number of differentially expressed microRNAs targeted a high number of differentially expressed mRNAs (>20) in multiple processes. Our results suggest that miRNAs orchestrate expression of multiple genes in the major pathophysiological processes in CCC heart tissue. This may have a bearing on pathogenesis, biomarkers and therapy.     

The regulation of heat shock proteins in response to dehydration in <Emphasis Type="Italic">Xenopus laevis</Emphasis>

African clawed frogs (Xenopus laevis) endure bouts of severe drought in their natural habitats and survive the loss of approximately 30% of total body water due to dehydration. To investigate molecular mechanisms employed by X. laevis during periods of dehydration, the heat shock protein response, a vital component of the cytoprotective stress response, was characterized. Using western immunoblotting and multiplex technology, the protein levels of HSP27, HSP40, HSP60, HSP70, HSC70, and HSP90 were quantified in the liver, skeletal muscle, kidney, lung, and testes from control frogs and those that underwent medium or high dehydration (~16 or ~30% loss of total body water). Dehydration increased HSP27 (1.45–1.65-fold) in the kidneys and lungs, and HSP40 (1.39–2.50-fold) in the liver, testes, and skeletal muscle. HSP60 decreased in response to dehydration (0.43–0.64 of control) in the kidneys and lungs. HSP70 increased in the liver, lungs, and testes (1.39–1.70-fold) during dehydration, but had a dynamic response in the kidneys (levels increased 1.57-fold with medium dehydration, but decreased to 0.56 of control during high dehydration). HSC70 increased in the liver and kidneys (1.20–1.36-fold), but decreased in skeletal muscle (0.27–0.55 of control) during dehydration. Lastly, HSP90 was reduced in the kidney, lung, and skeletal muscle (0.39–0.69 of control) in response to dehydration, but rose in the testes (1.30-fold). Overall, the results suggest a dynamic tissue-specific heat shock protein response to whole body dehydration in X. laevis.     

Global MicroRNA Expression Profiling of Mouse Livers following Ischemia-Reperfusion Injury at Different Stages

Hepatic ischemia-reperfusion injury is a dynamic process consisting of two stages: ischemia and reperfusion, and triggers a cascade of physiological and biochemical events. Given the important role of microRNAs in regulating gene expression, we analyzed gene expression changes in mouse livers at sham control, ischemia stage, and reperfusion stage. We generated global expression profiles of microRNA and mRNA genes in mouse livers subjected to ischemia-reperfusion injury at the three stages, respectively. Comparison analysis showed that reperfusion injury had a distinct expression profile whereas the ischemia sample and the sham control were clustered together. Consistently, there are 69 differentially expressed microRNAs between the reperfusion sample and the sham control whereas 28 differentially expressed microRNAs between the ischemia sample and the sham control. We further identified two modes of microRNA expression changes in ischemia-reperfusion injury. Functional analysis of both the differentially expressed microRNAs in the two modes and their target mRNAs revealed that ischemia injury impaired mitochondrial function, nutrient consumption, and metabolism process. In contrast, reperfusion injury led to severe tissue inflammation that is predominantly an innate-immune response in the ischemia-reperfusion process. Our staged analysis of gene expression profiles provides new insights into regulatory mechanisms of microRNAs in mouse hepatic IR injury.     

RNA Expression Profiling of Human iPSC-Derived Cardiomyocytes in a Cardiac Hypertrophy Model

Cardiac hypertrophy is an independent risk factor for cardiovascular disease and heart failure. There is increasing evidence that microRNAs (miRNAs) play an important role in the regulation of messenger RNA (mRNA) and the pathogenesis of various cardiovascular diseases. However, the ability to comprehensively study cardiac hypertrophy on a gene regulatory level is impacted by the limited availability of human cardiomyocytes. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer the opportunity for disease modeling. Here we utilize a previously established in vitro model of cardiac hypertrophy to interrogate the regulatory mechanism associated with the cardiac disease process. We perform miRNA sequencing and mRNA expression analysis on endothelin 1 (ET-1) stimulated hiPSC-CMs to describe associated RNA expression profiles. MicroRNA sequencing revealed over 250 known and 34 predicted novel miRNAs to be differentially expressed between ET-1 stimulated and unstimulated control hiPSC-CMs. Messenger RNA expression analysis identified 731 probe sets with significant differential expression. Computational target prediction on significant differentially expressed miRNAs and mRNAs identified nearly 2000 target pairs. A principal component analysis approach comparing the in vitro data with human myocardial biopsies detected overlapping expression changes between the in vitro samples and myocardial biopsies with Left Ventricular Hypertrophy. These results provide further insights into the complex RNA regulatory mechanism associated with cardiac hypertrophy.     

Regulation of nuclear factor of activated T cells (NFAT) and downstream myogenic proteins during dehydration in the African clawed frog

Xenopus laevis, otherwise known as the African clawed frog, undergoes natural dehydration of up to 30% of its total body water during the dry season in sub-Saharan Africa. To survive under these conditions, a variety of physiological and biochemical changes take place in X. laevis. We were interested in understanding the role that the calcineurin-NFAT pathway plays during dehydration stress response in the skeletal muscles of X. laevis. Immunoblotting was performed to characterize the protein levels of NFATc1-4, calcium signalling proteins, in addition to myogenic proteins (MyoD, MyoG, myomaker). In addition, DNA–protein interaction ELISAs were used to assess the binding of NFATs to their consensus binding sequence, and to identify the effect of urea on NFAT-binding. Our results showed that NFATc1 and c4 protein levels decreased during dehydration, and there were no changes in NFATc2, c3, and calcium signalling proteins. However, MyoG and myomaker both showed increases in protein levels during dehydration, thus indicating that the late myogenic program involving myoblast differentiation, but not satellite cell activation and myoblast proliferation, could be involved in preserving the skeletal muscle of X. laevis during dehydration. In addition, we observed that urea seems to reduce NFATc3-binding to DNA during control, but not during dehydration, possibly indicating that NFATc3 is protected from the denaturing effects of urea as it accumulates during dehydration. These findings expand upon our knowledge of adaptive responses to dehydration, and they identify specific protein targets that could be used to protect the skeletal muscle from damage during stress.     

<Emphasis Type="Italic">Xenopus laevis</Emphasis> peroxiredoxins: Gene expression during development and characterization of the enzymes

Reactive oxygen species (ROS) are produced via catabolic and anabolic processes during normal embryonic development, and ROS content in the cell is maintained at a certain level. Peroxiredoxins are a family of selenium-independent peroxidases and play a key role in maintaining redox homeostasis of the cell. In addition to regulating the ROS level, peroxiredoxins are involved in intracellular and intercellular signaling, cell differentiation, and tissue development. The time course of peroxiredoxin gene (prx1–6) expression was studied in Xenopus laevis during early ontogeny (Nieuwkoop and Faber stages 10–63). The highest expression level was observed for prx1 at these developmental stages. The prx1, prx3, and prx4 expression level changed most dramatically in response to oxidative stress artificially induced in X. laevis embryos. In X. laevis adults, prx1–6 were all intensely expressed in all organs examined, the prx1 expression level being the highest. The X. laevis prx1–6 genes were cloned and expressed in Escherichia coli, and physico-chemical characteristics were compared for the recombinant enzymes. The highest peroxidase activity and thermal stability were observed for Prx1 and Prx2. It was assumed that Prx1 plays a leading role in X. laevis early development.     

MicroRNA Expression and Virulence in Pandemic Influenza Virus-Infected Mice

The worst known H1N1 influenza pandemic in history resulted in more than 20 million deaths in 1918 and 1919. Although the underlying mechanism causing the extreme virulence of the 1918 influenza virus is still obscure, our previous functional genomics analyses revealed a correlation between the lethality of the reconstructed 1918 influenza virus (r1918) in mice and a unique gene expression pattern associated with severe immune responses in the lungs. Lately, microRNAs have emerged as a class of crucial regulators for gene expression. To determine whether differential expression of cellular microRNAs plays a role in the host response to r1918 infection, we compared the lung cellular “microRNAome” of mice infected by r1918 virus with that of mice infected by a nonlethal seasonal influenza virus, A/Texas/36/91. We found that a group of microRNAs, including miR-200a and miR-223, were differentially expressed in response to influenza virus infection and that r1918 and A/Texas/36/91 infection induced distinct microRNA expression profiles. Moreover, we observed significant enrichment in the number of predicted cellular target mRNAs whose expression was inversely correlated with the expression of these microRNAs. Intriguingly, gene ontology analysis revealed that many of these mRNAs play roles in immune response and cell death pathways, which are known to be associated with the extreme virulence of r1918. This is the first demonstration that cellular gene expression patterns in influenza virus-infected mice may be attributed in part to microRNA regulation and that such regulation may be a contributing factor to the extreme virulence of the r1918.H1N1 influenza A viruses continue to pose serious threats to public health, as exemplified by the ongoing 2009 H1N1 influenza pandemic. The 1918-1919 H1N1 influenza pandemic was even deadlier in comparison, causing more than 20 million deaths worldwide. The keys to unlocking the mystery of the extreme virulence of the 1918 virus were provided with the reconstruction of the virus (reconstructed 1918 influenza virus [r1918]) by reverse genetics (37). The lethality of r1918 has since been examined in both mouse and macaque models (17, 18). Unlike the nonlethal infections of some other H1N1 influenza virus strains, such as A/Texas/36/91 (Tx/91) or A/Kawasaki/173/01 (K173), the r1918 causes severe and lethal pulmonary disease. We subsequently conducted functional genomics analyses that revealed that the extreme virulence of r1918 was correlated with atypical expression of immune response-related genes, including massive induction of cellular genes related to inflammatory response and cell death pathways (17, 18). In spite of these findings, the mechanistic basis for these atypical gene expression patterns remains unknown.Cellular gene expression is a complicated process and is subject to regulation by many cellular factors. As a group of newly identified cellular regulators, microRNAs are known to regulate the expression of a large number of targets, mainly cellular genes. Through mRNA degradation or translational repression of their targets, microRNAs regulate a wide range of crucial physiologic and pathological processes. For example, miR-34a acts as a tumor suppressor by inhibiting the expression of sirt1 (40), whereas miR-21 contributes to myocardial disease by inhibiting the expression of spry1 (36). By targeting zeb1/2, the miR-200 family members play roles in maintaining the epithelial phenotype of cancer cells (27). Furthermore, Let-7s regulates the expression of hbl-1, which drives the developmental progression of epidermal stem cells (5). Cellular microRNAs also play critical roles in virus-host interactions. The cellular microRNA miR-122 is an indispensable factor in supporting hepatitis C virus (HCV) replication (16), whereas miR-196 and miR-296 substantially attenuate viral replication through type I interferon (IFN)-associated pathways in liver cells (28). Furthermore, miR-125b and miR-223 directly target human immunodeficiency virus type 1 (HIV-1) mRNA, thereby attenuating viral gene expression in resting CD4⁺ T cells (14), and miR-198 modulates HIV-1 replication indirectly by repressing the expression of ccnt1 (34), a cellular factor necessary for HIV-1 replication. More importantly, viruses may promote their life cycles by modulating the intracellular environment through actively regulating the expression of multiple cellular microRNAs. For example, human T-cell lymphotropic virus type 1 (HTLV-1) modulates the expression of a number of cellular microRNAs in order to control T-cell differentiation (3). Similarly, human cytomegalovirus (HCMV) selectively manipulates the expression of miR-100 and miR-101 to facilitate its own replication (38). In contrast, the involvement of microRNAs during influenza A virus infection or pathogenesis is largely unknown.To determine whether cellular microRNAs play a role in the host response to influenza virus infection, we performed a systematic profiling of cellular microRNAs in lung tissues from mice infected with r1918 or a nonlethal seasonal influenza virus, Tx/91 (17). We identified a group of microRNAs whose expression patterns differentiated the host response to r1918 and Tx/91 infection. We assessed the potential functions of differentially expressed microRNAs by analyzing the predicted target genes whose expression was inversely correlated with the expression of these microRNAs. Our report provides a new perspective on the contribution of microRNAs to the pathogenesis of lethal 1918 influenza virus infection.