Hsiao-Ching Liu

Integrated analysis of microRNA expression and mRNA transcriptome in lungs of avian influenza virus infected broilers

BackgroundAvian influenza virus (AIV) outbreaks are worldwide threats to both poultry and humans. Our previous study suggested microRNAs (miRNAs) play significant roles in the regulation of host response to AIV infection in layer chickens. The objective of this study was to test the hypothesis if genetic background play essential role in the miRNA regulation of AIV infection in chickens and if miRNAs that were differentially expressed in layer with AIV infection would be modulated the same way in broiler chickens. Furthermore, by integrating with parallel mRNA expression profiling, potential molecular mechanisms of host response to AIV infection can be further exploited.ResultsTotal RNA isolated from the lungs of non-infected and low pathogenic H5N3 infected broilers at four days post-infection were used for both miRNA deep sequencing and mRNA microarray analyses. A total of 2.6 M and 3.3 M filtered high quality reads were obtained from infected and non-infected chickens by Solexa GA-I Sequencer, respectively. A total of 271 miRNAs in miRBase 16.0 were identified and one potential novel miRNA was discovered. There were 121 miRNAs differentially expressed at the 5% false discovery rate by Fisher’s exact test. More miRNAs were highly expressed in infected lungs (108) than in non-infected lungs (13), which was opposite to the findings in layer chickens. This result suggested that a different regulatory mechanism of host response to AIV infection mediated by miRNAs might exist in broiler chickens. Analysis using the chicken 44 K Agilent microarray indicated that 508 mRNAs (347 down-regulated) were differentially expressed following AIV infection.ConclusionsA comprehensive analysis combining both miRNA and targeted mRNA gene expression suggests that gga-miR-34a, 122–1, 122–2, 146a, 155, 206, 1719, 1594, 1599 and 451, and MX1, IL-8, IRF-7, TNFRS19 are strong candidate miRNAs or genes involved in regulating the host response to AIV infection in the lungs of broiler chickens. Further miRNA or gene specific knock-down assay is warranted to elucidate underlying mechanism of AIV infection regulation in the chicken.

Discovery of chicken microRNAs associated with lipogenesis and cell proliferation

The primary function of microRNA (miRNA, a class of small regulatory RNA) is to regulate gene expression. Studies of miRNA in mammals suggest that many liver-associated miRNAs are expressed, with a wide range of functions. To characterize miRNA expressed in the avian liver, we created two small RNA libraries from embryonic chick livers at embryonic day (E)15 and E20, a time at which the embryo begins to grow rapidly and so its energy demands increase. It is of interest to examine miRNAs expressed at these developmental stages because miRNAs involved in regulating metabolic pathways and cell proliferation are likely to be identified. The small RNA libraries were sequenced with 454 Life Sciences deep sequencing. Of the 49,937 sequences obtained, 29,390 represented known chicken miRNAs and 1,233 reads represented homologous miRNAs that have not been previously identified in chickens. Additionally, 1,032 reads represented 17 potential novel miRNAs not previously identified in any species. To further investigate the possible functions of avian liver miRNAs we identified the potential targets of two differentially expressed novel miRNAs, nc-miR-5 and nc-miR-33. These two miRNAs were predicted to target metabolic genes, including the lipid metabolism-associated gene fatty acid synthase (FAS), and genes involved in the control of cell proliferation, such as peroxisome proliferator-activated binding protein (Pparbp) and bone morphogenetic protein 4 (BMP4). Our findings demonstrate that a diverse group of miRNAs are expressed in developing avian livers. In addition, some of the identified miRNAs have been suggested to play a key role(s) in regulating metabolic pathways.

Current knowledge of microRNA characterization in agricultural animals.

MicroRNA (miRNA) is a class of single-stranded small (19-24nt) regulatory RNA that silences gene expression post-transcriptionally. miRNAs regulate a wide range of biological processes through the recognition of complementary sequences between miRNAs and their target genes. Profiling studies in livestock have revealed that many miRNAs are species- and tissue-specific, indicating that miRNAs play important roles in essential biological processes in livestock, such as muscle and organ development, the immune response and metabolism. The allelic variation of miRNA target sites and possibly in miRNAs themselves are also likely to be contributing factors to many phenotypic differences in livestock. In this review, we summarize the current miRNA studies undertaken in livestock.

Proteins involved in iron metabolism in beef cattle are affected by copper deficiency in combination with high dietary manganese, but not by copper deficiency alone.

A 493-d study was conducted to determine the impact of a severe, long-term Cu deficiency on Fe metabolism in beef cattle. Twenty-one Angus calves were born to cows receiving one of the following treatments: 1) adequate Cu (+Cu), 2) Cu deficient (-Cu), and 3) Cu deficient plus high Mn (-Cu+Mn). Copper deficiency was induced through the addition of 2 mg of Mo/kg of DM. After weaning, calves remained on the same treatment as their dam through growing (basal diet analyzed 7 mg of Cu/kg of DM) and finishing (analyzed 4 mg of Cu/kg of DM) phases. Plasma Fe concentrations were positively correlated (P < 0.01; r = 0.49) with plasma Cu concentrations. Liver Fe concentrations were greater (P = 0.05) in -Cu vs. +Cu calves and further increased (P = 0.07) in -Cu+Mn vs. -Cu calves. There was a negative relationship (P < 0.01; r = -0.31) between liver Cu and Fe concentrations. This relationship is likely explained by less (P < 0.01) plasma ceruloplasmin activity in -Cu than +Cu calves. As determined by real-time reverse transcription-PCR, relative expression of hepatic hepcidin was significantly downregulated (>1.5 fold) in -Cu compared with +Cu calves (P = 0.03), and expression of hepatic ferroportin tended (P = 0.09) to be downregulated in -Cu vs. +Cu. In the duodenum, ferritin tended to be upregulated in -Cu. vs. +Cu calves (P < 0.06). No significant change (P > 0.2) due to Cu-deficiency was detected at the transcriptional level for either isoform of divalent metal transporter 1 (DMT1 mRNA with or without an iron responsive element; dmt1IRE and dmt1-nonIRE) in liver or intestine. Duodenal expression of hephaestin and ferroportin protein was not affected by dietary treatment (P > 0.20). However, duodenal expression of DMT1 protein was less (P = 0.04) in -Cu+Mn steers vs. -Cu steers. In summary, Cu deficiency alone did affect hepatic gene expression of hepcidin and ferroportin, but did not affect duodenal expression of proteins important in Fe metabolism. However, the addition of 500 mg of Mn/kg of DM to a diet low in Cu reduced duodenal expression of the Fe import protein DMT1.

Age and Dietary Iron Affect Expression of Genes Involved in Iron Acquisition and Homeostasis in Young Pigs

To investigate the effects of dietary iron (Fe) and age on Fe metabolism, we used 36 weaned barrows in a 2 x 3 design with 2 concentrations of dietary Fe [97 (control) and 797 (high Fe) mg Fe/kg dry matter] and 3 time points of tissue collection (after 21, 42, or 63 d on diets). Pigs were weighed and bled on d 0, 20, 41, and 62. High Fe reduced feed efficiency but did not affect pig weight gain. Blood hemoglobin concentrations and Fe concentrations of liver, intestine, and heart were increased by high dietary Fe on all days. Concentrations of liver and heart Fe increased with age. As determined by quantitative real-time PCR, hepatic expression of hepcidin (HAMP) in pigs given the high-Fe diet was 6.25-fold that of control pigs. In the intestine, relative mRNA levels of ferroportin, divalent metal transporter 1, and transferrin receptor were downregulated by high Fe. Expression of an alternative route of Fe absorption, solute carrier family 39 member 14 (SLC39A14), was downregulated in the intestine of pigs fed high dietary Fe. Additionally, duodenal mRNA level of certain genes including scavenger receptor class A, member 5, and frataxin decreased with age of the animal. Our findings indicate new roles in Fe metabolism for several mineral metabolism-associated genes and that some of these genes, such as SLC39A14, may be regulated in response to dietary Fe in pigs. Additionally, the expression of some genes examined in this study was affected by age, suggesting age dependency of Fe metabolism in pigs.

Current State of Marek's Disease Virus MicroRNA Research

SUMMARY.  MicroRNA (miRNA) is a major family of small RNAs that posttranscriptionally regulate gene expression. Small RNA profiling studies have revealed that some viruses, particularly large DNA viruses, such as Mareks disease virus (MDV), encode their own set of miRNAs. There are currently 406 viral miRNAs in miRBase, of which 392 are encoded by herpesviruses. To date, 26 MDV-1 miRNAs, 36 MDV-2 miRNAs, and 28 herpesvirus of turkeys miRNAs have been identified. Interestingly, herpesvirus miRNAs appear to have spatial conservation, located in clusters within repeat regions, but lack sequence conservation. Two clusters of MDV-1 miRNA have been identified, one located near the MEQ gene and one within the latency-associated transcript (LAT). miRNA profiling studies have shown that MDV miRNA are differentially expressed between strains and stages of infection. For example, mdv1-miR-M4 and mdv1-miR-M2-3p are three- and sixfold higher, expressed, respectively, in vv+ strains compared to vv strains. A recent study found that deletion or seed region mutation of mdv1-miR-M4 reduces viral oncogenicity, suggesting a link between mdv1-mir-M4 and lymphoma development in MDV-infected birds. Taken together, current research suggests that viral miRNAs are a key component of MDV pathogenesis. RESUMEN.  Estudio Recapitulativo—Estado actual de la investigación sobre micro ARN en la enfermedad de Marek. Las moléculas de micro ARN (miRNA) son una familia de moléculas pequeñas de ARN que regulan de manera postranscripcional la expresión de genes. Los estudios de los perfiles de moléculas pequeñas de ARN han revelado que algunos virus, particularmente virus ADN grandes como el virus de Marek codifican su propio conjunto de micro ARN. Actualmente existen 406 moléculas de micro ARN en la base de datos miRBase, de las cuales 392 están codificadas por herpesvirus. Hasta la fecha, se han identificado 26 moléculas de micro ARN del virus de Marek 1, 36 del virus de Marek 2 y 28 del herpesvirus de pavos. De manera interesante, las moléculas de micro ARN de los herpesvirus parecen ser conservadas de manera espacial, localizados en grupos dentro de regiones repetidas, pero carecen de secuencias conservadas. Dos grupos del virus de Marek 1 han sido identificados, uno se encuentra localizado cerca del gene MEQ y otro dentro del transcripto asociado con la latencia (LAT). Los estudios de perfiles de micro ARN han demostrado que las moléculas de micro ARN del virus de la enfermedad de Marek se expresan de manera diferente de acuerdo a las cepas o al estado de infección. Por ejemplo, las moléculas mdv1-miR-M4 y mdv1-miR-M2-3p se expresan de tres y seis veces más, respectivamente en las cepas muy virulentas plus en comparación con las cepas muy virulentas. Un estudio reciente demostró que la deleción o una mutación en la región de la semilla de mdv1-miR-M4 reduce la oncogénesis viral, lo que sugiere un vínculo entre mdv1-mir-M4 y el desarrollo de linfomas en las aves infectadas con Marek. Considerando todo, la investigación reciente sugiere que las moléculas de micro ARN virales son un componente clave en la patogénesis de la enfermedad de Marek.

Involvement of Eukaryotic Small RNA Pathways in Host Defense and Viral Pathogenesis

Post-transcriptional gene regulation by small RNAs is now established as an important branch of the gene regulatory system. Many different classes of small RNAs have been discovered; among these are short interfering RNAs (siRNAs) and microRNA (miRNAs). Though differences in the processing and function of small RNAs exist between plants and animals, both groups utilize small RNA-mediated gene regulation in response to pathogens. Host encoded miRNAs and siRNAs are generated from viral RNA function in host defense and pathogenic resistance in plants. In animals, miRNAs are key regulators in both immune system development and in immune function. Pathogens, in particular viruses, have evolved mechanisms to usurp the host’s small RNA-mediated regulatory system. Overall, small RNAs are a major component of host defense and immunity in eukaryotes. The goal of this review is to summarize our current knowledge of the involvement of eukaryotic small RNA pathways in host defense and viral pathogenesis.

The Interplay Between MDV and HVT Affects Viral miRNA Expression

SUMMARY.  It is well established that herpesviruses encode numerous microRNAs (miRNAs) and that these virally encoded small RNAs play multiple roles in infection. The present study was undertaken to determine how co-infection of a pathogenic MDV serotype one (MDV1) strain (MD5) and a vaccine strain (herpesvirus of turkeys [HVT]) alters viral miRNA expression in vivo. We first used small RNA deep sequencing to identify MDV1-encoded miRNAs that are expressed in tumorigenic spleens of MDV1-infected birds. The expression patterns of these miRNAs were then further assessed at an early time point (7 days postinfection [dpi]) and a late time point (42 dpi) in birds with and without HVT vaccination using real-time PCR (RT-PCR). Additionally, the effect of MDV1 co-infection on HVT-encoded miRNAs was determined using RT-PCR. A diverse population of miRNAs was expressed in MDV-induced tumorigenic spleens at 42 dpi, with 18 of the 26 known mature miRNAs represented. Of these, both mdv1-miR-M4-5p and mdv1-miR-M2-3p were the most highly expressed miRNAs. RT-PCR analysis further revealed that nine MDV miRNAs were differentially expressed between 7 dpi and 42 dpi infected spleens. At 7 dpi, three miRNAs were differentially expressed between the spleens of birds co-infected with HVT and MD5 compared with birds singly infected with MD5, whereas at 42 dpi, nine miRNAs were differentially expressed. At 7 dpi, the expression of seven HVT-encoded miRNAs was affected in the spleens of co-infected birds compared with birds only receiving the HVT vaccine. At 42 dpi, six HVT-encoded miRNAs were differentially expressed between the two groups. Target prediction analysis suggests that these differentially expressed viral miRNAs are involved in regulating several cellular processes, including cell proliferation and the adaptive immune response. RESUMEN.  La interacción entre el virus de Marek y el herpesvirus de los pavos afecta a la expresión de micro ARN viral. Está bien establecido que los herpesvirus codifican numerosos micro ARN (con las siglas en inglés miRNAs) y que estas pequeñas moléculas de ARN codificadas viralmente juegan múltiples papeles en la infección. El presente estudio se realizó para determinar como la co-infección entre una cepa patógena (MD5) del virus de Marek serotipo 1 (MDV1) y una cepa vacunal (herpesvirus de los pavos [HVT]) altera la expresión de los genes micro ARN viral in vivo. Se utilizó por primera vez la secuenciación profunda de moléculas pequeñas de ARN para identificar micro ARN codificado por el virus de Marek serotipo 1 que se expresan en los bazos con tumores en las aves infectadas por el virus de Marek 1. Los patrones de expresión de micro ARN fueron valorados mediante PCR en tiempo real (RT-PCR), de manera temprana (7 días después de la infección) y tardíamente (42 días después de la infección) en las aves con y sin vacunación con el herpesvirus de los pavos. Además se determinó el efecto de la co-infección con el virus de Marek serotipo 1 sobre el micro ARN codificado por el herpesvirus de los pavos mediante PCR en tiempo real. Se expresó una población diversa de micro ARN en los bazos con tumores inducidos por el virus de Marek a los 42 días después de la infección, con 18 micro ARNs maduros representados de los 26. De éstos, tanto el mdv1-miR-M4-5p y el mdv1-miR-M2-3p fueron los micro ARN más expresados. El análisis por PCR en tiempo real reveló que nueve micro ARN del virus de Marek se expresan diferencialmente entre 7 y 42 días después de la infección en los bazos infectados. A los siete días después de la infección, tres micro ARN fueron expresados diferencialmente en los bazos de las aves co-infectadas con el herpesvirus de los pavos y la cepa MD5 en comparación con las aves infectadas con el virus MD5 por separado, mientras que a los 42 días después de la infección, nueve micro ARNs fueron expresados diferencialmente. A los siete días después, la expresión de siete micro ARNs codificados por el herpesvirus de los pavos se vio afectada en los bazos de las aves co-infectadas en comparación con las aves que sólo recibieron la vacuna con el herpesvirus de los pavos. A los 42 días después de la infección, seis micro ARNs codificados por el herpesvirus de los pavos fueron expresados diferencialmente entre los dos grupos. El análisis de predicción de moléculas blanco sugiere que estos micro ARNs virales expresados diferencialmente están involucrados en la regulación de varios procesos celulares, incluyendo la proliferación celular y la respuesta inmune adaptativa.

Characterization of miR-10a mediated gene regulation in avian splenocytes.

It is well established that microRNAs (miRNAs) are an important class of post-transcriptional regulators of gene expression. Although numerous miRNA expression profiles have been generated for many eukaryotic organisms, little is known about the specific functions of individual miRNAs in regulating gene expression. We previously reported that the miRNA, miR-10a, is highly expressed during spleen development in embryonic chicks. In this current study we have identified genes and potential pathways that are both directly and indirectly influenced by miR-10a expression. To achieve this goal, miRNA Real-Time (RT) PCR analysis was first utilized to examine miR-10a expression across tissues during both embryonic and post-hatch chick development. Next, microarray analysis was employed to determine alterations in global gene expression associated with miR-10a in embryonic chick splenocytes subjected to an in vitro miR-10a inhibitor treatment. Finally the miRNA target prediction algorithm miRanda was used to predict potential chicken genes directly targeted by miR-10a. A select group of potential miR-10a target genes was validated using an RCAS-miRNA expression based luciferase assay. Our results indicate that miR-10a is highly expressed in the avian spleen, lung, kidneys, and fat tissues. Functional analysis suggests that miR-10a is involved in regulating gene expression in pathways associated with Ras signaling, intracellular trafficking, and development of immune functions. Additionally, we confirmed that chicken HOXA1 is a miR-10a target gene, suggesting a conserved role for miR-10a in the regulation of hematopoiesis across vertebrates.

Alterations in cellular and viral microRNA and cellular gene expression in Marek's disease virus-transformed T-cell lines treated with sodium butyrate

ABSTRACT A shared feature of herpesviruses is their ability to enter a latent state following an initially lytic infection. Mareks disease virus serotype 1 (MDV‐1) is an oncogenic avian herpesvirus. Small RNA profiling studies have suggested that microRNAs (miRNAs) are involved in viral latency. Sodium butyrate treatment is known to induce herpesvirus reactivation. The present study was undertaken to determine transcriptome and miRNome changes induced by sodium butyrate in 2 MDV‐transformed cell lines, RP2 and CU115. In the first 24 h post‐treatment, microarray analysis of transcriptional changes in cell lines RP2 and CU115 identified 137 and 114 differentially expressed genes, respectively. Small RNA deep‐sequencing analysis identified 17 cellular miRNAs that were differentially expressed. The expression of MDV‐encoded miRNAs was also altered upon treatment. Many of the genes and miRNAs that are differentially expressed are involved in regulation of the cell cycle, mitosis, DNA metabolism, and lymphocyte differentiation.