Peiqing Cong

Comparative Analyses by Sequencing of Transcriptomes during Skeletal Muscle Development between Pig Breeds Differing in Muscle Growth Rate and Fatness

Understanding the dynamics of muscle transcriptome during development and between breeds differing in muscle growth is necessary to uncover the complex mechanism underlying muscle development. Herein, we present the first transcriptome-wide longissimus dorsi muscle development research concerning Lantang (LT, obese) and Landrace (LR, lean) pig breeds during 10 time-points from 35 days-post-coitus (dpc) to 180 days-post-natum (dpn) using Solexa/Illuminas Genome Analyzer. The data demonstrated that myogenesis was almost completed before 77 dpc, but the muscle phenotypes were still changed from 77 dpc to 28 dpn. Comparative analysis of the two breeds suggested that myogenesis started earlier but progressed more slowly in LT than in LR, the stages ranging from 49 dpc to 77 dpc are critical for formation of different muscle phenotypes. 595 differentially expressed myogenesis genes were identified, and their roles in myogenesis were discussed. Furthermore, GSK3B, IKBKB, ACVR1, ITGA and STMN1 might contribute to later myogenesis and more muscle fibers in LR than LT. Some myogenesis inhibitors (ID1, ID2, CABIN1, MSTN, SMAD4, CTNNA1, NOTCH2, GPC3 and HMOX1) were higher expressed in LT than in LR, which might contribute to more slow muscle differentiation in LT than in LR. We also identified several genes which might contribute to intramuscular adipose differentiation. Most important, we further proposed a novel model in which MyoD and MEF2A controls the balance between intramuscular adipogenesis and myogenesis by regulating CEBP family; Myf5 and MEF2C are essential during the whole myogenesis process while MEF2D affects muscle growth and maturation. The MRFs and MEF2 families are also critical for the phenotypic differences between the two pig breeds. Overall, this study contributes to elucidating the mechanism underlying muscle development, which could provide valuable information for pig meat quality improvement. The raw data have been submitted to Gene Expression Omnibus (GEO) under series GSE25406.

Lipopolysaccharide-induced miR-1224 negatively regulates tumour necrosis factor-α gene expression by modulating Sp1.

The innate immune response provides the initial defence mechanism against infection by other organisms. However, an excessive immune response will cause damage to host tissues. In an attempt to identify microRNAs (miRNAs) that regulate the innate immune response in inflammation and homeostasis, we examined the differential expression of miRNAs using microarray analysis in the spleens of mice injected intraperitoneally with lipopolysaccharide (LPS) and saline, respectively. Following challenge, we observed 19 miRNAs up‐regulated (1·5‐fold) in response to LPS. Among these miRNAs, miR‐1224, whose expression level increased 5·7‐fold 6 hr after LPS injection and 2·3‐fold after 24 hr, was selected for further study. Tissue expression patterns showed that mouse miR‐1224 is highly expressed in mouse spleen, kidney and lung. Transfection of miR‐1224 mimics resulted in a decrease in basal tumour necrosis factor‐α (TNF‐α) promoter reporter gene activity and a down‐regulation of LPS‐induced TNF‐α mRNA in RAW264.7 cells. With public databases of miRNA target prediction, miR‐1224 was shown to bind to the 3′ untranslated region (UTR) of Sp1 mRNA, whose coding product controls TNF‐α expression at the transcriptional level. Furthermore, we found that in HEK‐293 cells, the activity of the luciferase reporter bearing Sp1 mRNA 3′ UTR was down‐regulated significantly when transfected with miR‐1224 mimics. After transfection of miR‐1224 in RAW264.7 cells, nucleus Sp1 protein level decreased, and when endogenous miR‐1224 was blocked, the decrease was abolished. Therefore, we initially speculated that miR‐1224 was a negative regulator of TNF‐α in an Sp1‐dependent manner, which was confirmed in vivo by chromatin immunoprecipitation assay, and might be involved in regulating the LPS‐mediated inflammatory responses.

MicroRNAome Comparison between Intramuscular and Subcutaneous Vascular Stem Cell Adipogenesis

Background As an important factor affecting meat quality, intramuscular fat (IMF) content is a topic of worldwide concern. Emerging evidences indicate that microRNAs play important roles in adipocyte differentiation. However, miRNAome has neither been studied during porcine intramuscular preadipocyte differentiation, nor compared with subcutaneous preadipocytes. The objectives of this study were to identify porcine miRNAs involved in adipogenesis in primary preadipocytes, and to determine whether intramuscular and subcutaneous adipocytes differ in the expression and regulation of miRNAs. Results miRNAomes in primary intramuscular and subcutaneous adipocytes during differentiation were first sequenced using the Solexa deep sequencing method. The sequences and relative expression levels of 224 known (98.2% in miRbase 18.0) and 280 potential porcine miRNAs were identified. Fifty-four of them changed in similar pattern between intramuscular vascular stem cells (IVSC) and subcutaneous vascular stem cells (SVSC) differentiation, such as miR-210, miR-10b and miR-99a. Expression levels of 10 miRNAs were reversely up-or down-regulated between IVSC and SVSC differentiation, 19 were up-or down-regulated only during IVSC differentiation and 55 only during SVSC differentiation. Additionally, 30 miRNAs showed fat-depot specific expression pattern (24 in cells of intramuscular origin and 6 in cells of subcutaneous origin). These adipogenesis-related miRNAs mainly functioned by targeting similar pathways in adipogenesis, obesity and syndrome. Conclusion Comparison of miRNAomes in IVSC and SVSC during differentiation revealed that many different miRNAs are involved in adipogenesis, and they regulate SVSC and IVSC differentiation through similar pathways. These miRNAs may serve as biomarkers or targets for enhancing IMF content, and uncovering their function in IMF development will be of great value in the near future.

Proteome changes of lungs artificially infected with H-PRRSV and N-PRRSV by two-dimensional fluorescence difference gel electrophoresis.

BackgroundPorcine reproductive and respiratory syndrome with PRRS virus (PRRSV) infection, which causes significant economic losses annually, is one of the most economically important diseases affecting swine industry worldwide. In 2006 and 2007, a large-scale outbreak of highly pathogenic porcine reproductive and respiratory syndrome (PRRS) happened in China and Vietnam. However little data is available on global host response to PRRSV infection at the protein level, and similar approaches looking at mRNA is problematic since mRNA levels do not necessarily predict protein levels. In order to improve the knowledge of host response and viral pathogenesis of highly virulent Chinese-type PRRSV (H-PRRSV) and Non-high-pathogenic North American-type PRRSV strains (N-PRRSV), we analyzed the protein expression changes of H-PRRSV and N-PRRSV infected lungs compared with those of uninfected negative control, and identified a series of proteins related to host response and viral pathogenesis.ResultsAccording to differential proteomes of porcine lungs infected with H-PRRSV, N-PRRSV and uninfected negative control at different time points using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mass spectrometry identification, 45 differentially expressed proteins (DEPs) were identified. These proteins were mostly related to cytoskeleton, stress response and oxidation reduction or metabolism. In the protein interaction network constructed based on DEPs from lungs infected with H-PRRSV, HSPA8, ARHGAP29 and NDUFS1 belonged to the most central proteins, whereas DDAH2, HSPB1 and FLNA corresponded to the most central proteins in those of N-PRRSV infected.ConclusionsOur study is the first attempt to provide the complex picture of pulmonary protein expression during H-PRRSV and N-PRRSV infection under the in vivo environment using 2D-DIGE technology and bioinformatics tools, provides large scale valuable information for better understanding host proteins-virus interactions of these two PRRSV strains.

Integrated miRNA and mRNA transcriptomes of porcine alveolar macrophages (PAM cells) identifies strain-specific miRNA molecular signatures associated with H-PRRSV and N-PRRSV infection

Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant viral diseases in swine, which causes large economic losses to the swine industry worldwide. There is considerable strain variation in PRRSV and two examples of this are the highly virulent Chinese-type PRRSV (H-PRRSV) and the classical North American type PRRSV (N-PRRSV), both with different pathogenesis. These differences may be due in part to genetic and phenotypic differences in virus replication, but also interaction with the host cell. MicroRNAs (miRNAs) are crucial regulators of gene expression and play vital roles in virus and host interactions. However, the regulation role of miRNAs during PRRSV infection has not been systematically investigated. In order to better understand the differential regulation roles of cellular miRNAs in the host response to PRRSV, miRNA expression and a global mRNA transcriptome profile was determined in primary cells infected with either H-PRRSV or N-PRRSV as multiple time points during the viral lifecycle. miRNA-mRNA interactome networks were constructed by integrating the differentially expressed miRNAs and inversely correlated target mRNAs. Using gene ontology and pathway enrichment analyses, cellular pathways associated with deregulated miRNAs were identified, including immune response, phagosome, autophagy, lysosome, autolysis, apoptosis and cell cycle regulation. To our knowledge, this is the first global analysis of strain-specific host miRNA molecular signatures associated with H- and N-PRRSV infection by integrating miRNA and mRNA transcriptomes and provides a new perspective on the contribution of miRNAs to the pathogenesis of PRRSV infection.

Effects of trichostatin A on histone acetylation and methylation characteristics in early porcine embryos after somatic cell nuclear transfer

Until now, the efficiency of animal cloning by somatic cell nuclear transfer (SCNT) has remained low. Efforts to improve cloning efficiency have demonstrated a positive role of trichostatin A (TSA), an inhibitor of deacetylases, on the development of nuclear transfer (NT) embryos in many species. Here, we report the effects of TSA on pre-implantation development of porcine NT embryos. Our results showed that treatment of reconstructed porcine embryos with 50 nmol/L TSA for 24 h after activation significantly improved the production of blastocysts (P < 0.05), while treating donor cells with the same solution resulted in increases in cleavage rates and blastomere numbers (P < 0.05). However, TSA treatment of both donor cells and SCNT embryos did not improve blastocyst production, nor did it increase blastomere numbers. Using indirect immunofluorescence, we found that TSA treatment of NT embryos could improve the reprogramming of histone acetylation at lysine 9 of histone 3 (H3K9) and affect nuclear swelling of transferred nuclei. However, no apparent effect of TSA treatment on H3K9 dimethylation (H3K9me2) was observed. These findings suggest a positive effect of TSA treatment (either treating NT embryos or donor cells) on the development of porcine NT embryos, which is achieved by improving epigenetic reprogramming.

Peripheral administration of TAT-obestatin can influence the expression of liporegulatory genes but fails to affect food intake in mice.

Obestatin is a 23-amino-acid peptide originally regarded as an anorexigenic factor. However, most of the subsequent studies failed to confirm the initially reported anorexigenic properties of obestatin. Obestatin is incapable of crossing the blood brain barrier (BBB), which may affect its biological function. Here, we report the physiological effects of obestatin in mice after intraperitoneal administration of obestatin conjugated to the cell-permeable peptide TAT, which is capable of delivering different types of proteins through the BBB. Acute peripheral administration of 1 μmol/kg of TAT-obestatin did not influence the 24 h cumulative food intake and body weight gain of mice that were fasted for 18 h. Fed mice were injected intraperitoneally with 100 nmol/kg of TAT-obestatin daily for 25 d. Compared with control groups, on day 3, the gain in body weight was significantly altered; on day 7, abdominal fat mass was remarkably reduced; however, on day 25, there was a surprisingly notable increase in abdominal and epididymal fat mass. In comparison with control groups, on day 25, the expression levels of adiponectin, ADD1, C/EBPα, PPARG and GLUT4 were significantly up-regulated in liver tissues; in white adipose tissue, the expression level of C/EBPα was significantly up-regulated, but adiponectin and GLUT4 were significantly down-regulated. In addition, GPR39, the suspected receptor of obestatin, was up-regulated in white adipose tissue on day 25. These findings suggest that TAT-obestatin might play a role in white adipose tissue metabolism, but its physiological effects on food intake and body weight gain regulation remain unclear.

Effect of miR-205 on 3T3-L1 preadipocyte differentiation through targeting to glycogen synthase kinase 3 beta

MiR-205 plays an important role during adipogenesis by modulating the Wnt signaling pathway. Here, we report that miR-205 can regulate the differentiation of 3T3-L1 preadipocyte cells by targeting glycogen synthase kinase 3 beta (GSK-3β), which is a negative regulatory factor of Wnt signaling. When transiently overexpressed in 3T3-L1 cells, miR-205 suppressed the translation of GSK-3β, resulting in increased expression of β-catenin, which can promote cell proliferation by facilitating the transcription of the Wnt target genes cyclin D1 and c-Myc. However, stable overexpression of miR-205 in 3T3-L1 cells did not show any apparent inhibitory effect on adipogenic differentiation. While endogenous miR-205 was inhibited in 3T3-L1 cells, the adipogenesis marker gene, C/EBPα, was significantly activated and more lipid droplets appeared in differentiated adipocytes. However, systemic silencing of miR-205 in mice by using a locked-nucleic-acid-modified oligonucleotide (LNA-antimiR) did not lead to any observable increase in adipose tissue differentiation, implying that, as opposed to the findings from 3T3-L1 cells, miR-205 is dispensable for adipose tissue development in mice.

Improvement of porcine cloning efficiency by trichostain A through early-stage induction of embryo apoptosis

Trichostain A (TSA), an inhibitor of histone deacetylases, improved developmental competence of SCNT embryos in many species, apparently by improved epigenetic reprogramming. The objective of the present study was to determine the effects of TSA-induced apoptosis in cloned porcine embryos. At various developmental stages, a comet assay and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining were used to detect apoptosis, and real-time polymerase chain reaction was used to assess expression of genes related to apoptosis and pluripotency. In this study, TSA significantly induced apoptosis (in a dose-dependent manner) at the one-, two-, and four-cell stages. However, in blastocyst stage embryos, TSA decreased the apoptotic index (P < 0.05). Expression levels of Caspase 3 were higher in TSA-treated versus control embryos at the two-cell stage (not statistically significant). The expression ratio of antiapoptotic Bcl-xl gene to proapoptotic Bax gene, an indicator of antiapoptotic potential, was higher in TSA-treated groups at the one-, two-, and four-cell and blastocyst stages. Furthermore, expression levels of pluripotency-related genes, namely, Oct4 and Nanog, were elevated at the morula stage (P < 0.05) in TSA treatment groups. We concluded that inducing apoptosis might be a mechanism by which TSA promotes development of reconstructed embryos. At the initial stage of apoptosis induction, abnormal cells were removed, thereby enhancing proliferation of healthy cells and improving embryo quality.

MiR-143 is not essential for adipose development as revealed by in vivo antisense targeting

MiR-143 plays an important role in promoting the adipogenic differentiation of pre-adipocytes. Here, we report that systematic silencing of miR-143 in mice by using a locked-nucleic-acid-modified oligonucleotide (LNA-antimiR) did not lead to any obvious abnormalities in the adipose tissue differentiation. Furthermore, there were no significant differences in the expression level of several adipogenic marker genes, such as PPARγ and C/EBPα, in these animals compared with the controls. Therefore, we hypothesize that miR-143 may function as a fine tuning molecule rather than as a switch in the adipogenic regulatory network in mice. In addition, the proposed miR-143 target, ERK5, which was previously identified in human preadipocytes, was not effectively inhibited by miR-143 either in the murine preadipocyte cell line, 3T3-L1, or in primary mouse adipose tissue. However, we did fibroblast growth factor 7 (Fgf7) was identified as a target of miR-143 in murine adipogenesis.