miR-199b-5p enhances the proliferation of medullary thymic epithelial cells via regulating Wnt signaling by targeting Fzd6.

Thymic epithelial cells (TECs) are essential regulators of T-cell development and selection. miRNAs play critical roles in regulating TEC proliferation during the process of thymic aging. Our previous studies revealed that miR-199b-5p was upregulated in TECs from 1- to 3-month-old mice. But its function and potential mechanism are not clear. We hypothesized that miR-199b-5p may play an important role in age-related thymus involution via targeting some genes. To confirm it, the murine thymic epithelial cell line 1 (MTEC1) cells were used. Our results showed that overexpression of miR-199b-5p can enhance MTEC1 cell proliferation. On the contrary, repression of miR-199b-5p can inhibit MTEC1 cell proliferation. Meanwhile, it was confirmed that frizzled receptor 6 (Fzd6) is the direct target gene of miR-199b-5p. Furthermore, overexpression of miR-199b-5p can upregulate the expressions of ß-catenin, Tcf7, Wnt4, and C-myc to activate Wnt signaling and cell cycle signaling. Silence of Fzd6 and co-transfection with siFzd6 and miR-199b-5p mimic/inhibitor confirmed that the biological function of miR-199b-5p is indeed by targeting Fzd6 in medullary TECs. Overall, miR-199b-5p is an important regulator in medullary TEC proliferation through targeting Fzd6 to activate Wnt signaling and cell cycle signaling. Our data indicate that miR-199b-5p may block the process of thymic aging and be a potential therapeutic target for thymus involution.

Profiling analysis of 17ß-estradiol-regulated lncRNAs in mouse thymic epithelial cells.

Thymus is the primary organ for T cell differentiation and maturation. Many studies have demonstrated that estrogen plays a crucial role in thymic epithelial cell (TEC) proliferation during thymic involution. LncRNAs are involved in various biological processes; however, estrogen-mediated lncRNA expression in TECs has not been yet reported. To address this question, the mouse medullary thymic epithelial cell line 1 (MTEC1) was treated with 17ß-estradiol (E2). By using CCK8 assay and flow cytometry, we found that E2 was able to inhibit viability and proliferation of MTEC1 cells. The expression profiles of lncRNAs in MTEC1 cells with or without E2 treatment were then measured by RNA-Seq, and a total of 962 lncRNAs and 2,469 mRNAs were shown to be differentially expressed. The reliability of RNA-Seq was confirmed by quantitative RT-PCR. Correlation analysis was conducted to investigate the potential function of lncRNAs. According to gene ontology (GO) analysis, differentially expressed lncRNAs were mainly related to cell proliferation, cell cycle and cell apoptosis. KEGG pathway analysis indicated that these lncRNAs were associated with several pathways, namely immunological activity, metabolism and cytokine-cytokine receptor interaction. In conclusion, our study provided a novel direction for studying the relationship between lncRNAs and E2 in the thymus.

Integrated microRNA and mRNA sequencing analysis of age-related changes to mouse thymic epithelial cells.

MicroRNAs (miRNAs) play key roles in the regulation of gene expression during multiple physiological processes, including early development, differentiation, and ageing. However, their involvement in age-related thymic involution is not clear. In this study, we profiled the global transcriptome and miRNAome of thymic epithelial cells in 1- and 3-month-old male and female mice, and predicted the possible transcription factors and target genes of the four most significantly differentially expressed miRNAs (DEMs) (miR-183-5p, miR-199b-5p, miR-205-5p, and miR-200b-3p) by performing bioinformatics analyses. We also evaluated the relationships between the significantly DEMs and mRNAs. We performed quantitative polymerase chain reaction to confirm the changes in the expression of the miRNAs and their predicted target genes. We found that miR-183-5p, miR-199b-5p, miR-205-5p, and miR-200b-3p can be used as a biomarker group for mouse thymus development and involution. In addition, the predicted target genes (Ptpn4, Slc2a9, Pkib, Pecam1, and Prkdc), which were identified by mRNA sequencing analysis, were mainly involved in growth, development, and accelerated senescence. In conclusion, miRNAs and their predicted target genes likely play important roles in thymus development and involution. To the best of our knowledge, this is the first study to systematically analyze the relevance of miRNAs and their targets by mRNA sequencing in mouse thymic epithelial cells. © 2018 IUBMB Life, 70(7):678-690, 2018.

Transcriptional profiling analysis of Zearalenone-induced inhibition proliferation on mouse thymic epithelial cell line 1.

Zearalenone (ZEA) was a mycotoxin biosynthesized by a variety of Fusarium fungi via a polypeptide pathway. ZEA has significant toxic reaction on immune cells. Thymic epithelial cells (TECs) as a crucial constituent of thymic stroma can provide unique microenvironment for thymocyte maturation, but the mechanism of ZEA affecting the TECs is poorly understood. The basic data about gene expression differences for the ZEA on thymic epithelial cell line 1 (MTEC1) will help us to elucidate this mechanism. Here, cell viability and proliferation assay and transcriptome sequencing on MTEC1 treated with ZEA were performed. 4188 differentially expressed genes (DEGs) between ZEA treated and control groups were identified, confirmed and analyzed. Our results showed that 10-50µg/ml ZEA significantly inhibited MTEC1 proliferation and arrested cell cycle at G2/M phase. Gene ontology and KEGG pathway analysis revealed that Chemokine, JAK-STAT and Toll-like receptor signaling pathway, were involved in the cell cycle pathway. 16 key genes involved in the cell cycle processes were validated and the results suggested that Mitotic catastrophe (MC) may take part in ZEA inhibition of METC1 cell proliferation. These data highlighted the importance of cell cycle pathway in MTEC1 treated with ZEA, and will contribute to get the molecular mechanisms of ZEA inhibition of MTEC1 cell proliferation.

Distinct expression profile and histological distribution of NLRP3 inflammasome components in the tissues of Hainan black goat suggest a site-specific role in the inflammatory response.

The NOD-like receptor protein 3 (NLRP3) inflammasome comprised of NLRP3, ASC and caspase-1 plays an important role in the inflammatory and innate immune response. However, little is known about the expression pattern and histological distribution of these genes in goat. Here, we first cloned the fulllength cDNAs of the NLRP3, ASC and caspase-1 genes of Hainan black goat and produced their polyclonal antibodies. Tissue-specific expression and histological distribution of these genes were analysed. Phylogenetic analysis revealed that these three goat genes had high homology with Bos taurus genes and low homology with avian or fish genes. After immunisations with these recombinant Histagged proteins, the titres of antiserum were higher than 1:1024 and purified IgG was obtained. These three genes were expressed in all examined tissues, the mRNA expression level of NLRP3 and caspase-1 was most abundant in the spleen and mesenteric lymph nodes (MLNs), while ASC was primary expressed in the liver, spleen and kidney. The histological distribution of NLRP3, ASC and caspase-1 was detected in myocardial cells, hepatocytes, focal lymphocytes, bronchiolar epithelial cells, renal tubular epithelial cells, cortical neurons and endothelial cells of the germinal centres in the MLNs. These results will be helpful in further investigations into the function of the NLRP3 inflammasome and in elucidating its role in caprine inflammatory diseases.

Expression of inflammation-related genes in the lung of BALB/c mice response to H7N9 influenza A virus with different pathogenicity.

H7N9 influenza A virus (IAV)-infected human cases are increasing and reported over 200 mortalities since its first emergence in 2013. Host inflammatory response contributes to the clearance of influenza virus; meanwhile, the induced "cytokine storm" also leads to pathological lesions. However, what inflammation-related response of the host for H7N9 influenza A virus infection to survival from injures of exuberant cytokine release is still obscure. In this research, expression pattern and histological distribution of inflammation-related genes, RIP3, NLRP3, IL-1ß, TNF-α, Slit2 and Robo4 in the lung of BALB/c mice infected with two H7N9 IAV strains with only a PB2 residue 627 difference were investigated, as well as the histopathological injury of the lung. Results showed that significantly higher expression level of NLRP3, RIP3, IL-1ß and TNF-α in H7N9-infected groups compared with the control would play a key role in driving lung pathological lesion. While the expression level of Slit2 and Robo4 in H7N9 rVK627E group had significantly increased trend than VK627 which might be the main factor to inhibit the interstitial pneumonia and infiltration. Also, H7N9 induced the histopathological changes in the lung of infected mice, and RIP3, NLRP3, IL-1ß, TNF-α, Slit2 and Robo4 showed cell-specific distribution in the lung. The results will provide basic data for further research on the mechanism of inflammatory response and understanding of the role of site 627 in PB2 in H7N9 IAVs infection. In addition, enhancing the resilience of the host vascular system to the inflammatory response by regulation of Slit2-Robo4 signaling pathway might provide a novel strategy for H7N9 IAVs infection.

Expression Profile and Tissue-Specific Distribution of the Receptor-Interacting Protein 3 in BALB/c Mice.

RIP3, a member of receptor-interacting protein family, is serine/threonine kinase that contributes to necrosis and promotes systematic inflammation. However, detailed information of the expression pattern and tissue distribution in BALB/c mice, a commonly used laboratory animal model, is still unavailable. Here, we provided the basic data of expression profile and histologic distribution of RIP3 in tissues of BALB/c mice. Rip3 mRNA expression levels and tissue distribution were detected by real-time quantitative PCR and immunohistochemical detection, respectively. Rip3 mRNA expression showed the highest level in the spleen and duodenum, while with the lowest level in brain. Immunohistochemical detection revealed this protein located in different type cells in different tissues. What's more, the obvious positive staining in nuclear was detected in liver cells and neurons in cerebral cortex of the brain, while cells in other organs, including heart, spleen, lung, kidney, stomach, duodenum and trachea, showed strong positive mainly in cytoplasm. The results will help us to further understand the site-specific functions of RIP3 in necrosis and inflammatory responses.

Expression profile and histological distribution of IFITM1 and IFITM3 during H9N2 avian influenza virus infection in BALB/c mice.

The H9N2 avian influenza virus is a pandemic threat which has repeatedly caused infection in humans and shows enhanced replication and transmission in mice. Previous reports showed that host factors, the interferon-inducible transmembrane (IFITM) protein, can block the replication of pathogens and affect their pathogenesis. BALB/c mice are routine laboratory animals used in influenza virus research, but the effects of H9N2 influenza virus on tissue distribution and expression pattern of IFITM in these mice are unknown. Here, we investigated the expression patterns and tissue distribution of IFITM1 and IFITM3 in BALB/c mice by infection with H9N2 AIV strains with only a PB2 residue 627 difference. The results showed that the expression patterns of ITITM1 and IFITM3 differ in various tissues of BALB/c mice at different time points after infection. IFITM1 and IFITM3 showed cell- and tissue-specific distribution in the lung, heart, liver, spleen, kidney and brain. Notably, the epithelial and neuronal cells all expressed the proteins of IFITM1 and IFITM3. Our results provide the first look at differences in IFITM1 and IFITM3 expression patterns in BALB/c mice infected by H9N2 influenza viruses. This will enhance research on the interaction between AIV and host and further will elucidate the pathogenesis of influenza virus infection based on the interferon-inducible transmembrane (IFITM) protein.

Expression pattern of NLRP3 and its related cytokines in the lung and brain of avian influenza virus H9N2 infected BALB/c mice.

BACKGROUND: H9N2 avian influenza virus (AIV) becomes the focus for its ability of transmission to mammals and as a donor to provide internal genes to form the new epidemic lethal influenza viruses. Residue 627 in PB2 has been proven the virulence factor of H9N2 avian influenza virus in mice, but the detailed data for inflammation difference between H9N2 virus strains with site 627 mutation is still unclear. The inflammasome NLRP3 is recently reported as the cellular machinery responsible for activation of inflammatory processes and plays an important role during the development of inflammation caused by influenza virus infection. METHODS: In this study, we investigated the expression pattern of NLRP3 and its related cytokines of IL-1ß and TNF-α in BALB/c mice infected by H9N2 AIV strains with only a site 627 difference at both mRNA and protein levels at different time points. RESULTS: The results showed that the expression level of NLRP3, IL-1ß and TNF-α changed in the lung and brain of BALB/c mice after infection by VK627 and rVK627E. The immunohistological results showed that the positive cells of NLRP3, IL-1ß and TNF-α altered the positive levels of original cells in tissues and infiltrated inflammatory cells which caused by H9N2 infection. CONCLUSIONS: Our results provided the basic data at differences in expression pattern of NLRP3 and its related cytokines in BALB/c mice infected by H9N2 influenza viruses with only a site 627 difference. This implied that NLRP3 inflammasome plays a role in host response to influenza virus infection and determines the outcome of clinical manifestation and pathological injury. This will explain the variable of pathological presentation in tissues and enhance research on inflammation process of the AIV H9N2 infection.

Forsythoside A protects against Zearalenone-induced cell damage in chicken embryonic fibroblasts via mitigation of endoplasmic reticulum stress.

Zearalenone (ZEA) is a non-steroidal estrogenic mycotoxin that exerts its toxic effects through various damage mechanisms such as oxidative stress, endoplasmic reticulum stress (ERS), mitochondrial damage, cell cycle arrest, and apoptosis. At present, there are few studies on drugs that can rescue ZEA-induced chicken embryonic fibroblasts damage. Forsythoside A (FA) is one of effective ingredients of traditional Chinese medicine that plays a role in various biological functions, but its antitoxin research has not been investigated so far. In this study, in vitro experiments were carried out. Chicken embryo fibroblast (DF-1) cells was used as the research object to select the appropriate treatment concentration of ZEA and examined reactive oxygen species (ROS), mitochondrial membrane potential, ERS and apoptosis to investigate the effects and mechanisms of FA in alleviating ZEA-induced cytotoxicity in DF-1 cells. Our results showed that ZEA induced ERS and activated the unfolded protein response (UPR) leading to apoptosis, an apoptotic pathway characterized by overproduction of Lactate dehydrogenase (LDH), Caspase-3, and ROS and loss of mitochondrial membrane potential. We also demonstrated that FA help to prevent ERS and attenuated ZEA-induced apoptosis in DF-1 cells by reducing the level of ROS, downregulating GRP78, PERK, ATF4, ATF6, JNK, IRE1, ASK1, CHOP, BAX expression, and up-regulating Bcl-2 expression. Our results provide a basis for an in-depth study of the mechanism of toxic effects of ZEA on chicken cells and the means of detoxification, which has implications for the treatment of relevant avian diseases.

miR-152-3p Represses the Proliferation of the Thymic Epithelial Cells by Targeting Smad2.

MicroRNAs (miRNAs) control the proliferation of thymic epithelial cells (TECs) for thymic involution. Previous studies have shown that expression levels of miR-152-3p were significantly increased in the thymus and TECs during the involution of the mouse thymus. However, the possible function and potential molecular mechanism of miR-152-3p remains unclear. This study identified that the overexpression of miR-152-3p can inhibit, while the inhibition of miR-152-3p can promote, the proliferation of murine medullary thymic epithelial cell line 1 (MTEC1) cells. Moreover, miR-152-3p expression was quantitatively analyzed to negatively regulate Smad2, and the Smad2 gene was found to be a direct target of miR-152-3p, using the luciferase reporter assay. Importantly, silencing Smad2 was found to block the G1 phase of cells and inhibit the cell cycle, which was consistent with the overexpression of miR-152-3p. Furthermore, co-transfection studies of siRNA-Smad2 (siSmad2) and the miR-152-3p mimic further established that miR-152-3p inhibited the proliferation of MTEC1 cells by targeting Smad2 and reducing the expression of Smad2. Taken together, this study proved miR-152-3p to be an important molecule that regulates the proliferation of TECs and therefore provides a new reference for delaying thymus involution and thymus regeneration.

Effects of Castration on miRNA, lncRNA, and mRNA Profiles in Mice Thymus.

Thymic degeneration and regeneration are regulated by estrogen and androgen. Recent studies have found that long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) are involved in organ development. In this study, RNA sequencing (RNA-seq) results showed that ovariectomy significantly affected 333 lncRNAs, 51 miRNAs, and 144 mRNAs levels (p < 0.05 and |log2fold change| > 1), and orchiectomy significantly affected 165 lncRNAs, 165 miRNAs, and 208 mRNA levels in the thymus. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that differentially expressed genes (DEGs) were closely related to cell development and immunity. Next, we constructed two lncRNA-miRNA-mRNA networks using Cytoscape based on the targeting relationship between differentially expressed miRNAs (DEMs) and DEGs and differentially expressed lncRNAs (DELs) analyzed by TargetScan and miRanda. Besides, we screened DEGs that were significantly enriched in GO and in ceRNA networks to verify their expression in thymocytes and thymic epithelial cells (TECs). In addition, we analyzed the promoter sequences of DEGs, and identified 25 causal transcription factors. Finally, we constructed transcription factor-miRNA-joint target gene networks. In conclusion, this study reveals the effects of estrogen and androgen on the expression of miRNAs, lncRNAs, and mRNAs in mice thymus, providing new insights into the regulation of thymic development by gonadal hormones and non-coding RNAs.

miR-205-5p inhibits thymic epithelial cell proliferation via FA2H-TFAP2A feedback regulation in age-associated thymus involution.

Thymic epithelial cells (TECs) are essential regulators of T cell development and selection. microRNAs (miRNAs) play critical roles in regulating TECs proliferation during thymus involution. miR-205-5p is highly expressed in TECs and increases with age. However, the function and potential mechanism of miR-205-5p in TECs are not clear. miRNA expression was profiled using TECs from male and female mice at 1 and 3 months old. A total of 325 differentially expressed miRNAs (DEMs) were detected at different ages in two sexes. 24 of the DEMs had the same trend between males and females. Among them, miR-205-5p had the highest fold change. Our results showed that the expression of miR-205-5p was dramatically increased in TECs from 1 to 9 months old mice. miR-205-5p mimic inhibited TECs proliferation. Moreover, we confirmed that Fa2h was the direct target gene of miR-205-5p and FA2H was significantly decreased in TECs with increased expression of miR-205-5p. Silencing of Fa2h inhibited TECs proliferation. Furthermore, we found that the expression of Tfap2a could be promoted by FA2H and that TFAP2A could interact with miR-205-5p in TECs. Overall, miR-205-5p is an important regulator of TECs proliferation and regulates age-associated thymus involution via the miR-205-5p-FA2H-TFAP2A feedback regulatory circuit. miR-205-5p might act as a potential biomarker in TECs for age-related thymus involution.

Phylogenetic analysis of three orf virus strains isolated from different districts in Shandong Province, East China.

Orf virus (ORFV) is the causative agent of contagious ecthyma, which is a zoonotic disease that affects sheep, goats, wild small ruminants and humans. Shandong Province in East China is one of the main producing areas in China for sheep and goats. Here, we conducted epidemiological surveys in different areas in this Province, isolated three orf virus strains, SDLC, SDTA and SDJN, from goat flocks and then analyzed the genetic evolution of these strains. The ORFV011, ORFV059, ORFV109, ORFV110 and ORFV127 genes of these three strains were amplified, sequenced and analyzed. Phylogenetic analysis showed that ORFV011 of the SDLC and SDTA strains cluster together with the Gansu, Liaoning, Shanxi, Nantou, Hoping and FJ-YX strains, while SDJN clusters with the FJ-GS and FJ-GO strains. ORFV059 of the SDLC and SDTA strains cluster together with the FJ-YX strain, while SDJN clusters with the FJ-GS and FJ-GO strains. ORFV059 and ORFV127 of these three strains were similar to those of the OV-SA00 strain. The results suggested that SDLC, SDTA and SDJN originated from Fujian Province and formed a complex group of viruses in Shandong Province. As the role of ORFV127 gene responsible for the immune evasion of ORFV, the pathogenesis of these three virus strains may similar to that of OV-SA00. These three strains first isolated in Shandong Province are novel ORFV strains, and the data reported here will be helpful for further research about ORFV and its comprehensive prevention and control.

Tissue-specific expression pattern and histological distribution of NLRP3 in Chinese yellow chicken.

Pathogen-associated molecular patterns (PAMPs) play important role in inflammation which means response of the host to stimuli. NOD-like receptor protein 3 (NLRP3) inflammasome is involved in the onset and development of inflammation. NLRP3, as one of the most important inflammasome sensors, has significant effect on the regulation of inflammasome activation to avoid the consequences of over activation. Up to date, there are no detailed tissue specific expression and distribution data about NLPR3 in chicken. Here, NLRP3 of Chinese yellow chicken was cloned and sequence analyzed, the polyclonal antibody was produced by purified protein of recombinant prokaryotic expression. Relative expression levels and tissue distribution of NLRP3 were investigated by real-time quantitative PCR and immunohistochemical analysis, respectively. The results showed that NLRP3 gene is highly variable between mammalian and avian. The nucleotide homology of NLRP3 between yellow chicken and Bos taurus, Hainan black goat, Sus scrofa, Callithrix jacchus, Homo sapiens, Macaca mulatta, Mus musculus and Rattus norvegicus were 54.2%, 53.9%, 53.7%, 55.4%, 54.3%, 54.5%, 53.5% and 53.7%. NLRP3 expressed in all detected tissues and higher in the trachea are lung than in other tissues. Cytoplasmic expression of NLRP3 was detected in ciliated epithelial cells, basal cells and cells in lamina propria of trachea, alveolar epithelial cells, cardiac muscle cells, cerebral cortex neurons, epithelial reticular cells of the spleen, and lymphocytes of medulla in stannius follicle, liver cells and the renal tubule epithelial cells. The results will help to elucidate the role of NLRP3 of different tissues in inflammatory diseases of chicken and provide a basis for further investigations in the function and evolution of NLRP3 in different species, which would be helpful for further research on avian inflammatory diseases.