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The present study aims to investigate the distribution and expression characteristics of HIF-1α, VEGF, VEGFR-2, VCAM-1, and IL-4 in the spleen of plateau yaks and plain yellow cattle and to speculate the possible regulatory role of HIF-1α and its related hypoxia-inducible factors in the adaptation of the yak spleen to the plateau hypoxic environment. Histological features were observed using H&E and PAS stains. Immunohistochemical staining and optical density analysis were applied to investigate the distribution and differences in the expression of HIF-1α, VEGF, VEGFR-2, VCAM-1, and IL-4 in the spleen of yaks and cattle. The results showed that the area of splenic trabeculae and splenic nodules was significantly larger in the yak than in yellow cattle (P<0.05). Glycogen was mainly distributed in splenic arterial endothelial cells, vascular smooth muscle cells, splenic blood sinusoidal endothelial cells, and fibroblasts, and the distribution was significantly higher in the spleen of yaks than in cattle (P<0.05). HIF-1α, VEGF, VEGFR-2, VCAM-1, and IL-4 were mainly expressed in lymphocytes, arterial endothelial cells, vascular smooth muscle cells, splenic blood sinusoidal endothelial cells, and fibroblast cytoplasm, with higher expression in yak spleen (P<0.05). In conclusion, combining the differences in spleen tissue structure, glycogen distribution, and expression distribution of several hypoxia-related factors between yaks and cattle, we suggest that HIF-1α, VEGF, VEGFR-2, VCAM-1, and IL-4 may be important factors in the adaptation of yak spleen to the plateau environment, which provides a theoretical basis for further exploring the adaptation mechanism of plateau hypoxia in yaks.
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Circular RNAs (circRNAs) are non-coding RNAs discovered in recent years, which are produced by back-splicing involving the 3' and 5' ends of RNA molecules. There is increasing evidence that circRNAs have important roles in cancer, neurological diseases, cardiovascular and cerebrovascular diseases, and other diseases. In addition, host circRNAs and virus-encoded circRNAs participate in the body's immune response, with antiviral roles. This review summarizes the mechanisms by which host and viral circRNAs interact during the host immune response. Comprehensive investigations have revealed that host circRNAs function as miRNA sponges in a particular manner, primarily by inhibiting viral replication. Viral circRNAs have more diverse functions, which generally involve promoting viral replication. In addition, in contrast to circRNAs from RNA viruses, circRNAs from DNA viruses can influence host cell migration, proliferation, and apoptosis, along with their effects on viral replication. In summary, circRNAs have potential as diagnostic and therapeutic targets, offering a foundation for the diagnosis and treatment of viral diseases.
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Apoptosis , ARN Circular , Movimiento Celular , Replicación ViralRESUMEN
BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a multifaceted metabolic disorder, whose global prevalence is rapidly increasing. Acetyl CoA carboxylases 1 (ACACA) is the key enzyme that controls the rate of fatty acid synthesis. Hence, it is crucial to investigate the function of ACACA in regulating lipid metabolism during the progress of NAFLD. METHODS: Firstly, a fatty liver mouse model was established by high-fat diet at 2nd, 12th, and 20th week, respectively. Then, transcriptome analysis was performed on liver samples to investigate the underlying mechanisms and identify the target gene of the occurrence and development of NAFLD. Afterwards, lipid accumulation cell model was induced by palmitic acid and oleic acid (PA ⶠOA molar ratio = 1â¶2). Next, we silenced the target gene ACACA using small interfering RNAs (siRNAs) or the CMS-121 inhibitor. Subsequently, experiments were performed comprehensively the effects of inhibiting ACACA on mitochondrial function and lipid metabolism, as well as on AMPK- PPARα- CPT1A pathway. RESULTS: This data indicated that the pathways significantly affected by high-fat diet include lipid metabolism and mitochondrial function. Then, we focus on the target gene ACACA. In addition, the in vitro results suggested that inhibiting of ACACA in vitro reduces intracellular lipid accumulation, specifically the content of TG and TC. Furthermore, ACACA ameliorated mitochondrial dysfunction and alleviate oxidative stress, including MMP complete, ATP and ROS production, as well as the expression of mitochondria respiratory chain complex (MRC) and AMPK proteins. Meanwhile, ACACA inhibition enhances lipid metabolism through activation of PPARα/CPT1A, leading to a decrease in intracellular lipid accumulation. CONCLUSION: Targeting ACACA can reduce lipid accumulation by mediating the AMPK- PPARα- CPT1A pathway, which regulates lipid metabolism and alleviates mitochondrial dysfunction.
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Acetil-CoA Carboxilasa , Metabolismo de los Lípidos , Enfermedad del Hígado Graso no Alcohólico , Animales , Ratones , Proteínas Quinasas Activadas por AMP/metabolismo , Dieta Alta en Grasa , Metabolismo de los Lípidos/genética , Hígado/metabolismo , Mitocondrias/metabolismo , Enfermedades Mitocondriales/metabolismo , Enfermedad del Hígado Graso no Alcohólico/genética , Ácido Palmítico/metabolismo , Ácido Palmítico/farmacología , PPAR alfa/metabolismo , Acetil-CoA Carboxilasa/metabolismo , Carnitina O-Palmitoiltransferasa/metabolismoRESUMEN
The aim of this study was to investigate the molecular mechanisms by which hypoxia affects the biological behavior of yak PASMCs, the changes in the histological structure of yak and cattle lungs, and the relationships and regulatory roles that exist regarding the differences in the distribution and expression of PDK1 and its hypoxia-associated factors screened for their role in the adaptation of yak lungs to the plateau hypoxic environment. The results showed that, at the level of transcriptome sequencing, the molecular regulatory mechanisms of the HIF-1 signaling pathway, glucose metabolism pathway, and related factors (HK2/PGK1/ENO1/ENO3/ALDOC/ALDOA) may be closely related to the adaptation of yaks to the hypoxic environment of the plateau; at the tissue level, the presence of filled alveoli and semi-filled alveoli, thicker alveolar septa and basement membranes, a large number of erythrocytes, capillary distribution, and collagen fibers accounted for all levels of fine bronchioles in the lungs of yaks as compared to cattle. A higher percentage of goblet cells was found in the fine bronchioles of yaks, and PDK1, HIF-1α, and VEGF were predominantly distributed and expressed in the monolayers of ciliated columnar epithelium in the branches of the terminal fine bronchioles of yak and cattle lungs, with a small amount of it distributed in the alveolar septa; at the molecular level, the differences in PDK1 mRNA relative expression in the lungs of adult yaks and cattle were not significant (p > 0.05), the differences in HIF-1α and VEGF mRNA relative expression were significant (p < 0.05), and the expression of PDK1 and HIF-1α proteins in adult yaks was stronger than that in adult cattle. PDK1 and HIF-1α proteins were more strongly expressed in adult yaks than in adult cattle, and the difference was highly significant (p < 0.01); the relative expression of VEGF proteins was not significantly different between adult yaks and cattle (p > 0.05). The possible regulatory relationship between the above results and the adaptation of yak lungs to the plateau hypoxic environment paves the way for the regulatory mechanisms of PDK1, HIF-1α, and VEGF, and provides basic information for studying the mechanism of hypoxic adaptation of yaks in the plateau. At the same time, it provides a reference for human hypoxia adaptation and a target for the prevention and treatment of plateau diseases in humans and plateau animals.
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BACKGROUND: The MDCK cell line is the primary cell line used for influenza vaccine production. Using genetic engineering technology to change the expression and activity of genes that regulate virus proliferation to obtain high-yield vaccine cell lines has attracted increasing attention. A comprehensive understanding of the key genes, targets, and molecular mechanisms of viral regulation in cells is critical to achieving this goal, yet the post-transcriptional regulation mechanism involved in virus proliferation-particularly the effect of lncRNA on influenza virus proliferation-is still poorly understood. Therefore, this study used high-throughput RNA-seq technology to identify H1N1 infection-induced lncRNA and mRNA expression changes in MDCK cells and explore the regulatory relationship between these crucial lncRNAs and their target genes. RESULTS: In response to H1N1 infection in MDCK cells 16 h post-infection (hpi) relative to uninfected controls, we used multiple gene function annotation databases and initially identified 31,501 significantly differentially expressed (DE) genes and 39,920 DE lncRNAs (|log2FC| > 1, p < 0.05). Among these, 102 lncRNAs and 577 mRNAs exhibited predicted correlations with viral response mechanisms. Based on the magnitude of significant expression differences, related research, and RT-qPCR expression validation at the transcriptional level, we further focused on 18 DE mRNAs and 32 DE lncRNAs. Among these, the differential expression of the genes RSAD2, CLDN1, HCLS1, and IFIT5 in response to influenza virus infection was further verified at the protein level using Western blot technology, which showed results consistent with the RNA-seq and RT-qPCR findings. We then developed a potential molecular regulatory network between these four genes and their six predicted lncRNAs. CONCLUSIONS: The results of this study will contribute to a more comprehensive understanding of the molecular mechanism of host cell non-coding RNA-mediated regulation of influenza virus replication. These results may also identify methods for screening target genes in the development of genetically engineered cell lines capable of high-yield artificial vaccine production.
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The brain is an important part of the mammalian nervous system, is highly sensitive to hypoxia, and plays an important role in the adaptation of the body to hypoxic environments. This study was conducted to study the distribution and expression of hypoxia-related factors (hypoxia-inducible factor 1α, HIF-1α; erythropoietin, EPO; vascular endothelial growth factor, VEGF; vascular cell adhesion molecule, VCAM) in the cerebellum, cerebrum, medulla oblongata, and corpora quadrigemina in yaks of different ages (4d, 6-months-old and adult). Paraffin sections were obtained from the cerebellum, cerebrum, medulla oblongata, and corpora quadrigemina of healthy yak for 4-day-old, 6-months-old and adult yaks. Histological characteristics were assessed by haematoxylin staining. Immunohistochemical staining was performed to detect the distribution and expression of HIF-1α, EPO, VEGF and VCAM proteins. Immunohistochemical results showed that HIF-1α, EPO, VEGF, and VCAM were expressed in the pyramidal cell layer of the yak cerebrum, and distributed in the cerebellum granulose cell layer, Purkinje cell layer and medulla layer, and were mainly positive in Purkinje cells and medulla. It is expressed in the cell bodies of the medulla oblongata and the quadrimatous neurons. The expression level in the medulla oblongata was higher, indicating may play a crucial role in functional cohesion. The expression of HIF-1α in 4 d cerebellar tissues was higher than that in other age groups, and the expression of HIF-1α in the medulla oblongata increased with age. In addition, the expression levels of EPO and VEGF in the 6-month-old group were slightly higher than those in the other age groups. It is speculated that EPO and VEGF have obvious protective effects on brain tissue in the 6-month-old age group; VCAM showed no significant differences in the cerebrum, cerebellum, medulla oblongata, or corpora quadrigemina of the yaks. This study provides basic data for further exploration of the adaptive mechanism of plateau yak brain tissue.
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Background: Madin-Darby canine kidney (MDCK) cells are a cellular matrix in the production of influenza vaccines. The proliferation rate of MDCK cells is one of the critical factors that determine the vaccine production cycle. It is yet to be determined if there is a correlation between cell proliferation and alterations in metabolic levels. This study aimed to explore the metabolic differences between MDCK cells with varying proliferative capabilities through the use of both untargeted and targeted metabolomics. Methods: To investigate the metabolic discrepancies between adherent cell groups (MDCK-M60 and MDCK-CL23) and suspension cell groups (MDCK-XF04 and MDCK-XF06), untargeted and targeted metabolomics were used. Utilizing RT-qPCR analysis, the mRNA expressions of key metabolites enzymes were identified. Results: An untargeted metabolomics study demonstrated the presence of 81 metabolites between MDCK-M60 and MDCK-CL23 cells, which were mainly affected by six pathways. An analysis of MDCK-XF04 and MDCK-XF06 cells revealed a total of 113 potential metabolites, the majority of which were impacted by ten pathways. Targeted metabolomics revealed a decrease in the levels of choline, tryptophan, and tyrosine in MDCK-CL23 cells, which was in accordance with the results of untargeted metabolomics. Additionally, MDCK-XF06 cells experienced a decrease in 5'-methylthioadenosine and tryptophan, while S-adenosylhomocysteine, kynurenine, 11Z-eicosenoic acid, 3-phosphoglycerate, glucose 6-phosphate, and phosphoenolpyruvic acid concentrations were increased. The mRNA levels of MAT1A, MAT2B, IDO1, and IDO2 in the two cell groups were all increased, suggesting that S-adenosylmethionine and tryptophan may have a significant role in cell metabolism. Conclusions: This research examines the effect of metabolite fluctuations on cell proliferation, thus offering a potential way to improve the rate of MDCK cell growth.
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Metabolómica , Triptófano , Animales , Perros , Células de Riñón Canino Madin Darby , Carcinogénesis , Proliferación Celular , RiñónRESUMEN
MDCK is currently the main cell line used for influenza vaccine production in culture. Previous studies have reported that MDCK cells possess tumorigenic ability in nude mice. Although complete cell lysis can be ensured during vaccine production, host cell DNA released after cell lysis may still pose a risk for tumorigenesis. Greater caution is needed in the production of human vaccines; therefore, the use of gene editing to establish cells incapable of forming tumors may significantly improve the safety of influenza vaccines. Knowledge regarding the genes and molecular mechanisms that affect the tumorigenic ability of MDCK cells is crucial; however, our understanding remains superficial. Through monoclonal cell screening, we previously obtained a cell line, CL23, that possesses significantly reduced cell proliferation, migration, and invasion abilities, and tumor-bearing experiments in nude mice showed the absence of tumorigenic cells. With a view to exploring tumorigenesis-related genes in MDCK cells, DIA proteomics was used to compare the differences in protein expression between wild-type (M60) and non-tumorigenic (CL23) cells. Differentially expressed proteins were verified at the mRNA level by RT-qPCR, and a number of genes involved in cell tumorigenesis were preliminarily screened. Immunoblotting further confirmed that related protein expression was significantly reduced in non-tumorigenic cells. Inhibition of CDC20 expression by RNAi significantly reduced the proliferation and migration of MDCK cells and increased the proliferation of the influenza virus; therefore, CDC20 was preliminarily determined to be an effective target gene for the inhibition of cell tumorigenicity. These results contribute to a more comprehensive understanding of the mechanism underlying cell tumorigenesis and provide a basis for the establishment of target gene screening in genetically engineered non-tumorigenic MDCK cell lines.
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Vacunas contra la Influenza , Ratones , Animales , Perros , Humanos , Células de Riñón Canino Madin Darby , Ratones Desnudos , Línea Celular , Carcinogénesis/genética , Proteínas Cdc20RESUMEN
Influenza is an acute respiratory infectious disease caused by influenza virus that seriously endangers people's health. Influenza vaccination is the most effective means to prevent influenza virus infection and its serious complications. MDCK cells are considered to be superior to chicken embryos for the production of influenza vaccines, but the tumorigenicity of cells is a concern over the theoretical possibility of the risk of adverse events. The theoretical risks need to be adequately addressed if public confidence in programs of immunization are to be maintained. In this paper, studies of the tumorigenic potential of cell lines, with MDCK cells as an example, published since 2010 are reviewed. The mechanism of tumorigenicity of MDCK cells is discussed with reference to cell heterogeneity and epithelial to mesenchymal transition (EMT). Understanding the mechanism of the acquisition of a tumorigenic phenotype by MDCK cells might assist in estimating potential risks associated with using tumorigenic cell substrates for vaccine production.
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Vacunas contra la Influenza , Gripe Humana , Animales , Perros , Embrión de Pollo , Humanos , Células de Riñón Canino Madin Darby , Transición Epitelial-Mesenquimal , Línea Celular , CarcinogénesisRESUMEN
Madin-Darby canine kidney (MDCK) cells are one of the main cell lines used for influenza vaccine production due to their high virus yield and low mutation resistance. Due to their high tumorigenicity, the safety of vaccines produced from these cells is controversial. TGM2 is a multifunctional protein that plays an important role in the adhesion and migration of cells and is associated with tumor formation. We found that the expression level of TGM2 was significantly up-regulated in low tumorigenic MDCK cells. We first analyzed TGM2-overexpressed and knockout MDCK cells in vitro. Scratch-wound assay and Transwell chamber experiments showed that TGM2 overexpression significantly inhibited the migration and invasion of MDCK cells and significantly reduced their proliferation. TGM2 knockout significantly enhanced cell migration, invasion, and proliferation. The tumorigenesis results in nude mice were consistent with those in vitro. TGM2 knockout significantly enhanced the tumorigenesis rate of MDCK cells in nude mice. We also investigated the effects of TGM2 gene expression on the replication of the H1N1 influenza A virus in MDCK cells. The results showed that TGM2 induced the negative regulation of H1N1 replication. These findings contribute to a comprehensive understanding of the tumor regulation mechanism and biological functions of TGM2.
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Subtipo H1N1 del Virus de la Influenza A , Virus de la Influenza A , Animales , Perros , Ratones , Carcinogénesis/genética , Proliferación Celular , Subtipo H1N1 del Virus de la Influenza A/fisiología , Células de Riñón Canino Madin Darby , Ratones Desnudos , Proteína Glutamina Gamma Glutamiltransferasa 2/metabolismoRESUMEN
Erythropoietin (EPO), hypoxia-inducible factor-1α (HIF-1α), hypoxia-inducible factor-2α (HIF-2α), and vascular endothelial growth factor (VEGF) are key factors in the regulation of hypoxia, and can transcriptionally activate multiple genes under hypoxic conditions, thereby initiating large hypoxic stress in the network. The liver and kidneys are important metabolic organs of the body. We assessed the expression of EPO, HIF-1α, HIF-2α, and VEGF in liver and kidney tissues of plain and Tibetan sheep using hematoxylin and eosin staining, immunohistochemistry, and RT-qPCR. The results showed that EPO, HIF-1α, HIF-2α, and VEGF were expressed in tubular epithelial cells, collecting duct epithelial cells, mural epithelial cells, and the glomerular cytoplasm of Tibetan sheep, and their expression was significantly higher in Tibetan sheep than in plain sheep (P<0.05). EPO, HIF-1α, HIF-2α, and VEGF are expressed in hepatocytes, interlobular venous endothelial cells, and interlobular bile duct epithelial cells. In plain sheep, positive signals for EPO, HIF-1α, HIF-2α, and VEGF were localized mainly in interlobular venous endothelial cells, whereas VEGF and HIF-2α were negatively expressed in interlobular bile duct epithelial cells and positively expressed in EPO and HIF-1α. The differences in EPO, HIF-1α, and HIF-2α in Tibetan sheep were significantly higher than those in plain sheep (P<0.001). In the liver and kidney tissues of Tibetan sheep, EPO was associated with HIF-1α, HIF-2α, and VEGF (P<0.05). RT-qPCR results showed that EPO was not expressed, and HIF-1α, HIF-2α, and VEGF were expressed (P<0.05). The results showed that the expression of EPO, HIF-1α, HIF-2α, and VEGF in the kidney and liver of Tibetan sheep was higher than that in of plain sheep. Therefore, EPO, HIF-1α, HIF-2α, and VEGF may be involved in the adaptive response of plateau animals, which provides theoretical clarity to further explore the adaptive mechanism of plateau hypoxia in Tibetan sheep.
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Eritropoyetina , Factor A de Crecimiento Endotelial Vascular , Animales , Ovinos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Células Endoteliales/metabolismo , Tibet , Riñón/metabolismo , Eritropoyetina/metabolismo , Hígado/metabolismo , Hipoxia/metabolismo , Factores de Crecimiento Endotelial Vascular/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismoRESUMEN
Background: Yaks are animals that have lived in plateau environments for generations. Yaks can adapt to the hypoxic plateau environment and also pass this adaptability on to the next generation. The lungs are the most important respiratory organs for mammals to adapt to their environment. Pulmonary artery smooth muscle cells play an important role in vascular remodeling under hypoxia, but the genetic mechanism underpinning the yak's ability to adapt to challenging plateau conditions is still unknown. Methods: A tandem mass tag (TMT) proteomics study together with an RNA-seq transcriptome analysis were carried out on pulmonary artery smooth muscle cells (PASMCs) that had been grown for 72 hours in both normoxic (20% O2) and hypoxic (1% O2) environments. RNA and TP (total protein) were collected from the hypoxic and normoxic groups for RNA-seq transcriptome sequencing and TMT marker protein quantification, and RT-qPCR validation was performed. Results: A total of 17,711 genes and 6,859 proteins were identified. Further, 5,969 differentially expressed genes (DEGs) and 531 differentially expressed proteins (DEPs) were identified in the comparison group, including 2,924 and 186 upregulated genes and proteins and 3,045 and 345 down-regulated genes and proteins, respectively. The transcriptomic and proteomic analyses revealed that 109 DEGs and DEPs were highly positively correlated, with 77 genes showing the same expression trend. Nine overlapping genes were identified in the HIF-1 signaling pathway, glycolysis / gluconeogenesis, central carbon metabolism in cancer, PPAR signaling pathway, AMPK signaling pathway, and cholesterol metabolism (PGAM1, PGK1, TPI1, HMOX1, IGF1R, OLR1, SCD, FABP4 and LDLR), suggesting that these differentially expressed genes and protein functional classifications are related to the hypoxia-adaptive pathways. Overall, our study offers abundant data for further analysis of the molecular mechanisms in yak PASMCs and their adaptability to different oxygen concentrations.
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Proteoma , Transcriptoma , Animales , Bovinos , Transcriptoma/genética , Proteoma/genética , Arteria Pulmonar , Proteómica , Hipoxia/genética , Miocitos del Músculo Liso , MamíferosRESUMEN
Increasingly, attention has focused on improving vaccine production in cells using gene editing technology to specifically modify key virus regulation-related genes to promote virus replication. In this study, we used DIA proteomics analysis technology to compare protein expression differences between two groups of MDCK cells: uninfected and influenza A virus (IAV) H1N1-infected cells 16 h post infection (MOI = 0.01). Initially, 266 differentially expressed proteins were detected after infection, 157 of which were upregulated and 109 were downregulated. We screened these proteins to 23 genes related to antiviral innate immunity regulation based on functional annotation database analysis and verified the mRNA expression of these genes using qPCR. Combining our results with published literature, we focused on the proteins RSAD2, KCNN4, IDO1, and ISG20; we verified their expression using western blot, which was consistent with our proteomics results. Finally, we knocked down RSAD2 using lentiviral shRNA expression vectors and found that RSAD2 inhibition significantly increased IAV NP gene expression, effectively promoting influenza virus replication with no significant effect on cell proliferation. These results indicate that RSAD2 is potentially an effective target for establishing high-yield vaccine MDCK cell lines and will help to fully understand the interaction mechanism between host cells and influenza viruses.
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Subtipo H1N1 del Virus de la Influenza A , Virus de la Influenza A , Vacunas contra la Influenza , Gripe Humana , Perros , Animales , Humanos , Células de Riñón Canino Madin Darby , Vacunas contra la Influenza/genética , Virus de la Influenza A/fisiologíaRESUMEN
Transglutaminase 2 (TGM2) is a ubiquitous multifunctional protein, which is related to the adhesion of different cells and tumor formation. Previous studies found that TGM2 is involved in the interaction between host cells and viruses, but the effect of TGM2 on the proliferation of influenza virus in cells has not been reported. To explore the effect of TGM2 during H1N1 subtype influenza virus infection, a stable MDCK cell line with TGM2 overexpression and a knockout cell line were constructed. The mRNA and protein expression levels of NP and NS1 as well as the virus titer were measured at 48 hours after pot-infection with H1N1 subtype influenza virus. The results showed that overexpression of TGM2 effectively inhibited the expression of NP and NS1 genes of H1N1 subtype influenza virus, while knockout of TGM2 up-regulated the expression of the NP and NS1 genes, and the expression of the NP at protein level was consistent with that at mRNA level. Virus proliferation curve showed that the titer of H1N1 subtype influenza virus decreased significantly upon TGM2 overexpression. On the contrary, the virus titer in TGM2 knockout cells reached the peak at 48 h, which further proved that TGM2 was involved in the inhibition of H1N1 subtype influenza virus proliferation in MDCK cells. By analyzing the expression of genes downstream of influenza virus response signaling pathway, we found that TGM2 may inhibit the proliferation of H1N1 subtype influenza virus by promoting the activation of JAK-STAT molecular pathway and inhibiting RIG-1 signaling pathway. The above findings are of great significance for revealing the mechanism underlying the interactions between host cells and virus and establishing a genetically engineering cell line for high-yield influenza vaccine production of influenza virus.
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Subtipo H1N1 del Virus de la Influenza A , Gripe Humana , Animales , Proliferación Celular , Perros , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Células de Riñón Canino Madin Darby , Proteína Glutamina Gamma Glutamiltransferasa 2RESUMEN
BACKGROUND: Madin-Darby canine kidney (MDCK) cells are widely used for vaccine production, however, the safety of MDCK cells needs to be considered seriously because of high tumorigenicity. Micro RNAs (miRNAs) that are involved in the tumorigenicity of MDCK cells have been never been reported. OBJECTIVE: To reveal the role of miRNA in the tumorigenic phenotype of MDCK cell line. METHODS: The miRNA expression profiles of two monoclonal MDCK cells (M09CL and M35CL) with low tumorigenicity and one MDCK cell line (M73P) with high tumorigenicity were characterized and investigated by using small RNA-seq technology. RESULTS: A total of 5 known miRNAs and 5 novel miRNAs were highly expressed in M73P. In addition, 4 known miRNAs and 4 novel miRNAs were highly expressed in M09CL and M35CL. The target genes of the differentially expressed miRNAs were significantly enriched in several biological processes, and the majority of these genes were involved in pathways in cancer and the MAPK signaling pathway. Through interaction analysis, 4 up-regulated miRNAs (cfa-miR-452, cfa-miR-8826, cfa-miR-224, and cfa-miR-2387) and their crucial target genes related to the tumor regulation network were identified. Results indicated these 4 miRNAs might play crucial roles in the tumorigenesis of MDCK cells. CONCLUSION: Our findings, which were based on the functional prediction of miRNAs and target genes, suggested that miRNAs might influence the tumorigenicity of MDCK cells by regulating target genes. Moreover, the results provided important data for understanding the miRNA-mediated regulatory networks that control the tumorigenicities of MDCK cells.
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MicroARNs , Animales , Carcinogénesis/genética , Perros , Riñón/metabolismo , Células de Riñón Canino Madin Darby , MicroARNs/genética , MicroARNs/metabolismo , Transducción de Señal/genéticaRESUMEN
A broad spectrum of health benefits from intermittent fasting have been reported in studies on animal models and human subjects. However, the underlying mechanisms of these beneficial effects remain largely elusive. The present study aimed to explore the effects and potential mode of action of intermittent fasting in mouse models with a focus on the liver. C57BL/6 mice were subjected to intermittent fasting or ad libitum feeding as controls. It was determined that 12 h of daily intermittent fasting for 30 days significantly reduced the cumulative food intake compared with that in mice with ad libitum feeding. Fasting resulted in a significantly reduced liver mass but only had a minimal effect on bodyweight. The effects on the liver by 30 days of fasting were not reversed by subsequent ad libitum refeeding for 30 days. Among the measured blood biochemical parameters, the levels of blood glucose were decreased, while the levels of alkaline phosphatase were increased in fasting mice. Of note, targeted metabolic profiling revealed global elevation of metabolites in the livers of fasting mice. These metabolic molecules included adenosine triphosphate, nicotinamide adenine dinucleotide phosphate (NADP), reduced NADP and succinate, which are essentially involved in the citric acid cycle and oxidative phosphorylation. Thus, it was concluded that daily 12 h of intermittent fasting for one month significantly reduced the liver weight of mice, which is associated with enhanced liver metabolism.
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MDCK cells are a key reagent in modern vaccine production. As MDCK cells are normally adherent, creation of suspension cells for vaccine production using genetic engineering approaches is highly desirable. However, little is known regarding the mechanisms and effectors underlying MDCK cell adhesion. In this study, we performed a comparative analysis of whole protein levels between MDCK adhesion and suspension cells using an iTRAQ-based (isobaric tags for relative and absolute quantitation) proteomics approach. We found that expression of several proteins involved in cell adhesion exhibit reduced expression in suspension cells, including at the mRNA level. Proteins whose expression was reduced in suspension cells include cadherin 1 (CDH1), catenin beta-1 (CTNNB1), and catenin alpha-1 (CTNNA1), which are involved in intercellular adhesion; junction plakoglobin (JUP), desmoplakin (DSP), and desmoglein 3 (DSG3), which are desmosome components; and transglutaminase 2 (TGM2) and alpha-actinin-1 (ACTN1), which regulate the adhesion between cells and the extracellular matrix. A functional verification experiment showed that inhibition of E-cadherin significantly reduced intercellular adhesion of MDCK cells. E-Cadherin did not significantly affect the proliferation of MDCK cells and the replication of influenza virus. These findings reveal possible mechanisms underlying adhesion of MDCK cells and will guide the creation of MDCK suspension cells by genetic engineering.
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Adhesión Celular/fisiología , Células de Riñón Canino Madin Darby/fisiología , Proteoma , Proteómica/métodos , Animales , Línea Celular , PerrosRESUMEN
Influenza is an acute respiratory infection caused by the influenza virus, and vaccination against influenza is considered the best way to prevent the onset and spread. MDCK (Madin-Darby canine kidney) cells are typically used to isolate the influenza virus, however, their high tumorigenicity is the main controversy in the production of influenza vaccines. Here, MDCK-C09 and MDCK-C35 monoclonal cell lines were established, which were proven to be low in tumorigenicity. RNA-seq of MDCK-C09, MDCK-C35, and MDCK-W73 cells was performed to investigate the putative tumorigenicity mechanisms. Tumor-related molecular interaction analysis of the differentially expressed genes indicates that hub genes, such as CUL3 and EGFR, may play essential roles in tumorigenicity differences between MDCK-C (MDCK-C09 and MDCK-C35) and MDCK-W (MDCK-W73) cells. Moreover, the analysis of cell proliferation regulation-associated molecular interaction shows that downregulated JUN and MYC, for instance, mediate increased proliferation of these cells. The present study provides a new low-tumorigenic MDCK cell line and describes the potential molecular mechanism for the low tumorigenicity and high proliferation rate.
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Transformación Celular Neoplásica/genética , Células Clonales/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes/genética , Animales , Línea Celular , Células Clonales/virología , Perros , Células HeLa , Humanos , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H1N1 del Virus de la Influenza A/fisiología , Vacunas contra la Influenza/inmunología , Vacunas contra la Influenza/metabolismo , Células de Riñón Canino Madin Darby , Ratones Desnudos , Cultivo de Virus/métodosRESUMEN
Chicken plasma protein hydrolysate (CPPH) was prepared by trypsin with angiotensin I-converting enzyme (ACE) inhibitory activity of 53.5% ± 0.14% and the degree of hydrolysis (DH) of 16.22% ± 0.21% at 1 mg·ml-1; then, five proteases, including pepsin, trypsin, papain, alcalase, and neutrase, were employed to improve ACE inhibitory ability by catalyzing plastein reaction. The results indicated that trypsin-catalyzed plastein reaction showed the highest ACE inhibitory activity. The exogenous amino acids of leucine, histidine, tyrosine, valine, and cysteine were selected to modify the CPPH. The leucine-modified plastein reaction released the highest ACE inhibitory activity. The effects of four reaction parameters on plastein reaction were studied, and the optimal conditions with the purpose of obtaining the most powerful ACE inhibitory peptides from modified products were obtained by response surface methodology (RSM). The maximum ACE inhibition rate of the modified hydrolysate reached 82.07% ± 0.03% prepared at concentration of hydrolysates of 30%, reaction time of 4.9 hr, pH value of 8.0, temperature of 40°C, and E/S ratio of 5,681.62 U·g-1. The results indicated that trypsin-catalyzed plastein reaction increased ACE inhibitory activity of chicken plasma protein hydrolysates by 28.57%.
RESUMEN
Madin-Darby canine kidney(MDCK) cells can be used to prepare cell-based influenza vaccines; however, little is known regarding the effect of lncRNA regulatorson tumorigenicity. In the present study, two cell lines with low tumorigenicity were screened from highly tumorigenic MDCK cell lines using monoclonal cell technology. Accordingly, three groups of lncRNAs were extracted from three cell lines and investigated using strand-specific Ribo-Zero RNA sequencing, detecting 1092 known and 619 novel lncRNAs. Moreover, in pairwise comparisons between the libraries of the nominally tumorigenic clones and the highly tumorigenic parent cell line, a total of 344 transcripts were expressed differentially, which were validated by qPCR using six randomly selected lncRNA genes. Furthermore, 63 target genes were identified in the upstream and downstream 100â¯kb of lncRNAs and their relative functions were analyzed. It was found that ten GO terms and twelve KEGG terms related to tumor by target genes and functional items. Five lncRNA transcripts and the corresponding differentially expressed target genes were used for co-expression network analysis. In addition, certain classical tumor pathways were also activated by target genes, among which, lncRNA MSTRG.1056.2 directly regulates ERBB3 to activate the PI3K-Akt pathway, contributing to tumorigenesis. Consequently, direct evidence was obtained that lncRNA regulates tumorigenesis, and a variety of target genes regulated by lncRNA were elucidated, which may be significant for non-tumorigenic MDCK cells lines acquisition.