Objective To explore the effect of the knockdown of transmembrane 9 superfamily protein member 2 (TM9SF2) on the replication of vesicular stomatitis virus (VSV), and investigate its role in the mechanism of antiviral innate immunity. Methods Small interfering RNA (siRNA) was used to knock down the TM9SF2 gene in human non-small cell lung cancer A549 cells. The CCK-8 method was used to assess cell proliferation. A VSV-green fluorescent protein (VSV-GFP) infected cell model was established. The plaque assay was used to measure the viral titer in the supernatant. RT-qPCR and Western blotting were employed to quantify the mRNA and protein levels of VSV genome replication in A549 cells following VSV infection, as well as the expression of interferon β (IFN-β) mRNA and interferon regulatory factor 3 (IRF3) protein phosphorylation following polyinosinic-polycytidylic acid (poly(I:C)) stimulation. Results Compared to the negative control, the knockdown of TM9SF2 exhibited a significant effect, with no observed impact on A549 cell proliferation. The VSV-GFP infected A549 cell model was successfully established. After viral stimulation, fluorescence intensity was reduced following TM9SF2 knockdown, and the mRNA and protein levels of VSV were significantly downregulated. The viral titer of VSV was decreased. After poly(I:C) stimulation, TM9SF2 knockdown significantly upregulated the mRNA level of IFN-β and the phosphorylation level of IRF3 protein. Conclusion The knockdown of TM9SF2 inhibits the replication of vesicular stomatitis virus, and positively regulates the type I interferon signaling pathway, thus enhancing the host's antiviral innate immune response.
Humans ; Virus Replication/genetics* ; Signal Transduction ; Membrane Proteins/metabolism* ; A549 Cells ; Vesiculovirus/physiology* ; Interferon-beta/metabolism* ; Interferon Regulatory Factor-3/genetics* ; Interferon Type I/metabolism* ; Vesicular Stomatitis/immunology* ; Gene Knockdown Techniques ; Vesicular stomatitis Indiana virus/physiology* ; RNA, Small Interfering/genetics*

OBJECTIVES: Esophageal squamous cell carcinoma (ESCC) is characterized by complex pathogenesis and poor prognosis. In recent years, epithelial-mesenchymal transition (EMT) in tumor initiation and progression has attracted increasing attention. Enhancer of zeste homolog 2 (EZH2), which is aberrantly expressed in various tumors, may be closely related to the EMT process. This study aims to examine the expression and correlation of EZH2 and EMT markers in ESCC cells and tissues, evaluate the effects of EZH2 knockdown on ESCC cell proliferation, invasion, and migration, and explore how EZH2 contributes to the malignant biological behavior of ESCC. METHODS: Bioinformatics analyses were used to assess EZH2 expression levels in ESCC. Small interfering RNA was used to knock down EZH2 in ESCC cell lines EC109 and EC9706. Cell proliferation, invasion, and migration were evaluated using cell counting kit-8 (CCK-8), wound healing, and Transwell assays. Protein and mRNA expression levels of EZH2, E-cadherin (E-cad), and vimentin (Vim) were detected by Western blotting and real time fluorogenic quantitative PCR (RT-qPCR), respectively. Immunohistochemical (IHC) staining was performed on 70 ESCC tissue samples and 40 paired adjacent normal tissues collected from the First Affiliated Hospital of Shihezi University between 2010 and 2016 to assess the expression of EZH2, E-cad, and Vim, and to analyze their associations with clinicopathological feature and patient prognosis. RESULTS: Bioinformatics analysis showed that EZH2 was highly expressed in ESCC (P<0.001), and high EZH2 expression was associated with worse prognosis (P<0.001). CCK-8, wound healing, and Transwell assays demonstrated that EZH2 knockdown significantly suppressed the proliferation, invasion, and migration of ESCC cells (P<0.001). In addition, Vim expression was significantly reduced, while E-cad expression was significantly increased at both protein and mRNA levels in EZH2-silenced cells (all P<0.05). IHC staining analysis revealed higher expression of EZH2 and Vim and lower expression of E-cad in ESCC tissues compared to adjacent normal tissues. Kaplan-Meier survival analysis showed that low expression of EZH2 and Vim and high expression of E-cad were associated with longer survival (all P<0.05). CONCLUSIONS EZH2 promotes malignant biological behavior in ESCC by mediating EMT. Elevated EZH2 expression is associated with poor prognosis in ESCC patients.
Humans ; Enhancer of Zeste Homolog 2 Protein/physiology* ; Esophageal Squamous Cell Carcinoma/pathology* ; Epithelial-Mesenchymal Transition/genetics* ; Esophageal Neoplasms/metabolism* ; Cell Proliferation ; Cell Line, Tumor ; Cell Movement ; Cadherins/genetics* ; Vimentin/genetics* ; Male ; Female ; Middle Aged ; Neoplasm Invasiveness ; Prognosis ; RNA, Small Interfering/genetics* ; Gene Expression Regulation, Neoplastic

The study aims to investigate the impacts of prolyl oligopeptidase (POP) on the replication of foot-and-mouth disease virus (FMDV) in BHK-21 cells. Firstly, the effects of FMDV replication on POP expression in BHK-21 cells were analyzed by Western blotting and Real-time reverse transcription polymerase chain reaction (RT-qPCR). Secondly, a eukaryotic expression plasmid for POP was constructed, and the effects of POP overexpression on the replication of two different serotypes of FMDV were assessed by Western blotting, RT-qPCR, and virus titer assays. Thirdly, specific small interfering RNAs (siRNAs) targeting POP were synthesized, and their efficiency in interfering with endogenous POP expression was identified by RT-qPCR. The impacts of downregulating endogenous POP expression on FMDV replication were further evaluated by Western blotting, RT-qPCR, and virus titer assays. The results indicated that FMDV infection did not significantly affect POP expression in BHK-21 cells. Overexpression of POP dose-dependently enhanced the replication of both FMDV/O and FMDV/A serotypes. Conversely, siRNA-mediated downregulation of endogenous POP expression markedly suppressed FMDV/O replication. This study is the first to demonstrated that the role of the host POP protein in promoting FMDV replication in BHK-21 cells, thereby providing a critical theoretical foundation and potential molecular targets for developing efficient candidate cell strains for foot-and-mouth disease inactivated vaccines.
Foot-and-Mouth Disease Virus/genetics* ; Virus Replication/genetics* ; Prolyl Oligopeptidases ; Serine Endopeptidases/physiology* ; Animals ; Cell Line ; RNA, Small Interfering/genetics* ; Foot-and-Mouth Disease/virology* ; Cricetinae

This work aims to explore the effect of glycolysis on the replication of porcine reproductive and respiratory syndrome virus (PRRSV) in porcine alveolar macrophages (PAMs). The changes of glucose metabolism, PRRSV protein levels, PRRSV titers, and the relative expression levels of genes and proteins in PAMs were analyzed by ELISA, qPCR, virus titration, and Western blotting after PRRSV infection. The effect of hypoxia-inducible factor-1α (HIF-1α) on PRRSV replication was subsequently assessed by specific siRNAs targeting to HIF-1α. The results showed that PRRSV infection enhanced glycolysis, elevated the levels of glucose uptake and lactate in the supernatant (P<0.05 and 0.01, respectively), reduced ATP production (P<0.05), and up-regulated the expression of hexokinase 2 (HK2), 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3 (PFKFB3), and pyruvate kinase isozyme type M2 (PKM2) in PAMs (P<0.05 and 0.01, respectively). Glycolysis inhibitors down-regulated the expression of PRRSV proteins and reduced virus titers (P<0.01). The knockdown of HIF-1α by siRNAs inhibited glycolysis and lowered PRRSV titers (P<0.05). In addition, the interferon pathways inhibited by PRRSV infection were reversed by the inhibition of glycolysis. These findings may facilitate further investigation of the role of glycolysis in PRRSV replication.
Porcine respiratory and reproductive syndrome virus/physiology* ; Glycolysis ; Swine ; Animals ; Virus Replication ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics* ; Macrophages, Alveolar/metabolism* ; Porcine Reproductive and Respiratory Syndrome/virology* ; Cells, Cultured ; RNA, Small Interfering/genetics*

OBJECTIVE: To screen for aberrantly expressed genes in osteosarcoma cells and investigate the role of RHPN2 in regulating the proliferation, apoptosis, migration and tumorigenic abilities of osteosarcoma cells. METHODS: We used GEO2R to analyze the differential gene expression profile between osteosarcoma cells and normal cells in the GSE70414 dataset. RTqPCR and Western blotting were performed to detect RHPN2 expression in osteosarcoma cell lines MG-63, 143B and SAOS2. Two RHPN2-shRNA and a control NC-shRNA were designed to silence the expression of RHPN2 in 143B cells, and CCK8 assay, colony-forming assay, annexin V-FITC/PI staining and scratch assays were carried out to examine the changes in proliferation, apoptosis and migration of the cells. We also established nude mouse models bearing osteosarcoma xenografts derived 143B cells and RHPN2-shRNA-transfected 143B cells, and assessed the effect of RHPN2 silencing on osteosarcoma cell tumorigenesis using HE staining. Kaplan-Meier survival curves were used to analyze the correlation between RHPN2 expression and survival outcomes of patients with osteosarcoma. RESULTS: RHPN2 expression was significantly upregulated in osteosarcoma cell lines MG-63, 143B and SAOS2 (P < 0.01). Silencing of RHPN2 significantly inhibited the proliferation and migration of 143B cells in vitro, promoted cell apoptosis (P < 0.01), and suppressed tumorigenic capacity of the cells in nude mice. A high expression of RHPN2 was significantly correlated with a poor prognosis of patients with osteosarcoma (P < 0.05). CONCLUSION RHPN2 is highly expressed in osteosarcoma cells to promote cell proliferation and migration and inhibits cell apoptosis. A high expression of RHPN2 is associated with a poorer prognosis of the patients with osteosarcoma.
Adaptor Proteins, Signal Transducing/metabolism* ; Animals ; Apoptosis ; Bone Neoplasms/metabolism* ; Carcinogenesis ; Cell Line, Tumor ; Cell Movement/physiology* ; Cell Proliferation/physiology* ; Humans ; Immediate-Early Proteins ; Mice ; Mice, Nude ; Osteosarcoma/metabolism* ; RNA, Small Interfering/genetics*

OBJECTIVE: To explore the role of SIRT1 in the occurrence of epithelial-mesenchymal transition (EMT) in EC-9706 and Eca-109 cells and the possible mechanism. METHODS: Three chemically synthesized siRNA targeting SIRT1 were transfected into EC-9706 and Eca-109 cells with the non-transfected cells and cells transfected with the negative siRNAs as controls. Real-time PCR and Western blotting were used to detect the expressions of SIRT1, E-cadherin, vimentin, Snail, Twist1 and ZEB in the cells. Transwell invasion assay and wounding healing assay were used to examine the changes in the invasion and metastasis abilities of the cells after transfection. RESULTS: EC-9706 and Eca-109 cells transfected with SIRT1 siRNA1 and SIRT1 siRNA3 showed significantly decreased mRNA and protein expressions of SIRT1 ( < 0.05). Transwell invasion assay and wounding healing assay showed that transfection with SIRT1 siRNA1 and SIRT1 siRNA3 caused significantly lowered invasion and metastasis abilities in EC-9706 and Eca-109 cells ( < 0.05). In EC-9706 and Eca-109 cells transfected with SIRT1 siRNA1 and SIRT1 siRNA3, the expression level of E-cadherin was significantly increased while the expressions of vimentin, Snail and Twist were significantly lowered ( < 0.05). CONCLUSIONS SIRT1 participates in the invasion and metastasis of EC-9706 and Eca- 109 cells probably by inducing EMT via regulating the expression of Snail.
Antigens, CD ; metabolism ; Cadherins ; metabolism ; Cell Line, Tumor ; Cell Movement ; Epithelial-Mesenchymal Transition ; physiology ; Humans ; Neoplasm Invasiveness ; Nuclear Proteins ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; metabolism ; Sirtuin 1 ; genetics ; metabolism ; Snail Family Transcription Factors ; metabolism ; Transfection ; Twist-Related Protein 1 ; metabolism ; Vimentin ; metabolism ; Zinc Finger E-box-Binding Homeobox 1 ; metabolism

Animals ; Carrier Proteins ; genetics ; Immunohistochemistry ; Lacrimal Apparatus ; metabolism ; Lung ; metabolism ; Mice ; Mice, Inbred NOD ; Microfilament Proteins ; genetics ; RNA, Small Interfering ; genetics ; physiology ; Random Allocation ; Sjogren's Syndrome ; genetics ; immunology ; therapy

Background: Focal segmental glomerulosclerosis (FSGS) is a kidney disease that is commonly associated with proteinuria and the progressive loss of renal function, which is characterized by podocyte injury and the depletion and collapse of glomerular capillary segments. The pathogenesis of FSGS has not been completely elucidated; however, recent advances in molecular genetics have provided increasing evidence that podocyte structural and functional disruption is central to FSGS pathogenesis. Here, we identified a patient with FSGS and aimed to characterize the pathogenic gene and verify its mechanism. Methods: Using next-generation sequencing and Sanger sequencing, we screened the causative gene that was linked to FSGS in this study. The patient's total blood RNA was extracted to validate the messenger RNA (mRNA) expression of coenzyme Q monooxygenase 6 (COQ6) and validated it by immunohistochemistry. COQ6 knockdown in podocytes was performed in vitro with small interfering RNA, and then, F-actin was determined using immunofluorescence staining. Cell apoptosis was evaluated by flow cytometry, the expression of active caspase-3 was determined by Western blot, and mitochondrial function was detected by MitoSOX. Results: Using whole-exome sequencing and Sanger sequencing, we screened a new causative gene, COQ6, NM_182480: exon1: c.G41A: p.W14X. The mRNA expression of COQ6 in the proband showed decreased. Moreover, the expression of COQ6, which was validated by immunohistochemistry, also had the same change in the proband. Finally, we focused on the COQ6 gene to clarify the mechanism of podocyte injury. Flow cytometry showed significantly increased in apoptotic podocytes, and Western blotting showed increases in active caspase-3 in si-COQ6 podocytes. Meanwhile, reactive oxygen species (ROS) levels were increased and F-actin immunofluorescence was irregularly distributed in the si-COQ6 group. Conclusions This study reported a possible mechanism for FSGS and suggested that a new mutation in COQ6, which could cause respiratory chain defect, increase the generation of ROS, destroy the podocyte cytoskeleton, and induce apoptosis. It provides basic theoretical basis for the screening of FSGS in the future.
Adolescent ; Animals ; Apoptosis ; genetics ; physiology ; Cell Line ; Female ; Flow Cytometry ; Glomerulosclerosis, Focal Segmental ; genetics ; Humans ; Immunohistochemistry ; Mice ; Mutation ; genetics ; Podocytes ; metabolism ; pathology ; RNA, Messenger ; genetics ; RNA, Small Interfering ; genetics ; metabolism ; Ubiquinone ; analogs & derivatives ; genetics ; metabolism

Brain damage can cause lung injury. To explore the mechanism underlying the lung injury induced by acute cerebral ischemia (ACI), we established a middle cerebral artery occlusion (MCAO) model in male Sprague-Dawley rats. We focused on glia maturation factor β (GMFB) based on quantitative analysis of the global rat serum proteome. Polymerase chain reaction, western blotting, and immunofluorescence revealed that GMFB was over-expressed in astrocytes in the brains of rats subjected to MCAO. We cultured rat primary astrocytes and confirmed that GMFB was also up-regulated in primary astrocytes after oxygen-glucose deprivation (OGD). We subjected the primary astrocytes to Gmfb RNA interference before OGD and collected the conditioned medium (CM) after OGD. We then used the CM to culture pulmonary microvascular endothelial cells (PMVECs) acquired in advance and assessed their status. The viability of the PMVECs improved significantly when Gmfb was blocked. Moreover, ELISA assays revealed an elevation in GMFB concentration in the medium after OGD. Cell cultures containing recombinant GMFB showed increased levels of reactive oxygen species and a deterioration in the state of the cells. In conclusion, GMFB is up-regulated in astrocytes after ACI, and brain-derived GMFB damages PMVECs by increasing reactive oxygen species. GMFB might thus be an initiator of the lung injury induced by ACI.
Animals ; Brain ; metabolism ; pathology ; Brain Ischemia ; complications ; pathology ; Bronchoalveolar Lavage Fluid ; Cell Hypoxia ; physiology ; Cells, Cultured ; Cerebrovascular Circulation ; physiology ; Chromatography, High Pressure Liquid ; Culture Media, Conditioned ; pharmacology ; Disease Models, Animal ; Endothelial Cells ; metabolism ; Gene Expression Regulation ; physiology ; Glia Maturation Factor ; metabolism ; In Situ Nick-End Labeling ; Lung Injury ; etiology ; metabolism ; pathology ; Male ; Neuroglia ; metabolism ; Neurologic Examination ; Peroxidase ; metabolism ; Proteome ; RNA Interference ; physiology ; RNA, Small Interfering ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism ; Tandem Mass Spectrometry

Autophagy is an evolutionarily-conserved self-degradative process that maintains cellular homeostasis by eliminating protein aggregates and damaged organelles. Recently, vesicle-associated membrane protein-associated protein B (VAPB), which is associated with the familial form of amyotrophic lateral sclerosis, has been shown to regulate autophagy. In the present study, we demonstrated that knockdown of VAPB induced the up-regulation of beclin 1 expression, which promoted LC3 (microtubule-associated protein light chain 3) conversion and the formation of LC3 puncta, whereas overexpression of VAPB inhibited these processes. The regulation of beclin 1 by VAPB was at the transcriptional level. Moreover, knockdown of VAPB increased autophagic flux, which promoted the degradation of the autophagy substrate p62 and neurodegenerative disease proteins. Our study provides evidence that the regulation of autophagy by VAPB is associated with the autophagy-initiating factor beclin 1.
Autophagy ; drug effects ; physiology ; Beclin-1 ; genetics ; metabolism ; Cell Line, Transformed ; Gene Expression Regulation ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Microtubule-Associated Proteins ; genetics ; metabolism ; R-SNARE Proteins ; genetics ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; metabolism ; RNA-Binding Proteins ; genetics ; metabolism ; Transfection