Regulation mechanism of EBV-encoded EBER1 and LMP2A on YAP1 and the impact of YAP1 on the EBV infection status in EBV-associated gastric carcinoma.

This study aims to explore the role and regulatory mechanism of Yes-associated protein 1 (YAP1) in the development of Epstein-Barr virus-associated gastric cancer (EBVaGC). Here we showed that EBV can upregulate the expression and activity of YAP1 protein through its encoded latent products EBV-encoded small RNA 1 (EBER1) and latent membrane protein 2A (LMP2A), enhancing the malignant characteristics of EBVaGC cells. In addition, we also showed that overexpression of YAP1 induced the expression of EBV encoding latent and lytic phase genes and proteins in the epithelial cell line AGS-EBV infected with EBV, and increased the copy number of the EBV genome, while loss of YAP1 expression reduced the aforementioned indicators. Moreover, we found that YAP1 enhanced EBV lytic reactivation induced by two known activators, 12-O-tetradecanoylhorbol-13-acetate (TPA) and sodium butyrate (NaB). These results indicated a bidirectional regulatory mechanism between EBV and YAP1 proteins, providing new experimental evidence for further understanding the regulation of EBV infection patterns and carcinogenic mechanisms in gastric cancer.

Effects of methylation and imprinting expression of Insulin-like growth factor 2 gene in gastric cancer.

BACKGROUND: Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC) is a common malignant tumor associated with EBV infection. Insulin-like growth factor 2 (IGF2) is an imprinted gene and a key protein that regulates growth, especially during normal fetal development. Loss of imprinting (LOI), is a common epigenetic anomaly in a variety of human cancers. However, the promoter methylation, imprinting status and function of IGF2 gene in GC are unclear. OBJECTIVE: To explore the role of IGF2 in the occurrence and development of gastric cancer. METHODS: The biological function of IGF2 in gastric cancer was investigated by Transwell, wound healing, CCK-8 and flow cytometry assays. IGF2 imprinting status and gene promoter methylation in gastric cancer tissues were detected by PCR-RFLP and BGS. RESULTS: The results showed that the expression of IGF2 was higher in GC tissues than adjacent tissues. IGF2 gene promoter methylation and LOI were significantly higher in EBVaGC tissues than in EBV-negative gastric cancer (EBVnGC) tissues. The high expression of IGF2 in gastric cancer can promote the migration and proliferation of gastric cancer cells. CONCLUSION: Our data suggest that IGF2 is involved in the occurrence and development of gastric cancer. Targeting IGF2 may be a potential therapeutic target for gastric cancer.

MiR-BART1-3p and BART18-5p inhibit cell migration, proliferation and activate autophagy in Epstein-Barr virus-associated gastric cancer by targeting erythropoietin-producing human hepatocellular 2.

Epstein-Barr virus (EBV) is a human tumor-associated virus that encodes various microRNAs. EBV infection causes a variety of malignant tumors, including nasopharyngeal carcinoma and gastric cancer, etc. EBV-associated gastric cancer (EBVaGC) has unique molecular characteristics from other gastric cancers, but its pathogenic mechanism remains unclear. In recent years, erythropoietin-producing human hepatocellular 2 (EphA2) has been reported to be highly expressed in various cancers and promote tumor growth and metastasis. As an important cancer oncogene, EphA2 is a potential therapeutic target. However, whether EBV is involved in the regulation of EphA2 and thus affects the progression of EBVaGC remains unclear. In this study, we found that the expression of EphA2 in EBVaGC cells was significantly lower than that in EBV-negative gastric cancer (EBVnGC) cells. Additionally, overexpression of EphA2 in EBVaGC cells promoted migration and proliferation, and inhibited autophagy. EBV-miR-BART1-3p and BART18-5p were found to target the 3'-UTR of EphA2 and down-regulate its expression. Our results suggest that EBV may be involved in gastric cancer progression by targeting EphA2 through BART1-3p and BART18-5p.

Downregulation of MUS81 expression inhibits cell migration and maintains EBV latent infection through miR-BART9-5p in EBV-associated gastric cancer.

Epstein-Barr virus (EBV) infection is associated with the occurrence and development of gastric cancer (GC). Methyl methanesulfonate and ultraviolet-sensitive gene 81 (MUS81) is the catalytic component of a structure-specific endonuclease and plays an important role in chromosomal stability. However, the link between EBV infection and MUS81 remains unclear. In the present study, we found that MUS81 expression was much lower in EBV-associated GC cells than in EBV-negative GC. MUS81 acts as an oncogene in GC by inducing the cell migration and proliferation. Western blot and luciferase reporter assays revealed that miR-BART9-5p directly targeted MUS81 and downregulated its expression. Additionally, overexpression of MUS81 in EBV-positive GC cells inhibited the expression of EBV nuclear antigen 1 (EBNA1). EBNA1 is critical for the pathogenesis of EBV-associated tumors and the maintenance of a stable copy number of the viral genomes. Altogether, these results indicated that the lowering MUS81 expression might be a mechanism by EBV to maintain its latent infection.

Imidazo[1,2-a]Pyridine Derivatives as Novel Dual-Target Inhibitors of ABCB1 and ABCG2 for Reversing Multidrug Resistance.

ABCB1 and ABCG2 are the important ATP-binding cassette (ABC) transporters associated with multidrug resistance (MDR). Herein, we designed a series of imidazo[1,2-a]pyridine derivatives as dual-target inhibitors of ABCB1 and ABCG2 through the scaffold hopping strategy. Compound Y22 displayed potential efflux function inhibitory toward both ABCB1 and ABCG2 (reversal fold: ABCB1 = 8.35 and ABCG2 = 2.71) without obvious cytotoxicity. Y22 also enhanced the potency of antiproliferative drugs in vitro. Mechanistic studies demonstrated that Y22 slightly suppressed ATPase activity but did not affect the protein expression of ABCB1 or ABCG2. Notably, Y22 exhibited negligible CYP3A4 inhibition and enhanced the antiproliferative activity of adriamycin in vivo by restoring the sensitivity of resistant cells. Thus, Y22 may be effective clinically in combination with common chemotherapy agents. In summary, Y22 is a potential dual-target inhibitor that reverses MDR by blocking the efflux function of ABCB1 and ABCG2.

Nuclear respiratory factor 1 promotes the progression of EBV-associated gastric cancer and maintains EBV latent infection.

This study aimed to investigate the association of Epstein-Barr virus (EBV) with nuclear respiratory factor 1 (NRF1) and the biological function of NRF1 in EBV-associated gastric cancer (EBVaGC). Western blot and qRT-PCR were used to assess the effect of latent membrane protein 2A (LMP2A) on NRF1 expression after transfection with LMP2A plasmid or siLMP2A. The effects of NRF1 on the migration and apoptosis ability of GC cells were investigated by transwell assay and flow cytometry apoptosis analysis in vitro, respectively. In addition, we determined the regulatory role of NRF1 in EBV latent infection by western blot and droplet digital PCR (ddPCR). LMP2A upregulated NRF1 expression by activating the NF-κB pathway. Moreover, NRF1 upregulated the expression of N-Cadherin and ZEB1 to promote cell migration. NRF1 promoted the expression of Bcl-2 to increase the anti-apoptotic ability of cells. In addition, NRF1 maintained latent infection of EBV by promoting the expression of the latent protein Epstein-Barr nuclear antigen 1 (EBNA1) and inhibiting the expression of the lytic proteins. Our data indicated the role of NRF1 in EBVaGC progression and the maintenance of EBV latent infection. This provided a new theoretical basis for further NRF1-based anti-cancer therapy.

LMP2A inhibits the expression of KLF5 through the mTORC1 pathway in EBV-associated gastric carcinoma.

OBJECTIVE: To investigate the expression and biological role of KLF5 in EBV-associated gastric carcinoma (EBVaGC) and EBV-negative gastric carcinoma (EBVnGC), and to clarify the relationship between EBV and KLF5. METHODS: The expression of KLF5 in GC tissues was detected by immunohistochemistry. Western blot and immunofluorescence assay were used to examine the expression and localization of KLF5 in EBV positive and negative GC cell lines. The effect of LMP2A on KLF5 was analyzed by transfection of LMP2A plasmid or siRNA. The function of KLF5 in GC was elucidated by molecular biology experiments. RESULTS: The expression of KLF5 was significantly reduced in EBVaGC tissues and cell lines. LMP2A inhibited KLF5 expression through inactivating mTORC1 pathway in EBV positive GC cell lines. Meanwhile, KLF5 could enhance the migration ability of GC cells and induce autophagy. CONCLUSION: LMP2A downregulated KLF5 expression by inhibiting the mTORC1 pathway in EBV positive GC cells. KLF5 might play an oncogene-like role by promoting the migration of GC cells and inducing autophagy.

The promoter aberrant methylation status of TMEM130 is associated with gastric cancer.

BACKGROUND AND AIMS: Gastric cancer (GC) is a malignant tumor that seriously affects human health and Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC) is a molecular subtype of GC. This study aims to determine the relationship between the methylation status of the TMEM130 gene and GC, and to explore the influence of EBV infection. METHODS: qRT-PCR was conducted to investigate the transcriptional expression of TMEM130 in GC. BSP and MSP assays were used to detect the methylation level of the TMEM130 promoter. The cell migration ability was detected by Transwell and western blot after transfection of TMEM130 plasmids in GC cells. RESULTS: The transcriptional expression of TMEM130 decreased in GC with hypermethylation of the promoter region. The DNA methyltransferase inhibitor could increase the mRNA expression of TMEM130. Moreover, hypermethylation of the TMEM130 promoter in GC tissues was associated with EBV infection. Overexpression of TMEM130 in GC cell lines suppresses cell migration ability. CONCLUSION: This study was the first to research the expression and function of TMEM130 and found that TMEM130 gene hypermethylation might contribute to GC migration and EBV infection as a cause of hypermethylation of the TMEM130 gene. TMEM130 is a promising biomarker for the diagnosis of GC.

Ferulic acid: A review of its pharmacology, pharmacokinetics and derivatives.

Ferulic acid, a kind of phenolic substance widely existing in plants, is an important active component of many traditional Chinese medicines. So far, it has been proved that ferulic acid has a variety of biological activities, especially in oxidative stress, inflammation, vascular endothelial injury, fibrosis, apoptosis and platelet aggregation. Many studies have shown that ferulic acid can inhibit PI3K/AKT pathway, the production of ROS and the activity of aldose reductase. The anti-inflammatory effect of ferulic acid is mainly related to the levels of PPAR γ, CAM and NF-κ B and p38 MAPK signaling pathways. Ferulic acid not only protects vascular endothelium by ERK1/2 and NO/ET-1 signal, but also plays an anti-fibrosis role by TGF-ß/Smad and MMPs/TIMPs system. Moreover, ferulic acid has ant-apoptotic and anti-platelet effects. In addition to the pharmacological effects of ferulic acid, its pharmacokinetics and derivatives were also discussed in this paper. This review provides the latest summary of the latest research on ferulic acid.

MiR-BART2-3p targets Unc-51-like kinase 1 and inhibits cell autophagy and migration in Epstein-Barr virus-associated gastric cancer.

ULK1 (Unc-51-like kinase 1) is an evolutionarily conserved serine/threonine kinase that plays a central role in the regulation of autophagy. ULK1 is associated with prognosis for metastasis and survival in several tumors. However, its relationship with Epstein-Barr virus (EBV) has not been studied. We found that the expression of ULK1 in EBV-associated gastric cancer cells was lower than that in EBV-negative gastric cancer cells. Further, a luciferase reporter gene assay showed that miR-BART2-3p directly targets ULK1. EBV-miR-BART2-3p attenuated endogenous protein expression levels of some autophagy-related genes. MiR-BART2-3p could thus be involved in the regulation of autophagy. Most important, our research indicates that miR-BART2-3p targets ULK1, resulting in downregulation of epithelial-mesenchymal transformation (EMT) -associated marker proteins and reducing EMT and cell migration. Our study shows that modulation of ULK1 is the likely mechanism by which miR-BART2-3p participates in the regulation of autophagy and cancer cell migration.

A constructing study on professional competency standards of school health education teachers / 中国学校卫生

Objective@#To construct a model of professional competency standard for school health education teachers in China, then to provide a framework for the professional development of health education teachers at primary and secondary schools.@*Methods@#Seventy-five indicators of professional competency of school health education teacher were identified through job task analysis, qualitative interview, expert consultation. A total of 282 school health administrators/researchers, school principals, health education teachers in Shanghai, and the first undergraduate students of health education progame in China were surveyed. Items analysis verified appropriateness, and exploratory factor analysis determined construct validity of the competency standards.@*Results@#The framework of competency standards consists of four major areas (general literacy, school health services, school health education, school health management), nine categories, and 70 competence standards. Nine categories include professional ethics, general literacy as teacher, assist to deal with emergency/accident health events and common diseases situation in school, assist vaccination and mental health assessment, monitoring/communication the health situation of students, knowledge of health education and teaching skills, implement health education activities effectively and continuously, deal with infectious diseases and environment/ water/food safety in school, monitor conditions of the school health and optimize health strategy continuously. Cronbach’s Alpha coefficient of total competence standards was 0.98, and those of the sub-dimensions ranged from 0.86 to 0.96 ; the split-half reliability of total system was 0.93 with sub-dimensions coefficient ranging from 0.83 to 0.95.@*Conclusion@#The model of competency standard developed in this study show good validity and reliability, which can provide theoretical framework for the training, using and evaluation of school health education teachers with the characteristics of combinating medicine and teaching.

RNA-seq based gene expression analysis of ovarian granulosa cells exposed to zearalenone in vitro: significance to steroidogenesis.

Zearalenone (ZEA) is a natural contaminant of various food and feed products representing a significant problem worldwide. Since the occurrence of ZEA in grains and feeds is frequent, the present study was carried out to evaluate the possible effects of ZEA on steroid production and gene expression of porcine granulosa cells, using RNA-seq analysis. Porcine granulosa cells were administered 10 µM and 30 µM ZEA during 72 h of culture in vitro. Following ZEA treatment the gene expression profile of control and exposed granulosa cells was compared using RNA-seq analysis. The results showed that in the exposed granulosa cells ZEA significantly altered the transcript levels, particularly steroidogenesis associated genes. Compared with the control group, 10 µM and 30 µM ZEA treatment significantly increased the mRNA expression of EDN1, IER3, TGFß and BDNF genes and significantly reduced the mRNA expression of IGF-1 and SFRP2 genes. In particular, ZEA significantly decreased the expression of genes essential for estrogen synthesis including FSHR, CYP19A1 and HSD17ß in granulosa cells. Furthermore, Q-PCR and Western-blot analysis also confirmed reduced expression of these genes in ZEA exposed granulosa cells. These effects were associated with a significant reduction of 17ß-estradiol concentrations in the culture medium of granulosa cells. Collectively, these results demonstrated a concretely deleterious effect of ZEA exposure on the mRNA expression of steroidogenesis related genes and the production of steroid hormones in porcine ovarian granulosa cells in vitro.

Inhibitory effect of dihydroartemisinin on chondrogenic and hypertrophic differentiation of mesenchymal stem cells.

Chondrocytes located in hyaline cartilage may maintain phenotype while the chondrocytes situated in calcified cartilage differentiate into hypertrophy. Chondrogenic and hypertrophic differentiation of mesenchymal stem cells (MSCs) are two subsequent processes during endochondral ossification. However, it is necessary for chondrocytes to hold homeostasis and to inhibit hypertrophic differentiation in stem cell-based regenerated cartilage. Dihydroartemisinin (DHA) is derived from artemisia apiacea which has many biological functions such as anti-malarial and anti-tumor. Whereas the effects of DHA on chondrogenic and hypertrophic differentiation are poorly understand. In this study, the cytotoxicity of DHA was determined by CCK8 assay and the cell apoptosis was analyzed by flow cytometry. Additionally, the effects of DHA on chondrogenic and hypertrophic differentiation of MSCs are explored by RT-PCR, western blotting and immunohistochemistry. The results showed that DHA inhibited expression of chondrogenic markers including Sox9 and Col2a1 by activating Nrf2 and Notch signaling. After induced to chondrogenesis, cells were treated with hypertrophic induced medium with DHA. The results revealed that hypertrophic markers including Runx2 and Col10a1 were down-regulated following DHA treatment through Pax6/HOXA2 and Gli transcription factors. These findings indicate that DHA is negative to chondrogenesis and is protective against chondrocyte hypertrophy to improve chondrocytes stability. Therefore, DHA might be not suited for chondogenesis but be potential as a new therapeutic candidate to maintain the biological function of regenerated cartilage.

Effectiveness of Antibiotic Use Management in Tianjin (2011-2013): A Quasi-Experimental Study.

BACKGROUND In this study we investigated changes in the status of antibiotic use in Tianjin since the implementation of the Antibiotic Stewardship Program (ASP) (2011-2013), as well as existing problems, strategies, and outcomes to promote rational clinical antibiotic use. MATERIAL AND METHODS A quasi-experimental study was performed to investigate situations of antibiotic use in secondary and tertiary general hospitals in Tianjin from April 2011 to 2013. Five major indicators were analyzed: percentage of antibiotic use in inpatient cases (%), antibacterial use density (AUD), proportion of prophylactic antibiotic application for type I surgical incision, compliance rate of medication administration 0.5-2.0 h before such procedures, and antibiotic prophylaxis for ≤24 h in patients receiving these surgeries. RESULTS There was a decrease in the percentage of antibiotic use across general hospitals (60.38% to 46.88%), in AUD (51.60% to 35.37%), and in the proportion of prophylactic antibiotic applications for type I incisions (86.67% to 25.08%). For patients undergoing these procedures, there was an increased compliance rate of medication administration of 0.5-2.0 h prior to surgery (86.38% to 100%), and of antibiotic prophylactic use for ≤24 h (40.30% to 96.37%). CONCLUSIONS Implementation of the ASP campaign has reduced irrational antibiotic use, promoted rational antibiotic use, and delayed antibiotic resistance.

Zearalenone exposure impairs ovarian primordial follicle formation via down-regulation of Lhx8 expression in vitro.

Zearalenone (ZEA) is an estrogenic mycotoxin mainly produced as a secondary metabolite by numerous species of Fusarium. Previous work showed that ZEA had a negative impact on domestic animals with regard to reproduction. The adverse effects and the mechanisms of ZEA on mammalian ovarian folliculogenesis remain largely unknown, particularly its effect on primordial follicle formation. Thus, we investigated the biological effects of ZEA exposure on murine ovarian germ cell cyst breakdown and primordial follicle assembly. Our results demonstrated that newborn mouse ovaries exposed to 10 or 30µM ZEA in vitro had significantly less germ cell numbers compared to the control group. Moreover, the presence of ZEA in vitro increased the numbers of TUNEL and γH2AX positive cells within mouse ovaries and the ratio of mRNA levels of the apoptotic genes Bax/Bcl-2. Furthermore, ZEA exposure reduced the mRNA of oocyte specific genes such as LIM homeobox 8 (Lhx8), newborn ovary homeobox (Nobox), spermatogenesis and oogenesis helix-loop-helix (Sohlh2), and factor in the germline alpha (Figlα) in a dose dependent manner. Exposure to ZEA led to remarkable changes in the Lhx8 3'-UTR DNA methylation dynamics in oocytes and severely impaired folliculogenesis in ovaries after transplantation under the kidney capsules of immunodeficient mice. In conclusion, ZEA exposure impairs mouse primordial follicle formation in vitro.

Transformation of trollioside and isoquercetin by human intestinal flora in vitro.

The present study was designed to determine the intestinal bacterial metabolites of trollioside and isoquercetin and their antibacterial activities. A systematic in vitro biotransformation investigation on trollioside and isoquercetin, including metabolite identification, metabolic pathway deduction, and time course, was accomplished using a human intestinal bacterial model. The metabolites were analyzed and identified by HPLC and HPLC-MS. The antibacterial activities of trollioside, isoquercetin, and their metabolites were evaluated using the broth microdilution method with berberine as a positive control, and their potency was measured as minimal inhibitory concentration (MIC). Our results indicated that trollioside and isoquercetin were metabolized by human intestinal flora through O-deglycosylation, yielding aglycones proglobeflowery acid and quercetin, respectively The antibacterial activities of both metabolites were more potent than that of their parent compounds. In conclusion, trollioside and isoquercetin are totally and rapidly transformed by human intestinal bacteria in vitro and the transformation favors the improvement of the antibacterial activities of the parent compounds.

[Apoptosis-inducing effect of tetrandrine and imatinib on K562/G01 cells and its related mechanism].

This study was purposed to explore the apoptosis-inducing effect of tetrandrine (Tet) and imatinib (IM) alone or both combined on K562/G01 cells and their mechanism. MTT assay was used to detect the inhibitory effect of drugs on cell growth, flow cytometry was used to detect the cell cycle and apoptosis rate. The expression of caspase-3/BCL-2 mRNA was determined by real time-PCR, and the expression of caspase-3/BCL-2 protein was assayed by Western blot. The results showed that after being treated by 1.0 µmol/L IM or 1.5 µmol/L Tet alone and combination of these two drugs for 48 h, the inhibitory rate was (22.74 ± 0.05)%, (20.34 ± 0.57)% and (44.28 ± 0.60)%, respectively, suggesting that inhibitory effect of two drug combination was more obvious. The arrest of cell cycle at G1/S phase could be observed after Tet treatment. Early apoptosis rate was (7.81 ± 0.16) %, (14.10 ± 0.28) % respectively after being treated by combination of 1.5 µmol/L and 3.0 µmol/L Tet with 1.0 µmol/L IM. After being treated with Tet alone, FQ-PCR and Western blot showed that the expressions of caspase-3 mRNA and caspase-3 protein were up-regulated, the expressions of BCL-2 mRNA and protein were down-regulated, the effect of both drug combination was more significant. It is concluded that IM or Tet alone can induce apoptosis of K562/G01. Combination of IM with Tet shows obvious synergistic effect, mechanism of which may associate with up-regulation of caspase-3 mRNA and protein expressions, and down-regulation of BCL-2 mRNA and protein expressions.

Identification of a functional nuclear localization signal mediating nuclear import of the zinc finger transcription factor ZNF24.

ZNF24 is a member of the SCAN domain family of Krüppel-like zinc finger (ZF) transcription factors, which plays a critical role in cell proliferation and differentiation. However, how ZNF24 enters the nucleus in order to exert its function remains unclear since its nuclear localization signal(s) (NLS) has not been identified. Here, we generated a series of GFP-tagged deletion and point mutants and assessed their subcellular localization. Our results delimit the NLS to ZF1-2. Deletion of ZF1-2 caused cytoplasmic accumulation of ZNF24. Fusion of the ZF1-2 to green fluorescent protein (GFP) targeted GFP to the nucleus, demonstrating that the ZF1-2 is both necessary and sufficient for nuclear localization. ZNF24 containing histidine to leucine mutations that disrupt the structure of ZF1 or/and ZF2 retains appropriate nuclear localization, indicating that neither the tertiary structure of the zinc fingers nor specific DNA binding are necessary for nuclear localization. K286A and R290A mutation led to partial cytoplasmic accumulation. Co-immunoprecipitation demonstrated that ZNF24 interacted with importin-ß and this interaction required the ZF motifs. The ß-Catenin (CTNNB1) luciferase assays showed that the ZNF24 mutants defective in nuclear localization could not promote CTNNB1 promoter activation as the wild-type ZNF24 did. Taken together, these results suggest that consecutive ZF1-2 is critical for the regulation of ZNF24 nuclear localization and its transactivation function.

MicroRNA-375 targets the 3-phosphoinositide-dependent protein kinase-1 gene in pancreatic carcinoma.

Pancreatic carcinoma (PC) is an aggressive malignancy with one of the poorest mortality rates. It is the sixth leading cause of mortality from malignant disease in China and the fourth leading cause of cancer-related mortality in the United States. The poor outcome reflects the requirement for an improved understanding of the transcriptional control of oncogenic signaling pathways. 3-phosphoinositide-dependent protein kinase-1 (PDK1) is a potent oncogenic driver of PC. The present study aimed to elucidate the transcriptional regulation of microRNA (miR)-375-targeted PDK1. miR-375 is a putative target and, in the present study, was observed to be significantly downregulated in the tumor compared with non-tumor tissues from patients with PC (n=44). As determined by a luciferase reporter assay, the ectopic expression of miR-375 was identified to diminish the transcriptional activity of PDK1. Furthermore, immunoblotting revealed that miR-375 suppressed endogenous PDK1 protein levels. Functional assays showed that miR-375 was able to inhibit proliferation and promote apoptosis of the PC cells. miR-375 is a significant regulator of the PDK1 oncogene, suggesting that it may have a potential therapeutic role in the treatment of PC.