Sex reporting of cells used in cancer research: A systematic review.

Sex and gender disparities in biomedical research have been emphasized to improve scientific knowledge applied for the health of both men and women. Despite sex differences in cancer incidence, prognosis, and responses to therapeutic agents, mechanistic explanations at molecular levels are far from enough. Recent studies suggested that cell sex is an important biological variable due to differences in sex chromosome gene expression and differences in events associated with developmental biology. The objective of this study was to analyze the reporting of sex of cells used in cancer research using articles published in Cancer Cell, Molecular Cancer, Journal of Hematology & Oncology, Journal for ImmunoTherapy of Cancer, and Cancer Research in 2020, and to examine whether there exists any sex bias. We found that the percentage of cells with sex notation in the article was 36.5%. Primary cells exhibited higher sex notation compared to cell lines. A higher percentage of female cells were used in cell cultures with sex notation. Also, sex-common cells omitted sex description more often compared to sex-specific cells. None of the cells isolated from embryo and esophagus reported the cell sex in the article. Our results indicate cell sex report in cancer research is limited to a small proportion of cells used in the study. These results call for acknowledging the sex of cells to increase the applicability of biomedical research discoveries.

Ceramide promotes lytic reactivation of Epstein-Barr virus in gastric carcinoma.

Epstein-Barr virus (EBV) has a lifelong latency period after initial infection. Rarely, however, when the EBV immediate early gene BZLF1 is expressed by a specific stimulus, the virus switches to the lytic cycle to produce progeny viruses. We found that EBV infection reduced levels of various ceramide species in gastric cancer cells. As ceramide is a bioactive lipid implicated in the infection of various viruses, we assessed the effect of ceramide on the EBV lytic cycle. Treatment with C6-ceramide (C6-Cer) induced an increase in the endogenous ceramide pool and increased production of the viral product as well as BZLF1 expression. Treatment with the ceramidase inhibitor ceranib-2 induced EBV lytic replication with an increase in the endogenous ceramide pool. The glucosylceramide synthase inhibitor Genz-123346 inhibited C6-Cer-induced lytic replication. C6-Cer induced extracellular signal-regulated kinase 1/2 (ERK1/2) and CREB phosphorylation, c-JUN expression, and accumulation of the autophagosome marker LC3B. Treatment with MEK1/2 inhibitor U0126, siERK1&2, or siCREB suppressed C6-Cer-induced EBV lytic replication and autophagy initiation. In contrast, siJUN transfection had no impact on BZLF1 expression. The use of 3-methyladenine (3-MA), an inhibitor targeting class III phosphoinositide 3-kinases (PI3Ks) to inhibit autophagy initiation, resulted in reduced beclin-1 expression, along with suppressed C6-Cer-induced BZLF1 expression and LC3B accumulation. Chloroquine, an inhibitor of autophagosome-lysosome fusion, increased BZLF1 protein intensity and LC3B accumulation. However, siLC3B transfection had minimal effect on BZLF1 expression. The results suggest the significance of ceramide-related sphingolipid metabolism in controlling EBV latency, highlighting the potential use of drugs targeting sphingolipid metabolism for treating EBV-positive gastric cancer.IMPORTANCEEpstein-Barr virus remains dormant in the host cell but occasionally switches to the lytic cycle when stimulated. However, the exact molecular mechanism of this lytic induction is not well understood. In this study, we demonstrate that Epstein-Barr virus infection leads to a reduction in ceramide levels. Additionally, the restoration of ceramide levels triggers lytic replication of Epstein-Barr virus with increase in phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and CREB. Our study suggests that the Epstein-Barr virus can inhibit lytic replication and remain latent through reduction of host cell ceramide levels. This study reports the regulation of lytic replication by ceramide in Epstein-Barr virus-positive gastric cancer.

Epstein-Barr Virus miR-BART1-3p Regulates the miR-17-92 Cluster by Targeting E2F3.

Epstein-Barr virus (EBV) is associated with several tumors and generates BamHI A rightward transcript (BART) microRNAs (miRNAs) from BART transcript introns. These BART miRNAs are expressed at higher levels in EBV-associated epithelial malignancies than in EBV-infected B lymphomas. To test the effects of EBV miRNA on the cell cycle and cell growth, we transfected miR-BART1-3p, a highly expressed EBV-associated miRNA, into gastric carcinoma cells. We found that miR-BART1-3p induced G0/G1 arrest and suppressed cell growth in gastric carcinoma cells. As our microarray analyses showed that E2F3, a cell cycle regulator, was inhibited by EBV infection, we hypothesized that miR-BART1-3p regulates E2F3. Luciferase assays revealed that miR-BART1-3p directly targeted the 3'-UTR of E2F3 mRNA. Both E2F3 mRNA and encoded protein levels were reduced following miR-BART1-3p transfection. In contrast, E2F3 expression in AGS-EBV cells transfected with a miR-BART1-3p inhibitor was enhanced. As E2F3 has been shown to regulate the expression of highly conserved miR-17-92 clusters in vertebrates, we examined whether this expression is affected by miR-BART1-3p, which can downregulate E2F3. The expression of E2F3, miR-17-92a-1 cluster host gene (MIR17HG), and miR-17-92 cluster miRNAs was significantly reduced in EBV-associated gastric carcinoma (EBVaGC) patients compared with EBV-negative gastric carcinoma (EBVnGC) patients. Further, miR-BART1-3p as well as the siRNA specific to E2F3 inhibited the expression of the miR-17-92 cluster, while inhibition of miR-BART1-3p enhanced the expression of the miR-17-92 cluster in cultured GC cells. Our results suggest a possible role of miR-BART1-3p in cell cycle regulation and in regulation of the miR-17-92 cluster through E2F3 suppression.

The GNAQ T96S Mutation Affects Cell Signaling and Enhances the Oncogenic Properties of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC), the most common malignant tumor in the liver, grows and metastasizes rapidly. Despite advances in treatment modalities, the five-year survival rate of HCC remains less than 30%. We sought genetic mutations that may affect the oncogenic properties of HCC, using The Cancer Genome Atlas (TCGA) data analysis. We found that the GNAQ T96S mutation (threonine 96 to serine alteration of the Gαq protein) was present in 12 out of 373 HCC patients (3.2%). To examine the effect of the GNAQ T96S mutation on HCC, we transfected the SK-Hep-1 cell line with the wild-type or the mutant GNAQ T96S expression vector. Transfection with the wild-type GNAQ expression vector enhanced anchorage-independent growth, migration, and the MAPK pathways in the SK-Hep-1 cells compared to control vector transfection. Moreover, cell proliferation, anchorage-independent growth, migration, and the MAPK pathways were further enhanced in the SK-Hep-1 cells transfected with the GNAQ T96S expression vector compared to the wild-type GNAQ-transfected cells. In silico structural analysis shows that the substitution of the GNAQ amino acid threonine 96 with a serine may destabilize the interaction between the regulator of G protein signaling (RGS) protein and GNAQ. This may reduce the inhibitory effect of RGS on GNAQ signaling, enhancing the GNAQ signaling pathway. Single nucleotide polymorphism (SNP) genotyping analysis for Korean HCC patients shows that the GNAQ T96S mutation was found in only one of the 456 patients (0.22%). Our data suggest that the GNAQ T96S hotspot mutation may play an oncogenic role in HCC by potentiating the GNAQ signal transduction pathway.

Celecoxib induces apoptosis through Akt inhibition in 5-fluorouracil-resistant gastric cancer cells.

Gastric cancer is the fifth leading cause of cancer and a global public health problem. 5-Fluorouracil (5-FU) is the primary drug chosen for the treatment of advanced gastric cancer, but acquired cancer drug resistance limits its effectiveness and clinical use. Proliferation assays showed that a gastric carcinoma cell line, AGS and 5-FU-resistant AGS cells (AGS FR) treated with 3-100 µM 5-FU for 48 h or 72 h showed different sensitivities to 5-FU. Immunoblot assay demonstrated that AGS FR cells expressed more COX-2 and PGE2-cognated receptor EP2 than AGS cells. AGS FR cells considerably produced PGE2 than AGS upon stimulation with 5-FU. These results suggest that COX-2 expression is associated with 5-FU resistance. Unlike AGS FR cells, AGS cells showed increased levels of both cleaved caspase-3 and Bax following 5-FU treatment. Treatment of cells with the COX-2 selective inhibitor celecoxib induced cell death of AGS FR cells in a time- and concentration-dependent manner. FACS analysis showed that celecoxib at high doses caused apoptotic cell death, demonstrating a concentration-dependent increase in the cell populations undergoing early apoptosis and late apoptosis. This apoptotic induction was strongly supported by the expression profiles of apoptosis- and survival-associated proteins in response to celecoxib; pro-apoptotic cellular proteins increased while expressions of COX-2 and p-Akt were downregulated in a concentration-dependent manner. An increase in PTEN expression was accompanied with downregulation of p-Akt. Based on the data that downregulation of COX-2 was correlated with the concentrations of celecoxib, COX-2 may play a key role in celecoxib-induced cell death of AGS FR cells. Butaprost, the EP2 agonist, promoted proliferative activity of AGS FR cells in a concentration-dependent manner compared with AGS cells. In cells exposed to butaprost, expressions of COX-2 and p-Akt were increased in a concentration-dependent manner with concomitantly reduced PTEN levels. Taken together, 5-FU-resistance in gastric cancer is correlated with COX-2 expression, and therefore the selective inhibition of COX-2 leads to suppression of cell proliferation of AGS FR cells. Modulation of COX-2 expression and its catalytic activity may be a potential therapeutic strategy to overcome 5-FU-resistant gastric cancer.

Potentiating activities of chrysin in the therapeutic efficacy of 5-fluorouracil in gastric cancer cells.

The incidence and mortality rates of gastric cancer rank among the highest five of all cancer types worldwide. The chemotherapeutic agent 5-fluorouracil (5-FU) is the gold standard for treating gastric cancer, but its efficacy is limited due to high rates of resistance. To improve the therapeutic efficacy of 5-FU and overcome its resistance, the synergistic effect of chrysin with 5-FU was investigated and its mechanism was elucidated. Chrysin was co-administered with 5-FU in AGS cells and 5-FU-resistant AGS cells (AGS/FR). Cytotoxicity was investigated using MTT assay, followed by calculating the combination index (CI). Several biomarkers were detected using western blotting analysis. Apoptosis and cell cycle distribution were measured by flow cytometry. The combination of chrysin and 5-FU significantly increased cytotoxicity more than chrysin or 5-FU alone. 5-FU induced apoptosis through p53-p21 activity, while chrysin arrested the cell cycle in the G2/M phase. The combination of chrysin and 5-FU showed an anticancer effect via S phase arrest. The results indicated that chrysin and 5-FU exhibited anticancer properties via different pathways. Furthermore, the present study found that chrysin enhanced the chemotherapeutic effect of 5-FU in AGS/FR cells. In the resistant cells, the combination of chrysin and 5-FU improved the anticancer effect via G2/M phase arrest. These findings indicated that chrysin potentiated the chemotherapeutic effect of 5-FU in gastric cancer AGS and AGS/FR cells via cell cycle arrest. Therefore, chrysin may be used to treat gastric cancers that have become resistant to 5-FU.

Sex-Biased Molecular Signature for Overall Survival of Liver Cancer Patients.

Sex/gender disparity has been shown in the incidence and prognosis of many types of diseases, probably due to differences in genes, physiological conditions such as hormones, and lifestyle between the sexes. The mortality and survival rates of many cancers, especially liver cancer, differ between men and women. Due to the pronounced sex/gender disparity, considering sex/ gender may be necessary for the diagnosis and treatment of liver cancer. By analyzing research articles through a PubMed literature search, the present review identified 12 genes which showed practical relevance to cancer and sex disparities. Among the 12 sex-specific genes, 7 genes (BAP1, CTNNB1, FOXA1, GSTO1, GSTP1, IL6, and SRPK1) showed sex-biased function in liver cancer. Here we summarized previous findings of cancer molecular signature including our own analysis, and showed that sexbiased molecular signature CTNNB1High, IL6High, RHOAHigh and GLIPR1Low may serve as a female-specific index for prediction and evaluation of OS in liver cancer patients. This review suggests a potential implication of sex-biased molecular signature in liver cancer, providing a useful information on diagnosis and prediction of disease progression based on gender.

Sex Chromosomes Are Severely Disrupted in Gastric Cancer Cell Lines.

Sex has not received enough attention as an important biological variable in basic research, even though the sex of cells often affects cell proliferation, differentiation, apoptosis, and response to stimulation. Knowing and considering the sex of cells used in basic research is essential as preclinical and clinical studies are planned based on basic research results. Cell lines derived from tumor have been widely used for proof-of-concept experiments. However, cell lines may have limitations in testing the effect of sex on cell level, as chromosomal abnormality is the single most characteristic feature of tumor. To examine the status of sex chromosomes in a cell line, 12 commercially available gastric carcinoma (GC) cell lines were analyzed using several different methods. Loss of Y chromosome (LOY) accompanied with X chromosome duplication was found in three (SNU-484, KATO III, and MKN-1) out of the six male-derived cell lines, while one cell line (SNU-638) showed at least partial deletion in the Y chromosome. Two (SNU-5 and MKN-28) out of six female-derived cell lines showed a loss of one X chromosome, while SNU-620 gained one extra copy of the X chromosome, resulting in an XXX karyotype. We found that simple polymerase chain reaction (PCR)-based sex determination gives a clue for LOY for male-derived cells, but it does not provide detailed information for the gain or loss of the X chromosome. Our results suggest that carefully examining the sex chromosome status of cell lines is necessary before using them to test the effect of sex on cell level.

Cell Cycle Dysregulation Is Associated With 5-Fluorouracil Resistance in Gastric Cancer Cells.

BACKGROUND/AIM: 5-Fluorouracil (5-FU) is an anticancer drug commonly used to treat gastric cancer; however, continuous 5-FU chemotherapy causes drug resistance. MATERIALS AND METHODS: We established five sublines of 5-FU-resistant AGS gastric cancer cells to investigate changes that may have occurred in the development of 5-FU resistance. Drug resistance to other chemotherapeutic reagents, proliferation, cell-cycle changes, and wound healing ability were assessed for each subline. RESULTS: Retarded cell growth, G0/G1 phase arrest, up-regulation of p57, and down-regulation of cyclin D1 were commonly observed in all five sublines. Resistance to paclitaxel and cisplatin was also observed in most of the sublines. CONCLUSION: Our data support the notion that G0/G1 arrest due to changes in p57 and cyclin D1 expression may confer drug resistance, while EMT seems non-essential to 5-FU resistance in AGS gastric carcinoma cells.

Epstein-Barr Virus miR-BART17-5p Promotes Migration and Anchorage-Independent Growth by Targeting Kruppel-Like Factor 2 in Gastric Cancer.

Epstein-Barr virus (EBV) infects more than 90% of the global population and is associated with a variety of tumors including nasopharyngeal carcinoma, Hodgkin lymphoma, natural killer/T lymphoma, and gastric carcinoma. In EBV-associated gastric cancer (EBVaGC), highly expressed EBV BamHI A rightward transcripts (BART) miRNAs may contribute to tumorigenesis with limited viral antigens. Despite previous studies on the targets of BART miRNAs, the functions of all 44 BART miRNAs have not been fully clarified. Here, we used RNA sequencing data from the Cancer Genome Atlas to find genes with decreased expression in EBVaGC. Furthermore, we used AGS cells infected with EBV to determine whether expression was reduced by BART miRNA. We showed that the expression of Kruppel-like factor 2 (KLF2) is lower in AGS-EBV cells than in the AGS control. Using bioinformatics analysis, four BART miRNAs were selected to check whether they suppress KLF2 expression. We found that only miR-BART17-5p directly down-regulated KLF2 and promoted gastric carcinoma cell migration and anchorage-independent growth. Our data suggest that KLF2 functions as a tumor suppressor in EBVaGC and that miR-BART17-5p may be a valuable target for effective EBVaGC treatment.

Epstein-Barr virus miR-BART1-3p suppresses apoptosis and promotes migration of gastric carcinoma cells by targeting DAB2.

Although Epstein-Barr virus (EBV) is known to encode over 40 different miRNAs of its own, the roles of most EBV miRNAs remain unknown. Disabled homolog 2 (DAB2) is a putative tumor suppressor, but its role in gastric carcinoma (GC), especially in EBV-associated GC, needs to be clarified. Our qRT-PCR and mRNA microarray results showed that DAB2 expression was down-regulated in EBV-positive GC cells compared to EBV-negative cells. Four BART miRNAs that might target DAB2 were predicted, and we found, using a luciferase reporter assay, that miR-BART1-3p directly targeted the 3'-UTR of DAB2. The miR-BART1-3p transfection decreased DAB2 expression at both mRNA and protein levels, while transfection of an inhibitor of miR-BART1-3p, miR-BART1-3p(i), increased DAB2 expression. In addition, miR-BART1-3p as well as siDAB2 increased migration and decreased apoptosis. Meanwhile, miR-BART1-3p(i) or pcDNA3.1-DAB2 transfection decreased migration and increased apoptosis in EBV-infected GC cells. Furthermore, decreased migration by miR-BART1-3p(i) was abrogated by co-transfected siDAB2, while decreased migration by miR-BART1-3p(i) was further suppressed by a co-transfected DAB2 over-expression vector. Our data suggest that miR-BART1-3p plays an important role in the tumorigenesis of EBV-associated GC by directly targeting DAB2.

Comprehensive Multi-Omics Analysis Reveals Aberrant Metabolism of Epstein-Barr-Virus-Associated Gastric Carcinoma.

The metabolic landscape of Epstein-Barr-virus-associated gastric cancer (EBVaGC) remains to be elucidated. In this study, we used transcriptomics, metabolomics, and lipidomics to comprehensively investigate aberrant metabolism in EBVaGC. Specifically, we conducted gene expression analyses using microarray-based data from gastric adenocarcinoma epithelial cell lines and tissue samples from patients with clinically advanced gastric carcinoma. We also conducted complementary metabolomics and lipidomics using various mass spectrometry platforms. We found a significant downregulation of genes related to metabolic pathways, especially the metabolism of amino acids, lipids, and carbohydrates. The effect of dysregulated metabolic genes was confirmed in a survival analysis of 3951 gastric cancer patients. We found 57 upregulated metabolites and 31 metabolites that were downregulated in EBVaGC compared with EBV-negative gastric cancer. Sixty-nine lipids, mainly ether-linked phospholipids and triacylglycerols, were downregulated, whereas 45 lipids, mainly phospholipids, were upregulated. In total, 15 metabolisms related to polar metabolites and 15 lipid-associated pathways were involved in alteration of metabolites by EBV in gastric cancer. In this work, we have described the metabolic landscape of EBVaGC at the multi-omics level. These findings could help elucidate the mechanism of EBVaGC oncogenesis.

EBV miR-BART10-3p Promotes Cell Proliferation and Migration by Targeting DKK1.

In Epstein-Barr virus (EBV)-infected epithelial cancers, BamHI A rightward transcript (BART) miRNAs are highly expressed. However, only a few target genes of BART miRNAs have been investigated. Our mRNA microarray data showed that DKK1 was markedly down-regulated in EBV-associated gastric carcinoma (EBVaGC) cells. Using luciferase reporter assay we tested whether miR-BART10-3p regulates DKK1 by directly targeting the 3'-UTR of DKK1 mRNA. We observed that miR-BART10-3p transfection decreased DKK1 expression, while an LNA inhibitor of miR-BART10-3p (LNA-miR-BART10-3p(i)) increased DKK1 expression. Furthermore, miR-BART10-3p and siDKK1 promoted cell proliferation and migration. In contrast, transfecting GC cells with LNA-miR-BART10-3p(i) or DKK1 over expression vector suppressed cell proliferation and migration. Our results suggest that miR-BART10-3p may be involved in the tumor progression of EBVaGC by targeting DKK1.

Regulation of proliferation and invasion by the IGF signalling pathway in Epstein-Barr virus-positive gastric cancer.

Several carcinomas including gastric cancer have been reported to contain Epstein-Barr virus (EBV) infection. EBV-associated gastric cancer (EBVaGC) is classified as one of four molecular subtypes of gastric cancer by The Cancer Genome Atlas (TCGA) group with increased immune-related signatures. Identification of EBV-dependent pathways with significant biological roles is needed for EBVaGC. To compare the biological changes between AGS gastric epithelial cells and EBV-infected AGS (AGS-EBV) cells, proliferation assay, CCK-8 assay, invasion assay, cell cycle analysis, RT-PCR, Western blot and ELISA were performed. BI836845, a humanized insulin-like growth factor (IGF) ligand-neutralizing antibody, was used for IGF-related signalling pathway inhibition. AGS-EBV cells showed slower proliferating rate and higher sensitivity to BI836845 compared to AGS cells. Moreover, invasiveness of AGS-EBV was increased than that of AGS, and BI836845 treatment significantly decreased the invasiveness of AGS-EBV. Although no apoptosis was detected, entry into the S phase of the cell cycle was delayed in BI836845-treated AGS-EBV cells. In conclusion, AGS-EBV cells seem to modulate their proliferation and invasion through the IGF signalling pathway. Inhibition of the IGF signalling pathway therefore could be a potential therapeutic strategy for EBVaGC.

Combinatorial Antitumor Activity of Oxaliplatin with Epigenetic Modifying Agents, 5-Aza-CdR and FK228, in Human Gastric Cancer Cells.

Epigenetic silencing is considered to be a major mechanism for loss of activity in tumor suppressors. Reversal of epigenetic silencing by using inhibitors of DNA methyltransferase (DNMT) or histone deacetylases (HDACs) such as 5-Aza-CdR and FK228 has shown to enhance cytotoxic activities of several anticancer agents. This study aims to assess the combinatorial effects of gene-silencing reversal agents (5-Aza-CdR and FK228) and oxaliplatin in gastric cancer cells, i.e., Epstein-Barr virus (EBV)-negative SNU-638 and EBV-positive SNU-719 cells. The doublet combinatorial treatment of 5-Aza-CdR and FK228 exhibited synergistic effects in both cell lines, and this was further corroborated by Zta expression induction in SNU-719 cells. Three drug combinations as 5-Aza-CdR/FK228 followed by oxaliplatin, however, resulted in antagonistic effects in both cell lines. Simultaneous treatment with FK228 and oxaliplatin induced synergistic and additive effects in SNU-638 and SNU-719 cells, respectively. Three drug combinations as 5-Aza-CdR prior to FK228/oxaliplatin, however, again resulted in antagonistic effects in both cell lines. This work demonstrated that efficacy of doublet synergistic combination using DNMT or HDACs inhibitors can be compromised by adding the third drug in pre- or post-treatment approach in gastric cancer cells. This implies that the development of clinical trial protocols for triplet combinations using gene-silencing reversal agents should be carefully evaluated in light of their potential antagonistic effects.

MiR-199a/b-5p Inhibits Lymphangiogenesis by Targeting Discoidin Domain Receptor 1 in Corneal Injury.

Discoidin domain receptor 1 (DDR1) is involved in tumorigenesis and angiogenesis. However, its role in lymphangiogenesis has been unknown. Here, we tested whether downregulation of DDR1 expression by miR-199a/b can suppress lymphangiogenesis. We also aimed to identify miRNA target site(s) in the 3' untranslated region (UTR) of DDR1. Transfection with miR-199a/b-5p mimics reduced expression of DDR1 and tube formation in primary human dermal lymphatic endothelial cells, whereas miR-199a/b-5p inhibitors showed the opposite effects. Critically, injection of miR-199a/b-5p mimics suppressed DDR1 expression and lymphangiogenesis in a corneal alkali-burn rat model. The three well-conserved seed matched sites for miR-199a/b-5p in the DDR1 3'-UTR were targeted, and miRNA binding to at least two sites was required for DDR1 inhibition. Our data suggest that DDR1 promotes enhanced lymphangiogenesis during eye injury, and miR-199a/b-5p suppresses this activity by inhibiting DDR1 expression. Thus, this miRNA may be useful for the treatment of lymphangiogenesis-related eye diseases.

High level of viral microRNA-BART20-5p expression is associated with worse survival of patients with Epstein-Barr virus-associated gastric cancer.

This study analyzed the relationship between several Epstein-Barr virus (EBV) microRNA (miRNA) expression profiles and the clinicopathologic features of patients with EBV-associated gastric cancer. The miRNA expression was examined in 59 tumor and 39 paired normal mucosal tissues from available formalin-fixed paraffin embedded tissue samples. The expression levels of miR-BamHI fragment A rightward transcript (BART)1-5p, miR-BART4-5p, and miR-BART20-5p were determined using a quantitative real-time polymerase chain reaction. The expression of all three analyzed EBV microRNAs was significantly higher in the tumor tissue than in the paired normal tissue (P < 0.001 for each). When the median value of the EBV microRNA expression levels was used as the cutoff point, a high BART20-5p expression was associated with worse recurrence-free survival (P = 0.034) in a multivariate analysis including age and pathologic stage. In conclusion, the expression level of BART20-5p may predict recurrence-free survival for patients with EBV-associated gastric cancer. Further studies are warranted to clarify the roles of EBV BART microRNAs in the carcinogenesis, and their potential as a biomarker and therapeutic target for EBV-associated gastric cancer.

TAX1BP1 downregulation by EBV-miR-BART15-3p enhances chemosensitivity of gastric cancer cells to 5-FU.

MicroRNAs (miRNAs) are a class of noncoding RNA molecules approximately 19 to 25 nucleotides in length that downregulate the expression of target genes at the post-transcriptional level by binding to the 3'-untranslated region (3'-UTR). Epstein-Barr virus (EBV) generates at least 44 miRNAs, but the functions of most of these miRNAs have not yet been identified. Previously, we reported BRUCE as a target of miR-BART15-3p, a miRNA produced by EBV, but our data suggested that there might be other apoptosis-associated target genes of miR-BART15-3p. Thus, in this study, we searched for new target genes of miR-BART15-3p using in silico analyses. We found a possible seed match site in the 3'-UTR of Tax1-binding protein 1 (TAX1BP1). The luciferase activity of a reporter vector including the 3'-UTR of TAX1BP1 was decreased by miR-BART15-3p. MiR-BART15-3p downregulated the expression of TAX1BP1 mRNA and protein in AGS cells, while an inhibitor against miR-BART15-3p upregulated the expression of TAX1BP1 mRNA and protein in AGS-EBV cells. Mir-BART15-3p modulated NF-κB activity in gastric cancer cell lines. Moreover, miR-BART15-3p strongly promoted chemosensitivity to 5-fluorouracil (5-FU). Our results suggest that miR-BART15-3p targets the anti-apoptotic TAX1BP1 gene in cancer cells, causing increased apoptosis and chemosensitivity to 5-FU.

Epstein-Barr Virus MicroRNA miR-BART20-5p Suppresses Lytic Induction by Inhibiting BAD-Mediated caspase-3-Dependent Apoptosis.

UNLABELLED: Epstein-Barr virus (EBV) is a human gammaherpesvirus associated with a variety of tumor types. EBV can establish latency or undergo lytic replication in host cells. In general, EBV remains latent in tumors and expresses a limited repertoire of latent proteins to avoid host immune surveillance. When the lytic cycle is triggered by some as-yet-unknown form of stimulation, lytic gene expression and progeny virus production commence. Thus far, the exact mechanism of EBV latency maintenance and the in vivo triggering signal for lytic induction have yet to be elucidated. Previously, we have shown that the EBV microRNA miR-BART20-5p directly targets the immediate early genes BRLF1 and BZLF1 as well as Bcl-2-associated death promoter (BAD) in EBV-associated gastric carcinoma. In this study, we found that both mRNA and protein levels of BRLF1 and BZLF1 were suppressed in cells following BAD knockdown and increased after BAD overexpression. Progeny virus production was also downregulated by specific knockdown of BAD. Our results demonstrated that caspase-3-dependent apoptosis is a prerequisite for BAD-mediated EBV lytic cycle induction. Therefore, our data suggest that miR-BART20-5p plays an important role in latency maintenance and tumor persistence of EBV-associated gastric carcinoma by inhibiting BAD-mediated caspase-3-dependent apoptosis, which would trigger immediate early gene expression. IMPORTANCE: EBV has an ability to remain latent in host cells, including EBV-associated tumor cells hiding from immune surveillance. However, the exact molecular mechanisms of EBV latency maintenance remain poorly understood. Here, we demonstrated that miR-BART20-5p inhibited the expression of EBV immediate early genes indirectly, by suppressing BAD-induced caspase-3-dependent apoptosis, in addition to directly, as we previously reported. Our study suggests that EBV-associated tumor cells might endure apoptotic stress to some extent and remain latent with the aid of miR-BART20-5p. Blocking the expression or function of BART20-5p may expedite EBV-associated tumor cell death via immune attack and apoptosis.

Inhibition of Lymphangiogenesis and Hemangiogenesis in Corneal Inflammation by Subconjunctival Prox1 siRNA Injection in Rats.

PURPOSE: Prospero homeobox 1 (Prox1) siRNA is a small interfering RNA that is designed to specifically bind Prox1 mRNA. We determined whether Prox1 siRNA inhibits lymphangiogenesis and hemangiogenesis after acute corneal inflammation. METHODS: Three Prox1 siRNAs were synthesized and investigated for their effects on Prox1 mRNA expression and tube formation in human dermal lymphatic endothelial cells (HDLECs) in vitro. The in vivo effects of Prox1 siRNA were assessed in alkali burn-induced inflammatory corneal neovascularization in rats. Prox1 siRNA was administered via subconjunctival injection. Corneal flat mounts were stained for lymphatic vessel endothelial hyaluronan receptor (LYVE)-1 to show lymphatic vessels. Lymphangiogenesis and hemangiogenesis were analyzed morphometrically using Image J software. Corneal inflammatory cell infiltration was evaluated by immunostaining for F4/80 and CD45. Protein levels of LYVE-1, podoplanin, VEGF receptor 2 (VEGFR2), and VEGFR3 were analyzed by Western blotting. RESULTS: Prox1 siRNA treatment decreased Prox1 mRNA expression and tube formation in cultured HDLECs. Subconjunctival injection of Prox1 siRNA significantly inhibited alkali burn-induced lymphangiogenesis and hemangiogenesis in the cornea compared with those of scrambled siRNA (negative control). This inhibition was comparable to that induced by bevacizumab (positive control). Prospero homeobox 1 knockdown by Prox1 siRNA also inhibited macrophage and leukocyte infiltration into the cornea. Prox1 siRNA downregulated the expression of all four proteins. CONCLUSIONS: Prox1 siRNA is a strong inhibitor of inflammatory corneal lymphangiogenesis and hemangiogenesis in vivo. Prox1 siRNA may be useful in preventing immune rejection after penetrating keratoplasty by suppressing lymphangiogenesis.