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J Virol 78:1697C1705

J Virol 78:1697C1705. membrane protein 2A (LMP2A) encoded by EBV plays a key role in ensuring the incubation period of EBV. Glycosylation modification is an important marker of cancer cells, and recent studies have reported that it is related to EBV. Our conclusions provide deeper theoretical support for the role of LMP2A and transforming growth factor (TGF)/Smad-mTORC1-GCNT3 in EBV-associated gastric cancer (EBVaGC) and help us to understand glycosylation abnormalities in cancer. Our results may provide novel therapeutic targets for the treatment of gastric cancer against the TGF/Smad-mTORC1-GCNT3 signaling cascade. for siGCNT3-1, for siGCNT3-2, for siLMP2A, for siRaptor, for simTOR, for sieIF4E, for siSmad2, for siSmad3, for siSmad4, and for control siRNA. Cells were transfected with the RNA oligoribonucleotides using Lipofectamine 2000 reagent (Invitrogen, Thermo Fisher Scientific, Germany) for 48 h at 50?nM per the manufacturers recommendations. Smad2, Smad3, and Smad4 plasmids were purchased from Genechem (China). LMP2A was cloned into the pcDNA3.1-EF1a-mcs-3flag-CMV-EGFP vector. Three micrograms of LMP2A (pcDNA3.1-LMP2A) or a negative-control vector (pcDNA3.1-vector) plasmid was transfected into SGC7901 cells using Lipofectamine 2000. Cells were harvested for assay at the indicated times. Stable transfection of LMP2A was selected by G418 and observed under a fluorescence microscope. Immunoprecipitation. Protein complexes were precipitated from whole-cell lysates with anti-eIF4E (Santa Cruz Biotechnology), anti-4EBP1 (Santa Cruz Biotechnology), anti-GCNT3 (Santa Cruz Biotechnology), anti-Smad2/3 (Santa Cruz Biotechnology), or anti-Smad4 antibodies (Cell Signaling Technology) and then precipitated with protein A/G PLUS-Agarose (Santa Cruz Biotechnology). Following the instructions, SDS-PAGE and Western blotting were performed after the sample was boiled. Chromatin immunoprecipitation. A chromatin immunoprecipitation (ChIP) kit was purchased from Cell Signaling Technology, and ChIP was performed according to the manufacturers recommendations. The antibodies used were normal rabbit IgG against Smad2/3 and Smad4. The primer pairs used for the GCNT3 promoter region were as follows: 5-test ( em P? ? /em 0.05 was considered significant; ns, nonsignificant; *, em P /em ? ?0.05; **, em P /em ? ?0.01). Statistical analyses were conducted using SPSS 19.0 statistical software (Chicago, IL). ACKNOWLEDGMENTS This research was supported by the National Natural Science Foundation of China (NSFC 81571995). J. Liu and B.L. conceived and designed the study. J. Liu, C.Y., and J. Li performed the experiments. J. Liu drafted the manuscript. J. Liu, Y.Z., and W.L. analyzed the experimental data. We declare no competing financial interests. REFERENCES 1. Young LS, Rickinson LPA receptor 1 antibody AB. 2004. Epstein-Barr virus: 40 years on. Nat Rev Cancer 4:757C768. 10.1038/nrc1452. [PubMed] [CrossRef] [Google Scholar] 2. Delecluse HJ, Feederle R, OSullivan B, Taniere P. 2007. Epstein Barr virus-associated tumours: an update for the attention of the working pathologist. J Clin Pathol 60:1358C1364. 10.1136/jcp.2006.044586. [PMC free article] [PubMed] [CrossRef] [Google Scholar] 3. Tempera I, Klichinsky M, Lieberman PM. 2011. EBV latency types adopt alternative chromatin conformations. PLoS Pathog 7:e1002180. 10.1371/journal.ppat.1002180. [PMC free article] [PubMed] [CrossRef] [Google Pyrantel tartrate Scholar] 4. Fukayama M, Hayashi Y, Iwasaki Y, Chong J, Ooba T, Takizawa T, Koike M, Mizutani S, Miyaki M, Hirai K. 1994. Epstein-Barr virus-associated gastric carcinoma and Epstein-Barr virus infection of the stomach. Lab Invest 71:73C81. [PubMed] [Google Scholar] 5. Shibata D, Weiss LM. 1992. Epstein-Barr virus-associated gastric adenocarcinoma. Am J Pathol 140:769C774. [PMC free article] [PubMed] [Google Scholar] 6. Sugiura M, Imai Pyrantel tartrate S, Tokunaga M, Koizumi S, Uchizawa M, Okamoto K, Osato T. 1996. Transcriptional analysis of Epstein-Barr virus gene expression in EBV-positive gastric carcinoma: unique Pyrantel tartrate viral latency in the tumour cells. Br J Cancer 74:625C631. 10.1038/bjc.1996.412. [PMC free article] [PubMed] [CrossRef] [Google Scholar] 7. Scholle F, Bendt KM, Raab-Traub N. 2000. Epstein-Barr virus LMP2A transforms epithelial cells, inhibits cell differentiation, and activates Akt. J Virol 74:10681C10689. 10.1128/jvi.74.22.10681-10689.2000. [PMC free article] [PubMed] [CrossRef] [Google Scholar] 8. Yeh JC, Ong E, Fukuda M. 1999. Molecular cloning and expression of a novel beta-1,6-N-acetylglucosaminyltransferase that forms core 2, core 4, and I branches..