CAJ | 학술논문

目的：研究BARF1表达下调对EBV阳性胃癌细胞凋亡的影响，以及BARF1基因沉默介导细胞凋亡的分子机制。方法：siRNA和NCsiRNA分别转染NUGC3和SNU719细胞，运用Western blot测定细胞中BARF1、Bcl－2、Bax、细胞色素C、caspase 3和caspase 9的蛋白表达；RT－PCR测定BARF1、Bcl－2和Bax mRNA的表达；台盼蓝染色法测定细胞存活率；Annexin V－FITC／PI染色法和流式细胞仪测定细胞凋亡；细胞凋亡因子抗体芯片分析细胞中凋亡相关蛋白的表达；线粒体膜电位检测试剂盒测定线粒体膜电位；免疫共沉淀检测细胞中Apaf－1和caspase 9的相互作用。结果：与空白对照组和阴性对照组相比， BARF1基因沉默显著诱导NUGC3和SNU719细胞凋亡，而线粒体膜电位显著降低。 BARF1沉默基因能促进促凋亡蛋白的表达并抑制抗凋亡蛋白的表达，Bcl－2／Bax比例显著降低；而caspase抑制剂能抑制由 BARF1基因沉默介导的细胞凋亡。在 siRNA 转染的细胞中， caspase 3和caspase 9蛋白发生裂解，细胞色素C的浓度显著高于阴性对照组，Apaf－1蛋白与caspase 9蛋白在细胞质中能够发生相互作用。结论：BARF1基因沉默通过线粒体途径调节Bcl－2和Bax蛋白的表达进而诱导NUGC3和SNU719细胞凋亡，并呈caspase通路依赖关系。
목적：연구BARF1표체하조대EBV양성위암세포조망적영향，이급BARF1기인침묵개도세포조망적분자궤제。방법：siRNA화NCsiRNA분별전염NUGC3화SNU719세포，운용Western blot측정세포중BARF1、Bcl－2、Bax、세포색소C、caspase 3화caspase 9적단백표체；RT－PCR측정BARF1、Bcl－2화Bax mRNA적표체；태반람염색법측정세포존활솔；Annexin V－FITC／PI염색법화류식세포의측정세포조망；세포조망인자항체심편분석세포중조망상관단백적표체；선립체막전위검측시제합측정선립체막전위；면역공침정검측세포중Apaf－1화caspase 9적상호작용。결과：여공백대조조화음성대조조상비， BARF1기인침묵현저유도NUGC3화SNU719세포조망，이선립체막전위현저강저。 BARF1침묵기인능촉진촉조망단백적표체병억제항조망단백적표체，Bcl－2／Bax비례현저강저；이caspase억제제능억제유 BARF1기인침묵개도적세포조망。재 siRNA 전염적세포중， caspase 3화caspase 9단백발생렬해，세포색소C적농도현저고우음성대조조，Apaf－1단백여caspase 9단백재세포질중능구발생상호작용。결론：BARF1기인침묵통과선립체도경조절Bcl－2화Bax단백적표체진이유도NUGC3화SNU719세포조망，병정caspase통로의뢰관계。
[ ABSTRACT] AIM:To investigate the effects of BARF1 down-regulation on EBV-positive gastric carcinoma cell apoptosis, and the molecular mechanisms by BARF1 silencing-mediated apoptosis.METHODS: After NUGC3 and SNU719 cells were transfected with NCsiRNA and siRNA, respectively, the protein levels of BARF1, Bcl-2, Bax, cyto-chrome C, caspase 3 and capase 9 were detected by Western blot, and the mRNA expression of BARF1, Bcl-2 and Bax was determined by RT-PCR.The cell viability was measured by the method of Trypan blue exclusion and the cell apoptosis was analyzed by flow cytometry analysis with Annexin V-FITC/PI staining.The expression of the apoptosis-related proteins in the cells transfected with siRNA and NCsiRNA was examined by human apoptosis antibody arrays.Mitochondrial mem-brane potential was determined by flow cytometry.The interaction between Apaf-1 and caspase 9 was confirmed by immuno-precipitation.RESULTS: Compared with untreated and NCsiRNA groups, BARF1 gene silencing significantly inhibited the cell viability, induced apoptosis, and reduced the mitochondrial membrane potential in the NUGC3 and SNU719 cells transfected with siRNA.BARF1 gene silencing up-regulated the expression of pro-apoptotic proteins and down-regulated the expression of anti-apoptotic proteins, and the Bcl-2/Bax ratio was significantly decreased.In BARF1 gene silencing cells, the caspase inhibitor z-VAD-fmk inhibited BARF1 silencing-mediated apoptosis, and significantly increased the levels of cleaved caspase 3 and caspase 9.The concentration of cytochrome C significantly increased as compared with NCsiRNA group, and Apaf-1 interacted with caspase 9 in the cytoplasm.CONCLUSION:BARF1 silencing induces apoptosis via the mitochondrial pathway through regulating the expression of Bcl-2 and Bax proteins in a caspase-dependent manner in the NUGC3 and SNU719 cells.