人巨细胞病毒UL138基因表达对胃癌细胞免疫相关功能的影响

The effects of human cytomegalovirus cancer suppressor UL138 gene expression on the immune-associated functions of gastric cancer cells

目的:探讨人巨细胞病毒UL138基因表达对胃癌细胞免疫相关功能的影响。方法:构建UL138基因真核表达重组质粒,转染BGC-823胃癌细胞,经Westernblot及免疫荧光验证UL138表达,通过基因芯片结合生物信息学技术分析UL138表达对胃癌细胞功能的影响,荧光定量PCR进一步验证胃癌细胞中免疫相关分子的表达,Westernblot分析免疫原性死亡相关蛋白的变化。结果:Westernblot及免疫荧光证实重组质粒转染后胃癌细胞表达UL138;基因芯片差异基因分析显示UL138的表达上调了370个基因表达,下调了206个基因的表达;生物信息学分析提示这些差异基因的表达参与了胃癌细胞骨架重构、细胞黏附等并与消化系统疾病相关。荧光定量PCR分析显示UL138的表达上调了IL1A、IL6、IL11、IL8、IL1RL1、IL7R、IL13RA2、CCL3L1、CCL5、TNFAIP3、TNFRSF21、VEGFA、CD44、CD55、CD59、CD27416种炎症相关细胞因子基因表达并下调了CXCL10、IFNAR1基因的表达。此外,Westernblot结果显示UL138的表达上调了免疫原性死亡相关蛋白ERp57的表达(P<0.01),不影响HSP70的表达(P>0.05)。结论:UL138基因的表达能促进胃癌细胞促炎相关细胞因子表达并诱导免疫原性细胞死亡的特征。

Objective： To investigate the effects of HCMV tumor suppressor gene UL138 gene expression on immune-related function in gastric cancer cell. Methods： The recombinant eukaryotic expression plasmid of UL138 gene was constructed and transfected into BGC-823 gastric cancer cells. The expression of UL138 and immunogenic death related protein were verified by Western blot and immunofluorescence. The effect of UL138 expression on gastric cancer cells was analyzed by gene chip, combined with GO, Pathway and other bioinformatics techniques. Fluorescence quantitative PCR was used to further verify the expression of immune related molecules in gastric cancer cells. Results： After being transfected with recombinant plasmid, the expression of UL138 in gastric cancer cells was confirmed by Western blot and immunofluorescence; in addition, Western blot analysis showed that the expression of UL138 up-regulated the expression of ERp57, an immunogenic death associated protein, but did not affect the expression of HSP70. Microarray analysis showed that the expression of UL138 up-regulated 370 genes and down regulated 206 genes. Bioinformatics analysis suggested that these differentially expressed genes were involved in cytoskeletal remodeling, cell adhesion and other pathways involved in gastric cancer and digestive system diseases. Quantitative PCR analysis showed that the expression of UL138 up-regulated the 16 inflammatory related cytokines expression of IL1A, IL6, IL11, IL8, IL1 RL1, IL7 R, IL13RA2, CCL3L1, CCL5, TNFAIP3, TNFRSF21, VEGFA, CD44, CD55, CD59, CD274 gene and down-regulated CXCL10, IFNAR1 gene. Conclusion： The expression of UL138 was involved in the process of apoptosis of gastric cancer cells, up-regulated expression of IL1 A, IL6, IL8 gene of pro-inflammatory cytokines and immune related immunogenicity of death associated protein ERp57.