HCMV IE86对恶性胶质瘤细胞凋亡及p53表达活性的影响
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摘要
人巨细胞病毒(human cytomegalovirus,HCMV)能诱导肿瘤细胞恶性转化且抑制肿瘤细胞凋亡,但HCMV编码的主要即刻早期调控蛋白IE86在这一过程中是否发挥关键作用仍然未知。为探究IE86对基因修饰荷胶质瘤小鼠p53表达水平及恶性胶质瘤细胞凋亡情况的影响,本研究通过PCR技术鉴定基因修饰小鼠IE86表达情况;实时定量PCR技术检测IE86和p53mRNA表达水平变化;免疫组织化学方法检测p53和p21蛋白的表达水平;TUNEL检测肿瘤组织细胞凋亡情况。结果显示,成功构建了IE86基因修饰小鼠模型;与IE86阴性组相比IE86阳性组p53表达水平上升(P<0.05),但p21表达水平下降(P<0.05);IE86阳性组细胞抗凋亡能力增强(P<0.05)。以上结果表明,在基因修饰的小鼠中IE86持续表达但p53转录活性的指示标志p21下调,且IE86可提高恶性胶质瘤细胞抗凋亡能力。
Human cytomegalovirus(HCMV) can induce malignant transformation of tumor cells and inhibit tumor cell apoptosis, however, whether the key immediate regulatory protein IE86 encoded by HCMV plays a key role in this process remains unknown. The purpose of this study was to study the effect of IE86 on genetically modified glioma mice p53 expression as well as on the apoptosis in glioblastoma. The expression of IE86 in genetically modified mice was identified by PCR. The expression of IE86 and p53 mRNA was detected by real-time quantitative PCR. The expression of p53 and p21 protein was detected by Western blot and immunohistochemistry. The apoptosis of tumor tissues was detected by TUNEL. The results showed that the IE86 gene-modified mouse model was successfully constructed. As compared with IE86-negative group, the expression level of IE86-positive group p53 increased(P<0.05), however, the expression of p21 decreased(P<0.05). The anti-apoptosis ability of IE86-positive group cells enhanced(P<0.05). The above results indicated that IE86 is continuously expressed in genetically modified mice, however, the indicator p21 of p53 transcriptional activity down-regulated, and IE86 could increase anti-apoptosis ability of glioblastoma cells.
Human cytomegalovirus(HCMV) can induce malignant transformation of tumor cells and inhibit tumor cell apoptosis, however, whether the key immediate regulatory protein IE86 encoded by HCMV plays a key role in this process remains unknown. The purpose of this study was to study the effect of IE86 on genetically modified glioma mice p53 expression as well as on the apoptosis in glioblastoma. The expression of IE86 in genetically modified mice was identified by PCR. The expression of IE86 and p53 mRNA was detected by real-time quantitative PCR. The expression of p53 and p21 protein was detected by Western blot and immunohistochemistry. The apoptosis of tumor tissues was detected by TUNEL. The results showed that the IE86 gene-modified mouse model was successfully constructed. As compared with IE86-negative group, the expression level of IE86-positive group p53 increased(P<0.05), however, the expression of p21 decreased(P<0.05). The anti-apoptosis ability of IE86-positive group cells enhanced(P<0.05). The above results indicated that IE86 is continuously expressed in genetically modified mice, however, the indicator p21 of p53 transcriptional activity down-regulated, and IE86 could increase anti-apoptosis ability of glioblastoma cells.
引文
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[3] Liao X H,Dong X,Wu C,et al.Human cytomegalovirus immediate early protein 2 enhances myocardin-mediated survival of rat aortic smooth muscle cells[J].Virus Res,2014,192:85-91.
[4] Price RL,Chiocca EA.Modeling cytomegalovirus infection in mouse tumor models[J].Front Oncol,2015,5:61.
[5] Ranjan A,Iwakuma T.Emerging Non-Canonical Functions and Regulation of p53[J].Int J Mol Sci,2018,19(待发表).
[6] Martinez J,Georgoff I,Martinez J,et al.Cellular localization and cell cycle regulation by a temperature-sensitive p53 protein[J].Genes Dev,1991,5(2):151-159.
[7] Hannemann H,Rosenke K,O′Dowd JM,et al.The presence of p53 influences the expression of multiple human cytomegalovirus genes at early times postinfection[J].J Virol,2009,83(9):4316-4325.
[8] Ni Z,Wang X,Zhang T,et al.Comprehensive analysis of differential expression profiles reveals potential biomarkers associated with the cell cycle and regulated by p53 in human small cell lung cancer[J].Exp Ther Med,2018,15(4):3273-3282.
[9] Riley T,Sontag E,Chen,P,et al.Transcriptional control of human p53-regulated genes[J].Nat Rev Mol Cell Biol,2008,9(5):402-412.
[10] Shi B,Sharifi HJ,DiGrigoli S,et al.Inhibition of HIV early replication by the p53 and its downstream gene p21[J].J Virol,2018,15(1):53.
[11] Wang X,Hu M,Xing,F,et al.Human cytomegalovirus infection promotes the stemness of U251 glioma cells[J].J Virol,2017,89(5):878-886.
[12] Harris SM,Bullock B,Westgard E,et al.Functional properties of the human cytomegalovirus IE86 protein required for transcriptional regulation and virus replication[J].J Virol,2010,84(17):8839-8848.
[13] Hsu CH,Chang MDT,Tai KY,et al.HCMV IE2-mediated inhibition of HAT activity downregulates p53 function[J].Embo Journal,2004,23(11):2269-2280.
[14] Kwon Y,Kim MN,Young Choi E,et al.Inhibition of p53 transcriptional activity by human cytomegalovirus UL44[J].Microbiol Immunol,2012,56(5):324-331.
[15] Zhang Q,Shim K,Wright K,et al.Atypical role of sprouty in p21 dependent inhibition of cell proliferation in colorectal cancer[J].Mol Carcinog,2016,55(9):1355-1368.
[16] Yeou Ping Tsao,Long Yuan Li,Tzung Chieh Tsai,et al.Human Papillomavirus Type 11 and 16 E5 Represses p21WafI/SdiI/CipI Gene Expression in Fibroblasts and Keratinocytes[J].Journal of Virology,1996,70(11):7535-7539.