EBV抑制鼻咽癌细胞系中MAP9表达的机制  

The mechanism of Epstein-Barr virus inhibiting the expression of microtubule-associated protein 9 in nasopharyngeal carcinoma cell lines

作　　者：赵梓秀 赵燕 师朵 刘雯[1] ZHAO Zixiu;ZHAO Yan;SHI Duo;LIU Wen(Department of Pathogenic Biology,School of Basic Medicine,Qingdao University,Qingdao 266071,China)

机构地区：[1]青岛大学基础医学院病原生物学系,山东青岛266071 [2]淄博市中心医院医学检验科

出　　处：《青岛大学学报(医学版)》2025年第1期1-6,共6页Journal of Qingdao University(Medical Sciences)

基　　金：山东省自然科学基金面上项目(ZR2021MC-068)。

摘　　要：目的探讨鼻咽癌中EB病毒(EBV)对微管相关蛋白9(MAP9)基因表达的调控机制。方法应用Western blot和实时荧光定量PCR(qRT-PCR)方法,检测EBV阴性和EBV阳性鼻咽癌细胞系中MAP9的蛋白和mRNA表达;软件预测MAP9基因甲基化岛,采用甲基化特异性PCR(MSP)和亚硫酸氢盐基因组测序(BGS)技术检测MAP9启动子区甲基化情况;采用DNA甲基转移酶抑制剂5-氮杂-2′-脱氧胞苷(5-Aza-cdR)处理EBV阳性鼻咽癌细胞系C666-1,减少DNA的甲基化,检测经处理后细胞中MAP9基因表达情况;选取EBV阴性鼻咽癌细胞系HONE和CNE-1,建立稳定表达外源性EBV潜伏膜蛋白1(LMP1)的细胞模型,将其命名为HONE-LMP1和CNE-1-LMP1,使用Western blot法检测两种细胞相较于对照组细胞HONE-LMP1-NC和CNE-1-LMP1-NC的极光激酶A(Aurora A)和MAP9蛋白表达情况。结果EBV阳性鼻咽癌细胞系中MAP9的蛋白和mRNA表达水平显著低于EBV阴性鼻咽癌细胞系,差异均有统计学意义(t=11.63、7.76,P<0.05)。EBV阳性鼻咽癌细胞系中MAP9基因启动子甲基化程度高于EBV阴性鼻咽癌细胞系,而5-Aza-cdR处理后MAP9的表达水平并未显著升高(F=2.90,P>0.05)。与对照组比较,在EBV阴性鼻咽癌细胞系HONE和CNE-1中外源性稳定表达LMP1后,MAP9上游激酶Aurora A的表达明显上调(t=6.76、8.29,P<0.05),而MAP9蛋白表达则出现明显下调(t=4.76、7.21,P<0.05)。结论EBV编码的LMP1能够通过上调MAP9上游Aurora A的表达来抑制MAP9的表达,从而参与鼻咽癌的发生发展。Objective To investigate the regulatory effect of Epstein-Barr virus(EBV)on the expression of the microtubule-associated protein 9(MAP9)gene in nasopharyngeal carcinoma.Methods Western blot and qRT-PCR were used to measure the protein and mRNA expression levels of MAP9 in EBV-negative and EBV-positive nasopharyngeal carcinoma cell lines.Related software was used to predict the methylation island of the MAP9 gene,and methylation-specific PCR and bisulfite genome sequencing were used to detect the methylation of the MAP9 promoter region.The DNA methyltransferase inhibitor 5-azA-2′-deoxycytidine(5-Aza-cdR)was used for the treatment of the EBV-positive nasopharyngeal carcinoma cell line C666-1 to reduce DNA methylation,and MAP9 gene expression was measured after treatment.The EBV-negative nasopharyngeal carcinoma cell lines HONE and CNE-1 were selected to establish a cell model with the stable expression of exogenous EBV latent membrane protein 1(LMP1),which was named HONE-LMP1 and CNE-1-LMP1,respectively,and Western blot was used to measure the protein expression levels of Aurora kinase A(Aurora A)and MAP9 in these two types of cells in comparison with the control cells HONE-LMP1-NC and CNE-1-LMP1-NC.Results The protein and mRNA expression levels of MAP9 in EBV-positive nasopharyngeal carcinoma cell lines were significantly lower than those in EBV-negative nasopharyngeal carcinoma cell lines(t=11.63,7.76,P<0.05).The degree of MAP9 gene promoter methylation in EBV-positive nasopharyngeal carcinoma cell lines was higher than that in EBV-ne-gative nasopharyngeal carcinoma cell lines,while there was no significant increase in the expression level of MAP9 after 5-Aza-cdR treatment(F=2.90,P>0.05).Compared with the control group,after the stable expression of LMP1 in the EBV-negative nasopharyngeal carcinoma cell lines HONE and CNE-1,there was a significant increase in the expression of the MAP9 upstream kinase Aurora A(t=6.76,8.29,P<0.05)and a significant reduction in the protein expression of MAP9(t=4.76,7.21,P<0.05).C

关 键 词：疱疹病毒4型 人 微管相关蛋白质类 鼻咽癌 极光激酶A DNA甲基化 

分 类 号：R373.9[医药卫生—病原生物学]