摘要
目的探讨爱泼斯坦-巴尔病毒(Epstein-Barr virus,EBV)操控鼻咽癌细胞HONE-1的miR-590-5p以实现逃避免疫的机制研究.方法通过miRDB在线分析miR-590-5p与泛素E3连接酶(itchy E3 ubiq-uitin protein ligase,ITCH)的匹配情况,然后通过荧光素酶报告系统检测miR-590-5p是否靶向ITCH;使用miR-590-5p mimics过表达或者miR-590-5p inhibitor敲低miR-590-5p的情况下,通过免疫印迹检测ITCH与其底物MAVS的表达量,通过酶联免疫吸附试验检测鼻咽癌细胞HONE-1上清液中干扰素γ(interferon-γ,IFNγ)的表达量,同时检测EBV的复制情况.结果EBV感染鼻咽癌细胞HONE-1后miR-590-5p的表达量显著下降(P<0.05),并且miR-590-5p靶向ITCH的3′UTR.过表达miR-590-5p后,发现鼻咽癌细胞HONE-1的ITCH表达量降低,线粒体抗病毒信号蛋白(mitochondrial antiviral signaling protein,MAVS)泛素化水平下降,MAVS的表达量升高,IFNγ表达升高(P<0.05),EBV复制水平下降(P<0.05).敲低miR-590-5p后,发现鼻咽癌细胞HONE-1的ITCH表达量上升,MAVS泛素化水平上升,MAVS的表达量下降,IFNγ表达下降(P<0.05),EBV复制水平上升(P<0.05).过表达miR-590-5p的同时敲低MAVS或敲低miR-590-5p的同时过表达MAVS后,EBV病毒的复制没有明显差异(P>0.05).结论EBV通过抑制miR-590-5p的表达量以提高MAVS的泛素E3连接酶ITCH的表达量,降低鼻咽癌细胞HONE-1的IFNγ表达量,最终实现自身复制的增强.
Objective To investigate the mechanism by which Epstein-Barr virus ( EBV) controls miR-590-5p of nasopharyngeal carcinoma cell line HONE-1 to achieve immune eva-sion.Methods miRDB was used to analyze the matching of miR-590-5p with itchy E3 ubiquitin protein ligase (ITCH) online, and then detect whether miR-590-5p targeted ITCH through lucifer-ase reporter system.With miR-590-5p mimics overexpression or knockdown of miR-590-5p by miR-590-5p inhibitor, the expression of ITCH and its substrate MAVS was detected by immunoblotting, and the expression of interferon-γ ( IFNγ) in the supernatant of nasopharyngeal carcinoma cell line HONE-1 was detected by enzyme-linked immunosorbent assay.And EBV replication was detec-ted.Results The expression of miR-590-5p was significantly decreased after EBV infection of naso-pharyngeal carcinoma cell line HONE-1 ( P<0.05) , and miR-590-5p was targeted to the 3'UTR of ITCH.After overexpression of miR-590-5p, it was found that the expression of ITCH in HYE-1 was decreased, the level of ubiquitination of mitochondrial antiviral signaling protein ( MAVS) was de-creased, the expression of MAVS was increased, the expression of IFNγ was increased ( P <0.05) , and the level of EBV replication was decreased ( P<0.05) .After knocking down miR- 590-5p, it was found that the expression of ITCH in HYE-1 of nasopharyngeal carcinoma cells in-creased, the level of ubiquitination of MAVS was increased, the expression of MAVS decreased, the expression of IFNγ decreased ( P<0.05) , and the level of EBV replication increased ( P<0.05) .There was no significant difference in EBV virus replication after miR-590-5p over-expres-sion and MAVS over-expression or miR-590-5p over-expression ( P >0.05 ) .Conclusion EBV can increase the expression level of ubiquitin E3 ligase ITCH of MAVS by inhibiting the expression of miR-590-5p, and reduce the expression of IFNγ in nasopharyngeal carcinoma cell line HONE-1, and finally achieve the enhancement of self-replication.
作者
张昌明
胡鹏刚
ZHANG Changming;HU Penggang(Otolaryngology and Neck Surgery,Xijing Hospital,Air Force Medical University,Xi'an,710032,China)
出处
《医学分子生物学杂志》
CAS
2020年第1期63-68,共6页
Journal of Medical Molecular Biology
