乙型肝炎病毒X蛋白对Polo样激酶1表达的调控作用  

Hepatitis B virus X protein-regulated expression of Plkl

作　　者：屠静[1] 张婷[1] 程锦[1] 曾浈浈 鲁凤民[1] 陈香梅[1] 

机构地区：[1]北京大学医学部基础医学院病原生物学系,100191

出　　处：《中华肝脏病杂志》2016年第1期46-50,共5页Chinese Journal of Hepatology

基　　金：国家自然科学基金（30771099、81372603）

摘　　要：目的探索乙型肝炎病毒x蛋白（HBx）对Polo样激酶1（Plkl）的表达调控作用。方法pCMV-HA—HBx表达质粒瞬时或稳定转染HepG2细胞，Westernblot检测HBx对Plkl蛋白水平的影响；采用荧光素酶活性实验检测HBx对Plkl基因启动子活性的影响，实时荧光定量PCR检测HBx对PlklmRNA水平的影响；采用Cycloheximide阻断新蛋白合成后检测HBx对Plkl蛋白半衰期的影响；流式细胞仪技术检测HBx对细胞周期的影响。组间数据比较采用t检验。结果Westernb10t结果显示，瞬时和稳定表达外源HBx蛋白都能显著上调S期HepG2细胞的Plkl蛋白相对水平（1．242±0．133与2．683±0．396，P〈0．05；1．340±0．128与3．683±0．349，P〈0．01）。荧光素酶活性和实时荧光定量PCR实验证实，HBx对Plkl基因的转录水平无影响，而Western1910t实验证实HBx能抑制S期P1R1蛋白的泛素化降解，使Plkl蛋白半衰期由30min延长至90min。与对照组相比，HBx过表达能促进S期和G：／M期细胞比例的增加（分别为31．65％与24．56％，9．43％与4．47％），G0／G1期细胞比例的减少（58．92％与70．97％）。结论HBx能通过抑制S期Plkl蛋白的降解进而上调Plkl蛋白的水平。Objective To investigate the ability and underlying mechanism of hepatitis B virus X protein （HBx） regulation of Polo-like kinase 1 （Plkl） expression. Methods The human HCC cell line HepG2 was transfected （transiently and stably） with an HBx plasmid expression vector （pCMV-HA-HBx） or empty plasmid vector （cona-ol）, with and without expression plasmids with the Plkl promoter. Effects on Plkl expression were assessed by western blotting. Functional effects on the Plkl promoter were assessed by luciferase reporter assay. Effects on the mRNA level of Plkl in S phase HepG2 cells were assessed by quantitative real-time reverse transcriptase polymerase chain reaction. After blocking protein synthesis by trealment with cycloheximide （CHX）, the turnover rate of Plkl was assessed by western blotting. Lastly, the effect of HBx on cell cycle was assessed by flow cytomelry. Results I-IBx did not increase the protein expression of Plkl in non-synchronized HepG2 cells, but did significantly up-regulate the Plkl protein level in the synchronized S phase cells （P = 0.026 andP= 0.003, respectively）. Ectopic expression of HBx did not increase the mRNA level of Plkl in HepG2 cells, but did inhibit the degradation of Plkl, as evidenced by an increased half-life of Plkl protein （from 30 to 90 minutes）. The HBx-expressing HepG2 ceils showed more frequent enlry into the S or Gz/IVl phase than the conlrol cells （31.65% vs. 24.56% or 9.43% vs. 4.47%, respectively） and less in the G0/G1 phase （decrease from 70.97% to58.92% for the HBx-expressmg HepG2 cells）. Conclusion HBx is able to up-regulate the expression of Plkl in HepG2 cells by a mechanism involving stabilization of the Plk 1 protein primarily in the S phase of the cell cycl.

关 键 词：肝炎病毒 乙型 X蛋白 POLO样激酶1 蛋白稳定性 表达 

分 类 号：R373.21[医药卫生—病原生物学]