靶向着丝粒蛋白A的小RNA干扰对肝癌细胞株HepG2细胞生物学行为的影响  

Effect of siRNA targeting centromere protein-A gene on biological behavior of HepG2 cells

作　　者：李咏梅[1] 祝峙[1] 陈颖[1] 罗志刚[1] 史敏[1] 朱明华[1] 

机构地区：[1]第二军医大学长海医院病理科,上海200433

出　　处：《中华病理学杂志》2008年第2期124-128,共5页Chinese Journal of Pathology

基　　金：国家自然科学基金资助项目（30070344,30070839）

摘　　要：目的研究靶向着丝粒蛋白A（CENP-A）的siRNA对肝癌细胞株HepG2细胞生物学行为的影响。方法设计并合成三对CENP-A编码基因的反向重复序列，中间间隔9个核苷酸序列，通过定向克隆至载体pSilencer^TM2．1-U6 neo，构建siRNA真核表达质粒，经稳定转染HepG2细胞后检测肝癌细胞中CENP-A基因mRNA及蛋白质表达的抑制情况；通过观察HepG2细胞生长、凋亡、细胞周期和平板克隆形成能力评价CENP-A基因干扰对细胞生物学行为的影响；通过检测bcl-2、Bax、p21^wafl、mdm2、p53蛋白表达水平初步探讨CENP-A生物学作用的可能机制。结果三对靶向CENP-A的siRNA干扰片段中有二对抑制效果明显。与未转染组及空载体组相比，转染CENP-A干扰片段的细胞生长减慢，平板克隆形成率下降。细胞周期检测显示，G1期阻滞（P〈0．01），S期细胞比例减少（P〈0．001）。细胞凋亡比例增加（P=0．003），并伴随bcl-2蛋白表达明显下降（P=0．000），Bax蛋白表达明显增高（P=0．001）。还可致p21^wafl表达增高，mdm2表达下降，但对野生型p53表达未显示明显影响。结论CENP-A通过参与细胞周期调控而密切相关于细胞恶性增殖和凋亡抑制，其机制可能涉及野生型p53非依赖性通路和凋亡相关基因bcl-2／Bax的表达异常。Objective To study the influence of siRNA inhibition of CENP-A expression on the biological behavior of HepG2 cells. Methods Three pairs of 21 bp reverse repeated motifs of CENP-A target sequence with 9 spacer were synthesized and inserted into vector pSilencerTM 2. 1-U6 neo to generate siRNA eukaryotic expression plasmids. After stable transfection into HepG2 cells, cell growth, apoptosis, cell cycles and plate clone forming efficiency were investigated. Expressions of CENP-A mRNA was monitored by the reverse transcriptase polymerase chain reaction （RT-PCR）. The protein expression of CENP-A, bel-2, Bax, p53, p21^wafl and mdm2 were detected by Western-blotting. Results Two eukaryotic expression plasmids with significant siRNA specific inhibition to the CENP-A gene were created. Compared with control cells, HepG2 cells transfeeted with the constructs showed G1 phase delay （ P 〈 0.01 ） and cell number decrease in the S phase （ P 〈 0. 001 ） , along with an increased apoptotic rate （ P = 0. 003 ） , significant increase of Bax expression and decreased bcl-2 expression （P≤ 0. 001 ）. The protein expressions of p21^wafl was higher and mdm2 was lower than those of the control groups. However, the wild type p53 protein expression was not effeeted by CENP-A siRNA. Conclusions An altered expression of CENP-A may be related to the proliferation of hepatocellular carcinoma through cell cycle regulation involving an altered bcl-2/Bax expression, that may be p53 independent.

关 键 词：肝肿瘤 RNA 小分子干扰 细胞凋亡 基因表达调控 

分 类 号：R686[医药卫生—骨科学]