慢病毒载体介导SHIP基因抑制K562细胞增殖及对PI3K/Akt信号通路的调控

Inhibitory effect of lentiviral vector-mediated SHIP gene transfection on proliferation of leukemia K562 cells and PI3K / Akt pathway regulation

背景与目的:SHIP基因主要表达于造血细胞,通过降解PIP3抑制PI3K/Akt信号通路而在造血细胞增殖和存活中起重要负调控作用。本研究通过慢病毒载体介导SHIP基因转染K562细胞,探讨SHIP基因改变及功能丢失与白血病发病的关系。方法:将携带SHIP基因的慢病毒感染K562细胞,FQ-PCR法检测SHIP转录水平,Westernblot法检测转染后SHIP蛋白表达及Akt磷酸化水平的变化;比较SHIP基因表达前后细胞增殖、形态的变化。结果:K562细胞中SHIP蛋白阴性。以携带SHIP基因的慢病毒载体转染K562细胞后,细胞增殖抑制率升高,K562-wtSHIP-FIV-G细胞的增殖抑制率由转染第3天的(9.9±1.5)%升到第5天的(40.6±2.3)%;伴有Akt磷酸化水平明显减弱;转染后,K562-wtSHIP-FIV-G组细胞p-Akt的表达水平由0.533降低到0.245(P<0.01),细胞增殖明显被抑制。此外,SHIP蛋白还能使K562细胞出现凋亡特征,Hoechst33342染色结果转染wtSHIP基因组K562细胞第5天早期凋亡率[(38.3±4.3)%]明显高于K562-FIV-G组细胞[(8.2±0.9)%]和未转染组K562细胞[(7.7±0.8)%]。结论:SHIP基因具有重要的抑制细胞增殖和促进凋亡的能力,SHIP基因缺失导致K562细胞PI3K/Akt信号途径失调,促进K562细胞增殖。

Background and Objective. The hemopoietic-restricted Src homology 2-containing inositol 5＇-phosphatase （SHIP） acts as a negative regulator for the proliferation and survival of hematopoietic cells by hydrolysing the phosphoinositide 3-kinase （PI3K）-generated second messenger, Ptdlns（3, 4,5）-P3 （Pl-3,4,5-P3） to Ptdlns（3,4）-P2 （Pt-3,4-P2）. This study was to investigate the biological function of SHIP gene in pathogenesis of leukemia cells by lentiviral vector-mediated SHIP transfection. Methods. Ectopic SHIP gene was transfected into leukemia K562 ceils by the mediation of lentiviral vector. The mRNA level of SHIP was detected by fluorescent quantitative reverse transcription-polymerase chain reaction （FQ-PCR）. The expression of SHIP, APt, and phosphorylated Akt （p-Akt） was detected by Western blot. The proliferation and morphology of K562 cells before and after SHIP gene transfection were compared. Results： The proliferation of K562 cells was inhibited after transfection： the proliferation inhibition rate was increased from （9.9±1.5）% on Day 3 to （40.6±2.3）% on Day 5. K562 cells were SHIP- negative but expressed high level of p-Akt which was down-regulated from 0.533 to 0.245 （P〈0.01） after SHIP transfection. Apoptotic characteristics were showed in K562 cells after SHIP transfection. The early apoptosis rate was significantly higher in K562-wtSHIP-FIV-G cells than in K562-FIV-G cells and untransfected K562 cells [（38.3±4.3）% vs. （8.2±0.9）% and （7.7±0.8）%, P〈0.05]. Conclusions： SHIP gene can inhibit cell proliferation and promote cell apoptosis via inactivating PI3K/Akt pathway. Loss of SHIP might activate PI3K/Akt pathway and promote the proliferation of K562 cells.