TRIM32(tripartite motif protein 32)在细胞分化和增殖过程中发挥重要作用,并且参与了多种生物学过程,包括信号转导、细胞凋亡和基因表达调控等.本课题组前期研究发现,TRIM32基因敲除小鼠对甲基苯丙胺(methamphetamine,MA)不易成瘾,但具体机制尚不清楚.本研究旨在探讨siRNA(small interfering RNA)转染沉默TRIM32基因对甲基苯丙胺成瘾致神经细胞凋亡的影响及其机制.本文以PC12细胞为研究对象,利用siRNA转染技术沉默TRIM32基因的表达,建立分组:正常对照组,MA处理组,沉默TRIM32组,沉默TRIM32+MA处理组.采用原位末端转移酶标记技术(terminal deoxynucleotyl transferase-mediated dUTP-biotin nick end labeling assay,TUNEL 染色)检测各组细胞的凋亡情况,结果显示,沉默TRIM32+MA处理组细胞较MA处理组凋亡比例下降(P＜0.01),说明沉默TRIM32基因可以抑制MA诱导的细胞凋亡.采用线粒体膜电位试剂盒检测各组线粒体膜电位的变化,结果显示,沉默TRIM32+MA处理组细胞较MA处理组线粒体膜电位升高(P＜0.05),表明沉默TRIM32基因可以调节MA诱导的线粒体膜电位的降低.细胞免疫荧光和West-ern 印迹结果显示,沉默TRIM32+MA处理组较MA处理组胱天蛋白酶-3(caspase-3)、剪切胱天蛋白酶-3(cleaved-caspase-3)和细胞色素C(Cytochrome c,Cyt-C)蛋白质水平显著下调(P＜0.01),磷脂酰肌醇 3-激酶(phosphatidylinositol 3-kinase,PI3K)、磷酸化蛋白质激酶 B(phospho-protein kinase B,p-AKT)显著上调(P＜0.01),说明沉默TRIM32基因抑制细胞凋亡可能通过调控PI3K/AKT信号通路来实现.在此基础上深入研究,实验中加入PI3K/AKT抑制剂LY294002,Western印迹检测结果证实,LY294002可部分阻断沉默TRIM32基因对细胞凋亡的调控作用(P＜0.05),进一步证实了沉默TRIM32基因通过激活PI3K/AKT信号通路抑制MA诱导的线粒体途径凋亡.综上,沉默TRIM32基因可抑制MA成瘾引发的线粒体凋亡途径,从而发挥神经保护作用,其机制可能与PI3K/AKT信号通路有关.
Methamphetamine Addiction-induced Apoptosis Is Inhibited by PI3K/AKT-Mediated Silencing of TRIM32
TRIM32(tripartite motif protein 32,TRIM32)plays an important role in cell differentiation and proliferation and is involved in a variety of biological processes,including signal transduction,apop-tosis,and gene expression regulation.In the previous study,our group found that TRIM32 knockout mice are less susceptible to methamphetamine(MA)addiction.The aim of this study was to investigate the effect of silencing TRIM32 by siRNA(small interfering RNA)transfection on the apoptosis of neuronal cells caused by MA addiction and its mechanism.In this paper,the expression of TRIM32 was silenced by siRNA in PC 12 cells,and subgroups were established:the normal control group,MA-treated group,TRIM32-silencing group,and TRIM32-silenceing+MA-treated group.The apoptosis of cells in each group was detected using in situ terminal deoxynucleotyl transferase-mediated dUTP-biotin nick end labe-ling assay(TUNEL staining),and the results showed that the percentage of cells in the TRIM32-silence-ing+MA-treated group was lower than the MA-treated group(P＜0.01),indicating that TRIM32 silen-cing could inhibit MA-induced apoptosis.We also detected the changes of mitochondrial membrane po-tential in each group,which was increased in the TRIM32-silenceing+MA-treated group compared with the MA treatment group(P＜0.05),indicating that TRIM32 silencing could regulate the MA-induced de-crease in mitochondrial membrane potential.The results of cellular immunofluorescence and Western blotting showed that the protein levels of cystatinase-3(caspase-3),cleaved-caspase-3(cleaved-caspase-3),and cytochrome c(Cyt-C)were significantly down-regulated in the TRIM32-silencing+MA-treated group compared with the MA-treated group(P＜0.01)and phosphatidylinositol 3-kinase(PI3K)and phospho-protein kinase B(p-AKT)were significantly up-regulated(P＜0.01),suggesting that silencing of TRIM32 to inhibit apoptosis may be achieved by modulating the PI3K/AKT signaling pathway.On the basis of this in-depth study,the PI3K/AKT inhibitor LY294002 was added to the experiment,and the results confirmed that LY294002 could partially block apoptosis by silencing TRIM32(P＜0.05),which further confirmed that TRIM32 silencing inhibited MA through activation of the PI3K/AKT signaling.In conclusion,silencing TRIM32 could inhibit the mitochondrial apoptotic pathway triggered by MA addic-tion,thus exerting a neuroprotective effect,and the mechanism may be related to the PI3K/AKT signa-ling pathway.
tripartite motif protein 32(TRIM32)methamphetamineapoptosisaddictionPI3K/AKT
NETL
NSTL
万方数据
