HCMV感染对恶性胶质瘤U87细胞增殖及ATF5表达的影响

人巨细胞病毒(HCMV)能够诱导肿瘤细胞的恶性转化,但其分子机制尚有待进一步探索。探讨HCMV是否通过调控转录激活因子5(ATF5)的表达变化促进胶质瘤细胞的增殖。采用HCMV AD169株(MOI=5)感染神经胶质瘤U87细胞株,MTT方法观察HCMV感染0、12、24、48 h后细胞的增殖活性。Real-time PCR及Western-blot检测HCMV感染U87细胞后ATF5基因及蛋白的表达水平变化。以慢病毒为载体的靶向ATF5小干扰RNA构建载体,敲低ATF5表达水平后感染HCMV,MTT检测病毒感染细胞的增殖活性变化。HCMV感染神经胶质瘤U87细胞后,与未感染组比较,增值活性明显升高(P0.05),ATF5表达水平上升,表明HCMV感染使胶质瘤细胞增殖活性提高,细胞抗凋亡能力增强。成功构建沉默ATF5细胞系siATF5 U87,HCMV感染siATF5 U87细胞后使细胞增殖活性减弱,抗凋亡能力下降。以上实验结果表明,HCMV感染上调胶质瘤U87细胞ATF5的表达水平,促进细胞的增殖。因此HCMV感染可能通过调控ATF5信号通路增加细胞恶性性状,为治疗胶质瘤提供一个新的思路。     

神经胶质瘤细胞ATF5表达水平对HCMV复制的影响

人巨细胞病毒(human cytomegalovirus,HCMV)在神经胶质瘤细胞中的复制水平不一,其机制尚不清楚。本研究通过下调转录激活因子5(ATF5)在神经胶质瘤细胞中的表达,检测HCMV感染神经胶质瘤细胞后病毒复制水平的变化。首先用HCMV AD169(MOI=5)分别感染U87、SY5Y及A172细胞,观察细胞形态变化,分别在24、48、72、96、120 h取各时间点上清液检测病毒滴度;Real-time PCR检测HCMV即刻早期基因(IE2)、早期基因(UL44)、晚期基因(UL99)及ATF5的表达情况;Western-blot检测病毒基因编码蛋白及ATF5表达的情况。结果显示HCMV在U87、SY5Y细胞中复制水平与病毒在A172细胞中复制水平相比,U87、SY5Y细胞组明显高于A172细胞组(P0.05),ATF5表达在U87、SY5Y细胞组与A172细胞组相比,U87、SY5Y细胞组ATF5表达明显高于A172组(P0.05);利用慢病毒介导的RNA干扰技术下调ATF5在U87、SY5Y细胞的表达,用HCMV感染细胞检测病毒基因及蛋白的表达,结果ATF5表达下调可抑制HCMV的复制(P0.05)。以上结果表明,在胶质瘤细胞中下调ATF5表达水平可以抑制HCMV的复制水平。     

HPV-18 E7蛋白通过SIRT1对宫颈癌细胞增殖、凋亡和自噬的影响

该研究旨在探究HPV-18 E7蛋白通过SIRT1在HPV感染的宫颈癌细胞增殖、凋亡、自噬中的作用。利用HPV-18 E7靶向小干扰RNA(E7-siRNA)抑制HPV-18 E7的表达, CCK-8法检测细胞增殖情况,流式细胞仪测定细胞凋亡情况, Western blot检测细胞自噬相关蛋白Beclin1和LC3表达水平, qRT-PCR和Western blot分别检测SIRT1的mRNA和蛋白表达水平。利用表达载体在HaCaT细胞表达HPV-18 E7蛋白,再分别通过qRT-PCR和Western blot检测SIRT1的mRNA和蛋白表达水平。最后,利用SIRT1激动剂(SRT1720)预处理细胞, CCK-8法检测细胞增殖情况,流式细胞仪测定细胞凋亡情况, Western blot检测细胞自噬相关蛋白Beclin1和LC3表达水平。结果发现, E7-siRNA沉默HPV-18 E7的表达后,细胞增殖和自噬显著减弱,凋亡水平升高, SIRT1的mRNA和蛋白表达水平显著降低。在HaCaT细胞表达HPV-18 E7蛋白后,发现HPV-18 E7可促进SIRT1的表达。SIRT1激...     

自噬在声动力疗法抑制C6胶质瘤细胞增殖中的作用

目的:探讨自噬在血卟啉单甲醚(Hematoporphyrin monomethyl ether,HMME)介导的声动力疗法(Sonodynamic therapy,SDT)抑制C6胶质瘤细胞增殖中的作用。方法:选取对数期生长的C6胶质瘤细胞并随机分为四组:对照组(未予处理)、超声组(单独超声照射)、HMME组(单独加入HMME)、SDT组(超声照射+HMME)。透射电镜观察SDT处理的C6胶质瘤细胞中自噬体数量的改变。应用qRT-PCR和免疫印迹分析SDT处理对C6胶质瘤细胞中的LC3、Beclin1、Bcl-2 m RNA及蛋白表达水平的影响。MTT检测C6胶质瘤细胞的活力变化。结果:透射电子显微镜显示SDT组自噬体数量较对照组明显增多。SDT组C6胶质瘤细胞中微管相关蛋白1轻链3 (Microtubule associated protein 1 light chain 3, LC3)、Beclin1 m RNA和蛋白水平高于对照组,B细胞淋巴瘤-2(B cell lymphoma-2, Bcl-2) m RNA和蛋白水平低于对照组。与对照组相比,SDT组C6胶质瘤细胞存活率从0 h至6 h逐渐下降,从12 h至72 h逐渐升高。3-甲基腺嘌呤(3-Methyladenine,3-MA)+SDT、氯喹(Chloroquine,CQ)+SDT处理后C6胶质瘤细胞存活率较SDT组明显降低。结论:SDT可能通过诱导自噬抑制C6胶质瘤细胞增殖。     

BST-2参与HCMV感染诱导的恶性胶质瘤细胞增殖和迁移

为探讨BST-2蛋白是否参与人巨细胞病毒(HCMV)感染导致的恶性胶质瘤细胞增殖和迁移,以HCMV AD169株感染U251细胞,通过细胞划痕愈合实验检测HCMV感染对U251迁移的影响;通过Western-blot方法检测HCMV感染对BST-2蛋白表达的影响;通过CCK-8、细胞划痕愈合和transwell方法检测HCMV感染后下调BST-2对U251细胞增殖和迁移能力的影响。结果显示,HCMV感染可促进U251细胞迁移并高表达BST-2,沉默BST-2后可抑制由HCMV感染诱导的细胞增殖和迁移。结果证实HCMV感染可促进胶质瘤细胞U251增殖迁移,BST-2参与了HCMV感染导致的恶性胶质瘤细胞增殖和迁移。     

大鼠骨髓间充质干细胞向神经细胞分化后自噬水平的变化

目的:探讨骨髓间充质干细胞(BMSCs)向神经细胞分化前后自噬水平的变化,进一步研究BMSCs神经分化机制。方法:原代培养大鼠BMSCs,三代后用L-DMEM(2%DMSO+200μmol/L BHA)诱导其向神经细胞分化,采用免疫细胞化学方法检测神经元特异标记物NSE表达,采用RT-PCR方法检测自噬相关基因LC3B、Beclin1、Atg5、Atg7、Atg10的表达,用Western blot及流式细胞术检测了自噬标志蛋白LC3B的表达。结果:大鼠BMSCs传3代后,贴壁的BMSCs可达90%以上,类成纤维细胞样梭形生长,经诱导剂作用5 h后,细胞呈现神经细胞样改变,NSE阳性率为(83±5)%。RT-PCR结果表明BMSCs向神经细胞分化后LC3B,Beclin1,Atg5,Atg7,Atg10的表达升高,流式细胞术检测LC3B的平均荧光强度升高,Western blot结果表明神经分化后LC3B-Ⅱ表达增加。结论:大鼠BMSCs向神经细胞分化后自噬水平增加。     

神经胶质瘤U251细胞氧化损伤中自噬和凋亡过程的时间顺序

目的：探讨过氧化氢（H2O2）诱导神经胶质瘤U251细胞损伤中自噬和凋亡发生的时间顺序。方法：实验分为4组：正常对照组、1mmol／L H2O2作用（6h、12h、24h）组。应用MTF法检测H202对神经胶质瘤U251细胞生存率的影响;MDC染色检测自噬空泡的变化;流式细胞仪检测细胞凋亡率变化。Western blot检测Beclin1和胞浆cyt c蛋白的表达。结果：与对照组相比,1mmol／L H2O2作用下,U251细胞存活率明显降低,并呈时间依赖性。与对照组相比,1mmol／L H2O2作用后,6h时U251细胞自噬空泡明显增加,自噬相关蛋白Beclin1表达明显增加,12h、24h细胞自噬水平逐渐增强;而6h时未见细胞凋亡率明显变化及cyt c由线粒体向胞浆的释放,12h、24h时细胞凋亡率明显增加,胞浆中cyt c蛋白表达明显增强（P〈0．05）。结论：氧化损伤能够诱导神经胶质瘤U251细胞发生自噬和凋亡,并且自噬发生于凋亡之前。     

二氢生物喋呤还原酶参与调控HEK293T细胞自噬作用的初步研究

目的探讨二氢生物喋呤还原酶（dihydropteridine reductase,QDPR）对HEK293T细胞自噬作用的影响。方法构建野生型QDPR和突变型QDPR重组质粒分别转染HEK293T细胞,并设空载体对照组。采用RT-PCR及Western blot方法检测空载体组,野生型QDPR组和突变型QDPR组自噬相关蛋白LC3和Beclin 1的表达量变化。结果 1）测序结果证实PCR扩增得到编码正常QDPR的cDNA序列正确以及突变的cDNA也在正确的位置突变;2）磷酸钙共沉淀法转染HEK293T细胞后,野生型QDPR和突变型QDPR融合蛋白成功表达;3）RT-PCR结果显示,与对照组相比,野生型QDPR组LC3基因水平明显上调（P〈0.05）,突变型QDPR组LC3基因水平与对照组相比无统计学差异;与对照组相比,野生型和突变型组Beclin1基因水平无统计学差异;4）Western blot结果显示,与对照组相比,野生型QDPR组LC3-II和Beclin1的蛋白表达量明显上调（P〈0.05）,但LC3-I的蛋白表达量无统计学差异,突变型QDPR组与对照组相比LC3-I,II和Beclin1的蛋白表达量均没有统计学差异（P〉0.05）。结论二氢生物喋呤还原酶能增强HEK293T细胞自噬相关基因LC3和Beclin 1的表达,提示其可能具有激活自噬作用的功能;二氢生物喋呤还原酶93位氨基酸的突变影响了其对细胞自噬作用的调控,降低了自噬标志分子LC3-I和Beclin1的基因表达。     

miRNA-383对急性胰腺炎腺泡细胞自噬的影响作用

目的:检测转染mi RNA-383的急性胰腺炎细胞模型中自噬标志蛋白LC3及Beclin1的表达情况,研究mi RNA-383对急性胰腺炎腺泡细胞自噬功能的影响作用。方法:在前期工作中利用生物信息技术筛选出在急性胰腺炎中表达下调的mi RNA-383。培养AR42J细胞,转染mi RNA-383类似物、抑制物以及阴性对照。使用牛磺胆酸钠盐以浓度200μM刺激细胞20 min制造急性胰腺炎细胞模型,使用BZi PAR检测胰酶激活程度证明此造模方法成功后,以此条件在转染完成后刺激AR42J细胞制造急性胰腺炎细胞模型。牛磺胆酸钠盐以浓度200μM刺激细胞20 min后提取细胞蛋白,利用western blot技术检测各组自噬标志蛋白Beclin1的表达情况及LC3-Ⅱ与LC3-Ⅰ比值的变化。结果:急性胰腺炎细胞模型造模成功,western blot技术检测mi RNA-383抑制物转染组AR42J细胞自噬标志蛋白Beclin1表达水平及LC3-Ⅱ/LC3-Ⅰ的值上调(P0.05),mi RNA-383类似物转染组细胞Beclin1表达水平及LC3-Ⅱ/LC3-Ⅰ的值下调(P0.05)。结论:mi RNA-383可以降低急性胰腺炎时细胞自噬水平,下调mi RNA-383的表达可以提高急性胰腺炎时细胞自噬水平。     

乳酸脱氢酶A通过AMPK信号通路抑制人脑胶质瘤细胞线粒体自噬

该研究旨在探讨乳酸脱氢酶A(lactate dehydrogenase A,LDHA)对人脑胶质瘤细胞线粒体自噬的影响。用质粒sh-EGFP或sh-LDHA转染人脑胶质瘤细胞株U87MG,qRT-PCR和Western blot检测干扰效率,荧光染色技术检测线粒体ROS水平及线粒体膜电位,Western blot检测线粒体自噬相关蛋白及AMPK信号通路相关蛋白表达。结果表明,与sh-EGFP组相比较,sh-LDHA组人脑胶质瘤细胞U87MG中LDHA的mRNA及蛋白质水平均显著降低,线粒体ROS的产生增加,线粒体膜电位明显降低,线粒体自噬相关蛋白PINK1、Parkin及BNIP3、BNIP3L的表达增高,AMPK的磷酸化水平明显升高,而mTOR的磷酸化水平降低。研究结果表明,LDHA能够通过抑制AMPK信号通路,降低线粒体ROS水平,提高线粒体膜电位,抑制线粒体自噬。     

Stimulation of autophagic activity in human glioma cells by anti-proliferative ardipusilloside I isolated from Ardisia pusilla

Aims

Ardipusilloside I (ADS-I), a triterpenoid saponin isolated from Ardisia pusilla A.DC (Myrsinaceae), has been recently tested for cancer treatment including brain cancer. However, the mechanism of its action remains elusive. The present study was to investigate the role of autophagy activation in the anti-tumor activities of ADS-I in human glioma cells.

Main methods

The tetrazolium dye (MTT) colorimetric assay was used for the measurement of cell proliferation in cultured glioma cells, transmission electron microscopy (TEM) for the examination of autophagic activity, flow cytometric analysis for the determination of cell cycle and apoptotic cells, and immunocytochemistry and Western blot for protein expression of microtubule-associated protein light-chain 3 (LC3) and Beclin 1.

Key findings

ADS-I significantly inhibited the proliferation of both U373 and T98G glioma cells in cultures in a dose-dependent manner. The cytotoxic activity of ADS-I against glioma cell growth was associated not only with the induction of cell cycle arrest at G₂/M phase and cell apoptosis in flow cytometric analysis, but also with the activation of autophagy, indicated by the formation of autophagosomes and up-regulated expression of both autophagic protein Beclin 1 and LC3 in glioma cells. Additionally, the treatment with chloroquine, an autophagy inhibitor, reduced ADS-1-mediated cell death.

Significance

These data suggest that the anti-proliferative activity of ADS-I in human glioma cells is associated with the activation of autophagy in addition to cell cycle arrest and apoptosis, and the antagonistic effect of chloroquine suggests an important role of autophagy in ADS-I-mediated cell death against tumor growth.     

The tricyclic antidepressant imipramine induces autophagic cell death in U-87MG glioma cells

In this study, we investigated the antitumor effects of the tricyclic antidepressant 3-(10,11-dihydro-5H-dibenzo[b,f]azepin-5-yl)-N,N-dimethylpropan-1-amine (imipramine) on glioma cells. We found that exposure of U-87MG cells to imipramine resulted in the inhibition of PI3K/Akt/mTOR signaling, reduction of clonogenicity, and induction of cell death. Imipramine stimulated the formation of acidic vesicular organelles, the conversion of LC3-I to LC3-II, and the redistribution of LC3 to autophagosomes, suggesting that it stimulates the progression of autophagy. It did not, however, induce apoptosis. We further showed that knockdown of Beclin-1 using siRNA abrogated imipramine-induced cell death. These results suggest that imipramine exerts antitumor effects on PTEN-null U-87MG human glioma cells by inhibiting PI3K/Akt/mTOR signaling and by inducing autophagic cell death.     

Early induction of autophagy in human fibroblasts after infection with human cytomegalovirus or herpes simplex virus 1

The infection of human fetal foreskin fibroblasts (HFFF2) with human cytomegalovirus (HCMV) resulted in the induction of autophagy. This was demonstrated by the increased lipidation of microtubule-associated protein 1 light chain 3 (LC3), a hallmark of autophagy, and by the visualization of characteristic vesicles within infected cells. The response was detected first at 2 h postinfection and persisted for at least 3 days. De novo protein synthesis was not required for the effect, since HCMV that was irradiated with UV light also elicited the response, and furthermore the continuous presence of cycloheximide did not prevent induction. Infection with herpes simplex virus type 1 (HSV-1) under conditions that inhibited viral gene expression provoked autophagy, whereas UV-irradiated respiratory syncytial virus did not. The induction of autophagy occurred when cells were infected with HCMV or HSV-1 that was gradient purified, but HCMV dense bodies and HSV-1 light particles, each of which lack nucleocapsids and genomes, were inactive. The depletion of regulatory proteins Atg5 and Atg7, which are required for autophagy, reduced LC3 modification in response to infection but did not result in any detectable difference in viral or cellular gene expression at early times after infection. The electroporation of DNA into HFFF2 cultures induced the lipidation of LC3 but double-stranded RNA did not, even though both agents stimulated an innate immune response. The results show a novel, early cellular response to the presence of the incoming virion and additionally demonstrate that autophagy can be induced by the presence of foreign DNA within cells.     

TRPM3表达上调通过调控Beclin1的表达促进卵巢癌细胞自噬

目的:探讨瞬时受体电位离子通道3(TRPM3)和Beclin1在卵巢癌中的表达和对卵巢癌细胞自噬的影响。方法:收集6例正常卵巢组织标本和20例卵巢癌组织标本,应用免疫组织化学染色法检测TRPM3和Beclin1在卵巢癌组织中的表达,采用western blotting法检测TRPM3和Beclin1在正常卵巢上皮细胞Moody和卵巢癌细胞Hey、ES-2中的表达差异。用TRPM3-siRNA瞬时转染细胞Hey和ES-2,通过western blotting法检测TRPM3基因沉默情况及Beclin1、p62和LC3的蛋白表达变化。结果:在正常卵巢组织和卵巢癌组织中,TRPM3的阳性表达率分别为33.3%、80.0%(P0.05),而Beclin1的阳性表达率分别为33.3%、65.0%(P0.05)。与正常卵巢上皮细胞Moody相比,卵巢癌细胞Hey和ES-2中TRPM3、Beclin1蛋白表达水平明显较高(P0.05)。沉默TRPM3基因表达的卵巢癌细胞中Beclin1和LC3蛋白表达与对照组相比明显降低,而p62表达升高(P0.05)。结论:卵巢癌组织和细胞中TRPM3蛋白呈高表达,可能通过调控Beclin1促进卵巢癌细胞的自噬。     

Autophagy: An overlooked mechanism of HIV-1 pathogenesis and NeuroAIDS?

Human immunodeficiency virus type 1 (HIV-1) establishes a persistent infection characterized by progressive depletion of CD4⁺ lymphocytes and immunosuppression. Although extensive research has examined the importance of apoptosis as a cause of cell death associated with HIV-1 infection, the role of autophagy has been largely ignored. Our laboratory has examined the autophagic process in HIV-1-infected cells. Following infection of human peripheral blood CD4+ T-cells or U937 cells with HIV-1 for 48 hours, the autophagy proteins Beclin 1 and LC3-II were found to be markedly decreased. Beclin 1 mRNA expression and autophagosomes were also reduced in HIV-1 infected cells. Thus, our data indicate that HIV-1 infection inhibits autophagy in infected cells in contrast to the previously described induction of autophagy by gp120 in uninfected bystander cells. It is likely that HIV-1 has evolved this mechanism as part of an elaborate attempt to evade the immune system while promoting its own replication. We believe that autophagy is an overlooked mechanism in HIV-1 pathogenesis and plays a particularly important role in the early cognitive impairment and dementia often associated with advanced AIDS. A model is presented that describes the potential role of autophagy in NeuroAIDS.

Addendum to: Zhou D, Spector SA. Human immunodeficiency virus type-1 infection inhibits autophagy. Aids 2008;22:695-9.