EphA2对神经胶质瘤细胞系U251的凋亡﹑增殖﹑迁移和侵袭的研究

为了研究EphA2对神经胶质瘤细胞系U251在增殖、凋亡、迁移和侵袭方面所起的作用,用RT-PCR方法检测正常脑组织标本与两种恶性胶质瘤细胞系中EphA2 mRNA表达水平,然后用化学合成的针对EphA2基因的小干扰RNA(siRNA)下调该基因的表达,以检测其在U251中的生物学功能．证实了EphA2基因在正常脑组织标本中的表达水平远低于两种恶性胶质瘤细胞系．把体外化学合成针对EphA2基因的小干扰RNA(siRNA- EphA2)转染入U251细胞后,Western blot, 实时定量 RT-PCR检测到U251细胞中EphA2蛋白及mRNA表达水平都明显降低,并且细胞增殖受到显著抑制,同时出现了明显的细胞凋亡．伤口愈合实验(检测细胞迁移能力),Transwell小室实验(检测细胞侵袭能力)均表明,下调EphA2的表达后,细胞的迁移和侵袭能力较阴性对照组显著减弱．上述结果表明,在神经胶质瘤U251细胞中,EphA2与其恶性增殖及高度侵染性相关,可作为分子治疗的有效靶点．     

B4GALT3促进肝癌细胞增殖和侵袭

β-1,4-半乳糖基转移酶III（β-1,4-galactosyltransferase III,B4GALT3）在肿瘤的作用正受到关注,但其在肝癌中的表达模式及其作用有待阐明。基于TCGA肿瘤组织数据库和GTEx正常组织数据库进行的生物信息学分析,发现相比于人正常肝组织,B4GALT3在人肝癌组织中的表达显著上调。实时荧光定量PCR结果发现肝癌细胞中B4GALT3的mRNA和Western 印迹检测蛋白质表达水平显著上调。其中肝癌细胞SMMC7721中B4GALT3的mRNA表达水平是正常肝细胞L-02的9.85倍。对TCGA数据库进行分析发现,B4GALT3表达水平与肝癌患者的生存率呈负相关。在内源性高表达B4GALT3的SMMC7721肝癌细胞中,干扰B4GALT3表达,可显著抑制该细胞的增殖能力和侵袭能力。干扰B4GALT3表达能显著上调SMMC7721细胞中p27和E-cadherin的蛋白质表达水平,干扰B4GALT3表达后SMMC7721细胞中,p27和E-cadherin的mRNA水平较对照组上调6.15倍和7.83倍。总之,B4GALT3在肝癌中表达上调,且促进肝癌细胞的增殖和侵袭。     

B4GALT3促进肝癌细胞的增殖和侵袭

β-1,4-半乳糖基转移酶III(β-1,4-galactosyltransferase III,B4GALT3)在肿瘤的作用正受到关注,但其在肝癌中的表达模式及其作用有待阐明。基于TCGA肿瘤组织数据库和GTEx正常组织数据库进行的生物信息学分析,发现相比于人正常肝组织,B4GALT3在人肝癌组织中的表达显著上调。实时荧光定量PCR结果发现肝癌细胞中B4GALT3的mRNA和Western印迹检测蛋白质表达水平显著上调。其中肝癌细胞SMMC7721中B4GALT3的mRNA表达水平是正常肝细胞L-02的9.85倍。对TCGA数据库进行分析发现,B4GALT3表达水平与肝癌患者的生存率呈负相关。在内源性高表达B4GALT3的SMMC7721肝癌细胞中,干扰B4GALT3表达,可显著抑制该细胞的增殖能力和侵袭能力。干扰B4GALT3表达能显著上调SMMC7721细胞中p27和E-cadherin的蛋白质表达水平,干扰B4GALT3表达后SMMC7721细胞中,p27和E-cadherin的mRNA水平较对照组上调6.15倍和7.83倍。总之,B4GALT3在肝癌中表达上调,且促进肝癌细胞的增殖和侵袭。     

慢病毒介导的TPX2沉默对人宫颈癌HeLa细胞凋亡和侵袭的影响及机制

该文的目的是研究慢病毒介导的Xklp2靶蛋白(targeting protein for Xklp2,TPX2)沉默对人宫颈癌He La细胞凋亡、侵袭的影响及机制。构建4种载有TPX2-sh RNA的重组慢病毒及其阴性对照,将5种重组慢病毒分别稳定感染人宫颈癌He La细胞,利用实时荧光定量PCR和Western blot筛选出TPX2沉默效果最佳的一组He La细胞作为干扰组进行后续实验。采用Transwell基底膜侵袭实验测定各组细胞的侵袭能力。采用流式细胞术检测各组细胞的凋亡情况。Western blot检测TPX2-sh RNA转染前后细胞凋亡及侵袭相关蛋白质Bcl-2、Bax、Caspase-3、MMP9、TIMP-1及nm23-H1水平。结果显示,筛选出的RNA干扰慢病毒载体LV-TPX2-sh RNA-1可有效抑制He La细胞TPX2的表达;与对照组相比,干扰组的He La细胞凋亡率明显增加(P0.01);穿过Transwell小室基底膜细胞明显减少(P0.01)。He La细胞感染LV-TPX2-sh RNA-1能下调凋亡相关蛋白Bcl-2的表达水平,上调Caspase-3及Bax的表达水平(P0.05);上调侵袭相关蛋白质nm23-H1及TIMP-1水平,下调MMP9水平(P0.05)。以上结果表明,沉默TPX2表达能增加宫颈癌细胞的凋亡,可能与其上调Caspase-3及Bax水平,下调Bcl-2水平有关;沉默TPX2表达能抑制宫颈癌细胞侵袭能力,可能与其上调TIMP-1及nm23-H1水平,下调MMP9的水平有关。     

KDR靶向RNA干扰对MCF一7细胞凋亡的影响

目的：研究KDR靶向RNA干扰对MCF-7细胞凋亡的影响,探讨其可能的机制。方法：采用阳离子脂质体Lipofecta．mine2000TM作为转染试剂将人KDR基因的siRNA转染人类乳腺细胞株MCF-7,诱RNAi,采用Hoechst33258染色和半定量RT—PCR检测Caspase-3、survivin的mRNA表达及细胞凋亡变化;比色法检测Caspase．3的活性;利用免疫组织化学方法检测survivin的表达,并用图像分析仪分析蛋白表达强度。结果：靶向KDR的siRNA转染MCF-7后,Caspase-3的mRNA表达上调,survivin基因mRNA及蛋白表达水平下调（P〈0．05）。结论：KDRsiRNA通过减少乳腺癌细胞survivin的表达,增加Caspase．3表达来促进肿瘤细胞凋亡,发挥其抗肿瘤作用。     

RMP在肝癌细胞基因组稳定性和凋亡中的作用

目的:研究RPB5调节蛋白(RMP)在正常肝细胞及肝癌细胞基因组稳定性中的作用,并探讨其与细胞凋亡的相关性。方法:采用逆转录-聚合酶链反应(RT-PCR)从mRNA 水平检测正常细胞系及多种肿瘤细胞系中RMP的表达。不同剂量⁶⁰Coγ射线照射肝癌细胞SMMC-7721细胞和正常肝细胞HL-7702细胞,RT-PCR法检测RMP的表达,流式细胞技术检测照射肝癌细胞周期变化及细胞凋亡。应用RNA干扰技术研究RMP在肝癌细胞基因组稳定性中的作用。结果:正常细胞系及多种肿瘤细胞系中RMP基因均有不同程度的表达。经⁶⁰Coγ射线诱导的肝癌细胞及正常肝细胞RMP表达水平明显升高,且有一定剂量依赖性。随着照射剂量增加细胞凋亡明显增多,细胞周期G₁期增高,而S期明显降低。RMP被干扰后, 电子显微镜观察细胞形态发生明显改变,p21基因表达减弱。结论:RMP具有维持细胞基因组稳定性的潜在作用。RMP的表达与p53、p21等具有一定相关性,可能与后者协同调节细胞凋亡过程。     

靶向survivin的siRNA对胃癌BGC-823细胞增殖的影响

目的探讨干扰RNA沉默生存素（survivin）基因表达对人胃癌BGC-823细胞增殖和凋亡的影响。方法设计并合成3条靶向survivin的小分子干扰RNA（siRNA）,构建表达性干扰RNA质粒（shRNA）——shRNA-survivin-1、shRNA-survivin-2和shRNA-survivin-3,分别转染胃癌BGC-823细胞,实时定量PCR检测干扰RNA沉默survivin mRNA表达效果,Westernblot观察对胃癌BGC-823细胞survivin蛋白质表达的抑制,MTT（四甲基偶氮唑盐）比色法分析检测细胞生长抑制率,流式细胞计数检测各组细胞周期和凋亡率,探讨干扰RNA对胃癌BGC-823细胞生长的影响。结果在体外,shRNA-survivin-1有效沉默人胃癌BGC-823细胞survivin mRNA的表达,使sur-vivin mRNA相对水平明显降低（P〈0.05）,survivin蛋白质表达抑制,72h细胞生长抑制率达74.92%（P〈0.05）,shRNA-survivin-1使G2/M期细胞百分比明显增加,凋亡率显著增加（P〈0.05）。结论 shRNA-survivin-1可以沉默survivin基因的表达,可以显著抑制胃癌BGC-823细胞的增殖,在一定程度上诱导其自发凋亡。本研究为靶向sur-vivin的RNA干扰在胃癌的基因治疗提供了有力的理论依据和技术储备。     

MiR-125b在肿瘤发生发展中作用的研究进展

MicroRNAs(微小RNAs)是新近发现的内源性高度保守的非蛋白质编码的长约22核苷酸单链小分子RNA,在转录后水平上调节基因表达的重要调节因子.MiR-125b在多种肿瘤组织中表达异常,既有上调发挥癌基因作用的报道,也有下调发挥抑癌基因作用的报道,miR-125b在肿瘤中作用的研究结果众说纷纭,涉及到肿瘤细胞生命活动周期的各个过程,以下就miR-125b与肿瘤细胞的增殖、凋亡、侵袭、转移、分化和耐药方面的最新研究进展作一综述.     

靶向VEGF小干扰RNA协同5-FU诱导人乳腺癌细胞MCF-7凋亡机制的研究

探讨慢病毒介导的靶向VEGF小干扰RNA联合应用化疗药物5 FU诱导人乳腺癌细胞MCF-7凋亡的机制。以携带VEGF siRNA的慢病毒载体感染MCF-7细胞,应用RT-PCR和Western blot分别检测各组VEGF mRNA、VEGF蛋白及凋亡相关蛋白的表达,流式细胞术检测细胞凋亡。结果表明,慢病毒VEGF siRNA干扰组细胞VEGF mRNA和蛋白表达水平明显低于对照组,凋亡相关蛋白P53及P21表达上调,而SIRT1、Bcl-2及Survivin表达下调。流式细胞术检测显示慢病毒干扰组及5-FU组细胞凋亡率显著升高,联合治疗组的协同作用更为明显。上述结果表明：慢病毒介导的RNA干扰能明显抑制MCF-7细胞VEGF的表达,通过下调SIRTI蛋白的表达,导致P53蛋白表达上调,并调控其下游P21、bcl-2和Survivin的表达,从而诱导MCF-7细胞的凋亡,并且提高了MCF-7对5-FU的敏感性。     

特异性下调葡萄糖调节蛋白78表达对肝细胞癌侵袭和转移能力的影响

为了研究特异性下调葡萄糖调节蛋白(Grp)78对肝细胞癌侵袭和转移能力的影响。通过小干扰RNA(siRNA)技术特异性下调人肝细胞癌细胞株BEL7402中Grp78的表达,并应用Transwell法和划痕法对肝细胞癌侵袭、转移能力的改变进行分析,应用免疫沉淀技术和GST-pulldown技术分别对黏着斑激酶(FAK)的磷酸化水平和小GTPase RhoA的活性进行研究,应用免疫印迹技术检测E-钙黏着蛋白、N-钙黏着蛋白和波形蛋白的表达。结果发现,Transwell实验和划痕实验结果显示特异性下调Grp78表达可以抑制肝细胞癌的侵袭和转移,免疫沉淀结果显示特异性下调Grp78表达可以降低FAK的磷酸化水平,GST-pulldown实验结果表明特异性下调Grp78表达可以上调RhoA的活性。免疫印迹实验结果表明特异性下调Grp78可以下调N-钙黏着蛋白、波形蛋白的表达,上调E-钙黏着蛋白的表达。结果表明特异性下调Grp78在体外可以抑制肝细胞癌的侵袭和转移,这种抑制作用是通过FAK脱磷酸化和抑制肿瘤的上皮-间叶转化实现的。     

Glucose effect on the expression of 150 kDa oxygen-regulated protein in HeLa cells

Chaperones assist in the correct folding of newly synthesised proteins in the endoplasmic reticulum (ER) of cells, this being essential for the translocation of protein molecules to specific subcellular compartments, extracellular matrix or to biological fluids. The biosynthesis of some ER chaperones is regulated by glucose. They are named "glucose-regulated proteins" (GRPs). The function of some GRPs depends on oxygen, a subgroup named "oxygen-regulated proteins" (ORPs). The biosynthesis of ORPs is induced by deprivation of glucose or oxygen. Exposure of HeLa cells to glucose starvation induces the biosynthesis of various GRPs including ORP 150. The expression of ORP 150 is regulated by the concentration of glucose in the culture medium, being induced by a shortage and repressed by a presence of glucose. We have shown that both glucose starvation and transfection of cells with siRNA (specific to ORP 150 mRNA) evoke similar, but quantitatively different, effects. The cells grown for 72 h in a 4.5 mg/ml glucose-containing medium demonstrated low apoptosis (3.7%) whereas in a 0.5 mg/ml glucose-containing medium the apoptosis was increased to 10%. The effect of transfection on apoptosis was distinctly higher with almost 22% of apoptotic cells detected in 72 h cultures. One may conclude that ORP 150 reduces the pro-apoptotic effects of glucose starvation. Such a hypothesis is supported by the observation that the transfection procedure makes HeLa cells resistant to the regulatory effect of glucose on ORP 150 production. The transfected cells do not respond to glucose starvation with an overexpression of ORP 150. It is apparent from our experiments that ORP 150 plays an important role in adaptation of cells to the shortage of glucose and reduces the pro-apoptotic effect of glucose starvation.     

The effect of hypoxia on the expression of 150 kDa oxygen-regulated protein (ORP 150) in HeLa cells.

Correct protein folding is an important factor, for the translocation of newly synthesised proteins to specific subcellular compartments, extracellular matrix or to biological fluids. This process is regulated by a group of specific proteins, referred to as chaperones. Many stress conditions, such as oxygen or glucose deprivation, slow down the folding process and cause accumulation of unfolded/misfolded proteins in the cell. Molecular chaperones are induced in these conditions; with some named as oxygen-regulated proteins (ORPs). These bind to unfolded / misfolded proteins to facilitate correct assembly. ORP 150 is the subject of this study. Hypoxia results in an enhancement of ORP 150 expression in several tumour cell lines cultured in vitro. HeLa cells grown in hypoxic conditions (despite an intensive expression of ORP 150) demonstrate higher rates of apoptosis in comparison to those cultured in normoxic conditions. Furthermore, the inhibition of ORP 150 synthesis by transfection of these cells with a specific siRNA resulted in an intensification of apoptosis, as indicated by specific markers of this process; the enhancement of poly ADP-ribose protein cleavage and the increase in Bim protein expression. We conclude from our study that the increase in ORP 150 synthesis protects the cells against the proapoptotic effect of hypoxia.     

Bub1基因对人肝癌MHCC97-H细胞增殖、周期和凋亡的影响

目的:研究Bub1基因在肝癌中的表达以及对肝癌细胞系MHCC97-H增殖、周期和凋亡的影响。方法:利用RNA干扰技术下调肝癌细胞系MHCC97-H中Bub1的表达;qRT-PCR和Western Blot分别检测Bub1在mRNA和蛋白水平表达的变化;CCK-8实验检测肿瘤细胞增殖能力的改变;流式细胞术检测细胞周期和凋亡的变化。结果:qRT-PCR和Western Blot结果显示si-Bub1能够成功下调Bub1的表达;下调Bub1后肝癌MHCC97-H细胞的增殖能力下降(P0.05),细胞的凋亡比例升高(P0.05),细胞发生S期阻滞。结论:Bub1基因在肝癌中高表达,下调Bub1的表达后能够降低肝癌细胞的增殖能力,促进细胞凋亡,诱导细胞发生S期阻滞。     

AMPK调控内质网应激抵抗COPD大鼠肺泡上皮细胞凋亡的实验研究

目的:初步探讨AMPK在内质网应激所致COPD大鼠肺泡上皮细胞凋亡中所起的作用及机制。方法:实验分三组:对照组,COPD模型组,AICAR干预组,以香烟烟雾烟熏加气管内滴注脂多糖方法构建COPD大鼠模型,取大鼠肺组织行HE染色病理观察,免疫组化,western blot检测p-AMPK/AMPK,ORP150,caspase-3及CHOP表达,TUNEL法检测各组凋亡情况。结果:病理HE染色提示模型组大量炎症细胞浸润,肺大疱形成,支气管壁发生狭窄;AICAR干预组炎症细胞较模型组减少。与正常对照组相比,免疫组化及western blot均提示模型组中p-AMPK和ORP150蛋白表达含量增强,差异有统计学意义(P0.05)。而AICAR干预组中p-AMPK/AMPK及ORP150蛋白表达较模型组明显上升,差异有统计学意义(P0.05)。内质网应激相关凋亡指标CHOP及caspase-3的表达在模型组明显增强,较正常组比较差异有显著性(P0.05),而AICAR组中凋亡指标较模型组明显下调。结论:AMPK可以保护肺泡上皮细胞免于香烟烟雾所致内质网应激凋亡,且有可能通过增加ORP150来实现其保护作用。     

Oxygen-regulated protein-150 prevents calcium homeostasis deregulation and apoptosis induced by oxidized LDL in vascular cells

Oxidized LDLs (oxLDLs) induce apoptosis, which contributes to the pathogenesis of atherosclerosis. The 150 kDa oxygen-regulated protein (ORP150), an endoplasmic reticulum (ER)-resident chaperone, is upregulated by hypoxia and prevents ischemia-induced cell death. The aim of this work was to investigate whether and how ORP150 can prevent apoptosis induced by oxLDLs in vascular cells. OxLDLs induced ORP150 expression in the ER of human microvascular endothelial cell line (HMEC-1). ORP150 expression was blocked by antioxidants, by the permeant calcium chelator BAPTA-AM, and by inhibitors of the inositol-1,4,5 trisphosphate (IP3) receptors, 2-aminoethyl diphenylborinate (2-APB) and xestospongin C. ORP150 silencing by siRNA-enhanced oxLDL-induced apoptosis, while forced ORP150 expression increased the resistance of cells via an inhibition of the oxLDL-induced calcium rise, and of subsequent calpain activation, cytochrome c release, caspase 3 activation and apoptosis. A similar protective effect was achieved by BAPTA-AM, 2-APB and xestospongin C. Altogether, these data indicate that (i)ORP150 inhibits oxLDL-induced apoptosis by blocking calcium signaling and subsequent apoptosis, (ii)calcium released from ER stores through IP3 channels is involved in the oxLDL-induced calcium rise and apoptosis, and is inhibited by ORP150. Finally, ORP150 is expressed in advanced atherosclerotic lesions, where it may locally participate to reduce the apoptotic effect of oxLDLs and the subsequent risk of plaque rupture.     

Retracted: miR-150 might inhibit cell proliferation and promote cell apoptosis by targeting LMO4 in Burkitt lymphoma

This study aimed at investigating the effect of microRNA-150 (miR-150) on cell proliferation of Burkitt lymphoma and its molecular mechanism. Gene expression analysis was applied to identify target genes of miR-150 in Burkitt lymphoma cell line ST486 based on the dataset from the Gene Expression Omnibus (GEO) datasets GSE86432. miRNA mimics, inhibitor and small interfering RNA (siRNA) were fluorescently labeled by Cy3, whereas plasmid vector was labeled by EGFP. Cells were viewed by fluorescence microscope and transfection efficiency was evaluated through fluorescent cell percentage. Quantitative real-time polymerase chain reaction analysis (qRT-PCR) and western blot were applied to detect the expression level of miR-150 and LMO4. Cell proliferation, cell cycle, and apoptosis were explored by CCK-8, flow cytometry. Targeting relationship was validated by the Luciferase reporter assay. Tumor xenograft and immunohistochemical analysis were conducted in nude mice model. In Burkitt lymphoma cells, miR-150 expression was significantly lower than normal ones, whereas the expression of LMO4 was upregulated. miR-150 might inhibit cell proliferation and promoted apoptosis in Burkitt lymphoma deterioration by downregulating LMO4. The results of tumor xenograft further confirmed the role of miR-150 in Burkitt lymphoma. Targeting LMO4 is a significant mechanism by which miR-150 suppresses cell growth and promotes apoptosis in Burkitt lymphoma cells, thus may provide a novel target for Burkitt lymphoma therapy in the future.