体外实验观察小分子干扰RNA对胶质瘤U251细胞血管内皮生长因子表达的影响

目的:血管内皮生长因子能促进血管内皮细胞增生和血管形成,而RNA干扰技术可高效特异地导致转录后基因沉默现象。实验针对人血管内皮生长因子基因化学合成小分子干扰RNA,观察其对胶质瘤U251细胞的体外干扰效应。方法:实验于2006-09/2007-02在中国科学院上海生物化学研究所分子生物学国家重点实验室完成。①实验材料:人类U251胶质母细胞瘤细胞株由上海市肿瘤研究所朱景德教授惠赠。非特异性小分子干扰RNA,绿色荧光标记的FAMnegativecontrolsiRNA(上海吉玛公司)。②实验方法:选择基因序列号为NM0033761的血管内皮生长因子基因cDNA序列上的3个位点设计3条不同序列的小分子干扰RNA,并应用BLAST技术排除其他同源基因。采用脂质体法转染U251细胞,以200nmol/L进行转染,以转染非特异性小分子干扰RNA作为阴性对照组,以未加入小分子干扰RNA作为空白对照组。48h后RT-PCR筛选最佳小分子干扰RNA,将抑制率最高的小分子干扰RNA再分别按50,100,200,300nmol/L进行转染。③实验评估:荧光显微镜及流式细胞仪检测转染效率,RT-PCR法半定量检测浓度梯度下小分子干扰RNA对U251细胞血管内皮生长因子mRNA表达的抑制效果。结果:①荧光显微镜及流式细胞术检测转染效率:镜下可见胞质内的绿色荧光,同一视野下细胞核呈蓝染,转染效率达95%以上。②RT-PCR检测血管内皮生长因子mRNA的表达水平:不同序列的小分子干扰RNA以200nmol/L转染细胞后血管内皮生长因子mRNA的表达水平均有所下调,以小分子干扰RNA1转染组最为明显(P<0.01),抑制率大于70%,选为最佳特异性小分子干扰RNA。U251细胞转染50,100,200,300nmol/L的小分子干扰RNA148h后,随着转染浓度的升高,小分子干扰RNA1的抑制效果逐渐增强,各浓度转染组血管内皮生长因子mRNA的表达水平可下调16%～73%。结论:以血管内皮生长因子为靶基因、化学修饰合成的特异性小分子干扰RNA,在体外可有效抑制目的基因mRNA的表达。     

血管内皮生长因子C靶向RNA干扰重组载体的构建和效应检测

目的:构建针对人血管内皮生长因子C小干扰RNA表达载体,并观察其在胃癌细胞中的干扰效果。方法:实验于2006-02/2007-02在河南省分子医学重点学科开放实验室完成。①实验材料:小干扰RNA表达载体pSilencer3.1为美国Ambion公司产品;胃癌细胞SGC7901为河南省分子医学重点学科开放实验室保存;血管内皮生长因子C基因的干扰片断和聚合酶链反应引物(152bp)由上海生工生物公司合成。②实验过程:根据pSilencer3.1-H1载体要求设计靶向血管内皮生长因子C基因的两对小干扰RNA,退火后连接入载体相应位点,构建重组表达质粒。经酶切和测序鉴定后分别转染胃癌细胞株SGC-7901,经G418筛选,获得稳定表达的细胞株。③实验评估:采用反转录-聚合酶链反应和Westernblot法检测血管内皮生长因子CmRNA和蛋白水平的表达。结果:①重组质粒的酶切、核苷酸序列分析结果:经酶切和测序鉴定证实成功构建了血管内皮生长因子C小干扰RNA表达载体,建立了稳定转染细胞株。②稳定转染的细胞株中血管内皮生长因子CmRNA和蛋白表达:反转录-聚合酶链反应和Western blot显示转染后血管内皮生长因子C表达与对照组相比有不同程度的降低,以pSilencer3.1-neo-VEGF-C1尤为明显,抑制血管内皮生长因子C表达,不影响细胞的生长。结论:成功构建了血管内皮生长因子C小干扰RNA表达载体及稳定转染的胃癌细胞株。     

靶向胆囊癌血管内皮生长因子基因短发夹样RNA表达质粒的构建与筛选

背景:已有研究通过RNA干扰技术,抑制结肠癌、前列腺癌和视网膜母细胞瘤细胞的血管内皮细胞生长因子基因的表达.尚未见关于RNA干扰血管内皮生长因子抑制胆囊癌肿瘤的相关研究.目的:创新性构建编码胆囊癌血管内皮生长因子基因的短发夹样RNA质粒表达载体,筛选出沉默血管内皮生长因子基因效果最明显的短发夹样RNA表达质粒.设计、时间及地点:基因工程观察实验,于2008/2009在中南大学湘雅医院卫生部肝胆肠外科研究中心实验室完成.材料:人胆囊癌细胞株GBC-SD购于同济大学肿瘤研究所.方法:设计4对针对血管内皮生长因子基因不同位点的短发夹样RNA片段.构建携带此短发夹样RNA片段的表达质粒(短发夹样RNA1-4)并行酶切鉴定分析.然后通过脂质体将重组质粒转染到胆囊癌细胞株GBC-SD中,48 h后测定转染率.主要观察指标:重组质粒酶切鉴定结果.转染效率.采用及荧光PCR检测血管内皮生长因子mRNA表达.结果:成功构建了靶向血管内皮生长因子基因的短发夹样RNA质粒表达载体,其中抑制效果最为明显的短发夹样RNA质粒表达载体为pDC316-EGFP-U6-shRNA2 质粒.重组质粒经酶切鉴定证实构建成功.质粒在GBC-SD细胞中的转染率约为58.6%.短发夹样RNA抑制血管内皮生长因子基因的mRNA表达,短发夹样RNA2抑制率最高,可达86%.结论:成功构建并筛选出的靶向血管内皮生长因子的短发夹样RNA表达质粒,其对胆囊癌GBC-SD细胞内的血管内皮生长因子表达具有明显抑制作用.短发夹样RNA2表达质粒抑制作用最明显.     

应用RNA干扰技术抑制白血病细胞血管内皮生长因子基因表达及功能的研究

目的探讨用RNA干扰技术下调血管内皮生长因子(VEGF)基因表达对白血病细胞生长的影响。方法设计3段分别针对VEGF第3~5外显子的短发夹状RNA(shRNA)并构建其表达载体,分别转染人白血病细胞株NB4,用实时定量PCR及Western blot方法观察3段shRNA对细胞内VEGF基因表达的影响。抗性筛选稳定抑制VEGF表达的永久细胞克隆,应用MTT法、甲基纤维素半固体培养法及细胞周期动力学检测了解VEGF基因缄默对白血病细胞生长的影响。结果成功构建分别携带3段shRNA及对照随机片段的重组质粒pGenesil-VR1,2,3和pGenesil-con,3种shRNA重组质粒中pGenesil-VR3可明显降低细胞内VEGF mRNA的丰度及VEGF蛋白表达,筛选出的NB4-VR3细胞株增殖能力明显下降,细胞集落形成率为(13.3±3.8)%,与NB4-con的(21.3±6.4)%和NB4组的(24.5±5.2)%相比,差异有统计学意义(P<0.05),细胞周期阻滞于G1期,3组细胞G1期比例分别为(53.2±4.6)%、(42.7±5.9)%和(40.5±5.3)%。结论应用RNA干扰技术能筛选出特异而高效阻断VEGF基因表达及功能的shRNA,VEGF基因表达下调能明显抑制白血病细胞生长。     

靶向血管内皮生长因子基因的微小RNA真核表达载体的构建

背景:有研究表明微小RNA及潜在靶基因在肝癌中起重要作用,但具体机制仍不清楚。目的:构建靶向血管内皮生长因子基因微小RNA真核表达载体,评估其转染人肝癌细胞株HepG2后血管内皮生长因子基因的干扰效果。方法:根据血管内皮生长因子序列设计合成4对微小RNA不同干扰片段,克隆到pcDNA6.2-GW/EmGFP-微小RNA真核表达载体上,测序分析鉴定插入序列的完整性;并将其转染至HepG-2细胞株中。采用实时荧光定量聚合酶链式反应分析血管内皮生长因子微小RNA干扰效果,蛋白印迹技术测定重组体对血管内皮生长因子基因蛋白表达情况。结果与结论:构建的4组重组体插入片段的碱基序列完全正确,重组体能干扰肝癌细胞HepG2细胞血管内皮因子基因的表达,4组重组体基因的mRNA的表达水平和蛋白表达水平与阴性对照组相比明显降低(P<0.05),其中血管内皮生长因子微小RNA-3表达水平最低,抑制率87%。结果证实,实验成功构建血管内皮生长因子微小RNA表达载体,在体外能有效抑制HepG2细胞血管内皮生长因子基因表达。     

慢病毒介导的血管内皮生长因子受体2小干扰RNA对鼠白血病的抑制

目的：拟建立慢病毒载体携带的血管内皮细胞生长因子受体2（VEGFR2）基因短发夹RNA（knti6／shVEGFR2）,研究其对鼠白血病的抑制作用。方法：用慢病毒载体系统构建Lenti6／shVEGFR2。检测pU6／shVEGFR2入门克隆转染、knti6／shVEGFR2表达克隆转导HL60细胞后的作用。流式细胞仪分析检测knti6／shVEGFR2对HL60鼠模型白血病的抑制效果。结果：pU6／shVEGFR2转染、Lenti6／shVEGFR2转导HL60细胞后48h细胞抑制率相近,之后pU6／shVEGFR2组细胞抑制率明显下降,而Lenti6／shVEGFR2组变化无显著差异。在异种移植白血病鼠模型中knti6／shVEGFR2感染显著抑制白血病细胞生长（P〈0．05）。结论：慢病毒介导的VEGFR2 RNA干扰有可能成为治疗白血病的有效方法。     

体外构建血管内皮生长因子短发夹状RNA表达载体及其对人骨肉瘤细胞相应因子mRNA及蛋白的抑制

目的：设计并构建血管内皮生长因子短发夹状RNA表达载体，检测其对人骨肉瘤细胞血管内皮生长因子mRNA及蛋白表达的影响。方法：实验于2006—01／07在青岛市市立医院试验中心完成。人骨肉瘤细胞系MG-63购自武汉大学生命科学院；pSilence2．1-neo载体带有U6启动子和neo基因（Ambin公司）。①以人血管内皮生长因子的mRNA序列5’-GAT AAC GCG GAT ACC TTG G-3’为靶点，体外构建血管内皮生长因子短发夹状RNA表达载体pSilence2．1-neo-VEGF。②人骨肉瘤细胞MG-63常规培养，消化传代后分为3组：短发夹状RNA载体组转染pSilence2，1-neo-VEGF载体，空载体组转染pSilence2．1-neo载体，空白组无特殊处理。③转染后48h收集各组细胞，采用RT—PCR检测血管内皮生长因子mRNA的表达，免疫组化法检测血管内皮生长因子蛋白的表达。结果：①各组细胞血管内皮生长因子mRNA的表达情况：短发夹状RNA载体组VEGF165 mRNA的相对含量显著低于空白组、空载体组（0．181&;#177;0.010，1．064&;#177;0.019，1．052&;#177;0.036，P〈0．01），而空白组和空载体组之间差异无显著性意义。②各组细胞血管内皮生长因子蛋白的表达：空白组、空载体组多数细胞中出现明显的棕黄色颗粒状细胞质着色，而短发夹状RNA载体组细胞着色较淡。短发夹状RNA载体组的阳性细胞率显著低于空白组、空载体组[（20．12&;#177;2．63）％，（92．45&;#177;7．56）％，（91．76&;#177;5．26）％，P〈0．011，而空白组和空载体组之间差异无显著性意义。结论：pSilence2．1-neo—VEGF可显著抑制人骨肉瘤细胞MG-63血管内皮生长因子mRNA及蛋白的表达，为靶向血管的骨肉瘤基因治疗提供了参考依据。     

沉默血管内皮生长因子及促血管生成素-2基因抑制裸鼠人肺腺癌移植瘤生长的实验研究

目的研究沉默血管内皮生长因子(VEGF)及促血管生成素-2(Ang-2)基因表达对裸鼠人肺腺癌移植瘤生物学特性的影响。方法利用基因重组技术构建H1启动子驱动表达针对VEGF及Ang-2siRNA的重组腺病毒Ad-VEGFshRNA及Ad-Ang-2shRNA。制备裸鼠人肺腺癌A549细胞移植瘤模型,观察RNA干扰后(RNAi)对肿瘤生物学特性的影响。结果重组腺病毒接种裸鼠30d后,Ad-VEGFshRNA、Ad-Ang-2shRNA干扰组与对照组比较,肿瘤体积及重量均明显减小,差异均有统计学意义(P<0.01),并且Ad-VEGFshRNA及Ad-Ang-2shRNA联合干扰组与Ad-VEGFshRNA、Ad-Ang-2shRNA干扰组比较能够更有效地抑制肿瘤细胞的生长,差异有统计学意义(P<0.05);而Ad-VEGFshRNA组与Ad-Ang-2shRNA组比较差异无统计学意义(P>0.05)。免疫组织化学染色显示:Ad-VEGFshRNA、Ad-Ang-2shRNA干扰组细胞增殖减慢,凋亡增加,微血管密度明显少于对照组,差异有统计学意义(P<0.01)。并且Ad-VEGF shRNA及Ad-Ang-2shRNA联合干扰组与单基因干扰比较,能够更有效地抑制肿瘤细胞增殖,促进凋亡,差异有统计学意义(P<0.05)。结论 VEGF及Ang-2基因靶向RNA干扰在体内有明显抑制肺腺癌细胞生长的作用,联合抑制VEGF及Ang-2基因的表达能够更有效地抑制肺腺癌的生长。     

体外构建血管内皮生长因子短发夹状RNA表达载体及其对人骨肉瘤细胞相应因子mRNA及蛋白的抑制

目的:设计并构建血管内皮生长因子短发夹状RNA表达载体,检测其对人骨肉瘤细胞血管内皮生长因子mRNA及蛋白表达的影响。方法:实验于2006-01/07在青岛市市立医院试验中心完成。人骨肉瘤细胞系MG-63购自武汉大学生命科学院;pSilence2.1-neo载体带有U6启动子和neo基因(Ambin公司)。①以人血管内皮生长因子的mRNA序列5’-GATAACGCGGATACCTTGG-3’为靶点,体外构建血管内皮生长因子短发夹状RNA表达载体pSilence2.1-neo-VEGF。②人骨肉瘤细胞MG-63常规培养,消化传代后分为3组:短发夹状RNA载体组转染pSilence2.1-neo-VEGF载体,空载体组转染pSilence2.1-neo载体,空白组无特殊处理。③转染后48h收集各组细胞,采用RT-PCR检测血管内皮生长因子mRNA的表达,免疫组化法检测血管内皮生长因子蛋白的表达。结果:①各组细胞血管内皮生长因子mRNA的表达情况:短发夹状RNA载体组VEGF165mRNA的相对含量显著低于空白组、空载体组(0.181±0.010,1.064±0.019,1.052±0.036,P<0.01),而空白组和空载体组之间差异无显著性意义。②各组细胞血管内皮生长因子蛋白的表达:空白组、空载体组多数细胞中出现明显的棕黄色颗粒状细胞质着色,而短发夹状RNA载体组细胞着色较淡。短发夹状RNA载体组的阳性细胞率显著低于空白组、空载体组[(20.12±2.63)%,(92.45±7.56)%,(91.76±5.26)%,P<0.01],而空白组和空载体组之间差异无显著性意义。结论:pSilence2.1-neo-VEGF可显著抑制人骨肉瘤细胞MG-63血管内皮生长因子mRNA及蛋白的表达,为靶向血管的骨肉瘤基因治疗提供了参考依据。     

靶向血管内皮生长因子siRNA对K562细胞凋亡及survivin表达的影响

目的 应用腺病毒介导的血管内皮生长因子(VEGF) siRNA沉默K562细胞中VEGF的表达,观察其对K562细胞凋亡及凋亡抑制基因survivin表达的影响.方法 应用成功构建的携带特异性VEGF siRNA的重组腺病毒(Ad5-VEGF siRNA)感染K562细胞,实验分为3组:实验组(K562/Ad5-VEGF siRNA组)、空载体组(K562/Ad5组)、对照组(K562组).通过RT-PCR法检测细胞内VEGF及survivin mRNA的表达,ELISA法检测细胞培养上清中VEGF蛋白表达,Western blot法检测细胞survivin蛋白表达,流式细胞术检测细胞凋亡.结果 实验组细胞VEGF及survivin mRNA表达水平较对照组均有明显降低(P＜0.01),且细胞培养上清中VEGF蛋白表达水平[(1121±15)pg/ml]低于空载体组[(1290±28)pg/ml]和对照组[(1303±28)pg/ml](P＜0.01).Western blot法检测结果显示,实验组survivin蛋白表达水平(0.26 ± 0.11)较对照组(0.74±0.10)也显著降低(P＜0.01).实验组细胞凋亡率与对照组比较明显增加(P＜0.01).结论 应用RNA干扰沉默K562细胞中VEGF基因表达后,survivin的表达也随之降低,同时细胞凋亡率相应增加.VEGF可诱导K562细胞中survivin基因表达,可能是survivin基因表达的上游调控因子.     

血管内皮细胞生长因子受体2基因短发夹RNA介导基因沉默对白血病的抑制

背景：血管内皮细胞生长因子受体2（vascular endothelial growth factor receptor2，VEGFR2）在血管内皮细胞生长因子的信号转导中有主导效应，并在某些疾病包括白血病的病理性血管生成中起关键作用。另外，白血病细胞分泌的血管内皮细胞生长因子还诱导自身表达VEGFR2．从而维持自身存活、增殖。目的：观察表达VEGFR2短发夹RNA（shorthairpin RNA，shRNA）的慢病毒载体Lenti6，shVEGFR2在全身性NOD／SCID白血病模型鼠中的作用，设计：随机、平行对照、开放性实验。单位：中山大学附属第二医院儿科，中山大学生物工程研究中心。材料：实验于2004-05／2006-01在中山大学附属第二医院医学研究中心、中山大学生物工程研究中心完成。分别以HL60，EA·hy926细胞株作为实验用白血病细胞、人血管内皮细胞的来源。Block-iT Lentiviral RNAi ExpressionSystem（Invitrogen）；humanVEGFR2Mcb（PE标记，R＆D）。CD31组化试剂盒（武汉博士德公司），CD33-PE流式细胞荧光标记抗体（BD公司）。方法：（D制备并筛选VEGFR2基因的最有效siRNA，并据此设计shRNA寡核苷酸链。②构建pU6／shVEGFR2入门克隆。瞬时转染细胞，构建表达克隆Lenti6／shVEGFR2，与VimPowed”PackagingMix共转染到293FT“细胞中，产生携带表达克隆的慢病毒载体。③pU6／shVEGFR2入门克隆转染和Lenti6／shVEGFR2转导HL60细胞，MTT法测定HL60细胞抑制率。④建立HL60白血病模型后观察注入人血管内皮细胞后小鼠骨髓微血管的分布。⑤将NODISCID小鼠20只随机分为4组，每组5只：白血病模型鼠组，白血病模型鼠静脉注射重组慢病毒组，白血病模型鼠静脉注射重组慢病毒和内皮细胞组及白血病模型鼠静脉注射内皮细胞组。检测各组小鼠的骨髓细胞CD33表达变化、骨髓微血管密度变化及外周血细胞涂片、肝、脾的肿瘤浸润情况，以了解Lenti6／shVEGFR2重组慢病毒对小鼠白血病的作用。主要观察指标：①VEGFR2siRNA鉴定结果。②pU6，shVEGFR2入门克隆转染HL60后对其VEGFR2表达的影响。③pU6，shVEGFR2入门克隆转染、Lenti6／shVEGFR2表达克隆转导HL60细胞后细胞抑制率比较。④慢病毒介导的shRNA对HL60模型鼠VEGFNEGFR2旁分泌和自分泌的影响。结果：①pU6，shVEGFR2入门克隆转染HL60后对其VEGFR2表达的影响：VEGFR2siRNAc抑制HL60细胞效率最高，利用其构建的pU6／shVEGFR2入门克隆并转染HL60，抑制细胞效率达84．9％；显著抑制HL60细胞VEGFR2基因mRNA和蛋白的表达。②pU6／shVEGFR2入门克隆转染、Lenti6／shVEGFR2表达克隆转导HL60细胞后细胞抑制率比较：pU6／shVEGFR2入门克隆转染、Lenti6／shVEGFR2表达克隆转导HL60细胞后48h细胞抑制率相近，48h后入门克隆转染组细胞抑制率明显下降（P〈0．01）；而表达克隆转导组细胞抑制率变化缓慢，在96h达高峰，120h稍降，变化无显著差异。③慢病毒介导的shRNA对HL60模型鼠VEGFNEGFR2旁分泌和自分泌的影响：白血病模型组、白血病模型鼠静脉注射重组慢病毒组、白血病模型鼠静脉注射重组慢病毒和内皮细胞组小鼠骨髓流式细胞仪HL60检测结果分别为（25．8％±4．9）％，（14．3％±5．1）％．（8．4＋2．6）％，三组比较，差异有显著性意义（P〈0．05）；白血病模型鼠静脉注射重组慢病毒和内皮细胞组微血管密度明显小于白血病模型鼠静脉注射内皮细胞组。白血病模型鼠静脉注射重组慢病毒和内皮细胞组与其他组相比HL60细胞数最低。结论：①pLenti6／shVEGFR2表达克隆转导HL60细胞后持续高水平抑制细胞。重组慢病毒Lenti6／shVEGFR2具有抑制白血病小鼠骨髓血管生成的作用。②VEGFR2siRNA可阻断白血病HL60细胞VEGF／VEGFR2自分泌的内环路，在体外、动物体内均得到验证。③VEGFNEGFR2自分泌链和旁分泌链的联合阻断加强了抗白血病效果：     

Pigment epithelium-derived factor inhibits vascular endothelial growth factor-induced vascular hyperpermeability both in vitro and in vivo

Administration of pigment epithelium-derived factor (PEDF) inhibits advanced glycation end products-elicited retinal vascular hyperpermeability, as well as cold injury-induced brain oedema in rats. However, the underlying molecular mechanism by which PEDF blocks the hyperpermeability in vivo is not fully understood. This study investigated whether PEDF could inhibit vascular endothelial growth factor (VEGF)-induced vascular hyperpermeability both in vitro and in vivo. The Miles assay revealed that, after intradermal injection of VEGF in nude mice, simultaneous administration of PEDF inhibited vascular hyperpermeability in a dose-dependent manner. The in vitro permeability assay also showed that PEDF blocked the VEGF-induced barrier dysfunction in endothelial cells. These results demonstrated that PEDF could inhibit the VEGF-induced vascular hyperpermeability both in vitro and in vivo, and suggest that PEGF may be suitable to be considered as a novel therapeutic agent for various vasopermeable disorders in which VEGF is involved.     

人血小板源性生长因子A基因发夹结构小干扰RNA真核表达载体的构建及鉴定

目的：血小板源性生长因子对诱发视网膜色素上皮细胞移行、最终导致增生性玻璃体视网膜病变起到了重要作用。构建人血小板源性生长因子A具有发夹结构小干扰RNA（shRNA）表达质粒，以观察其对人血小板源性生长因子A基因表达的沉默效果。 方法：实验于2007—04／2007—07在吉林大学第一临床医院传染科研究室完成。根据血小板源性生长因子A基因mRNA序列，设计有小发夹结构的3条寡核苷酸序列，克隆到空载体pGPU6／GFP／Neo中，构建重组质粒，同时设计构建分别针对人GAPDH干扰质粒作为阳性对照,不针对任何特异基因的质粒作为阴性对照。通过酶切鉴定和测序验证阳性克隆。 结果：经重组质粒筛选，重组质粒测序鉴定与设计的shRNA转录模板序列相同，均证实重组质粒构建成功。 结论：利用RNA干扰技术路线可成功构建靶向人血小板源性生长因子A的发夹结构小干扰RNA表达载体。     

Fasudil inhibits vascular endothelial growth factor-induced angiogenesis in vitro and in vivo

Vascular endothelial growth factor (VEGF)-induced endothelial cell migration is an important component of tumor angiogenesis. Rho and Rho-associated kinase (ROCK) are key regulators of focal adhesion, stress fiber formation, and thus cell motility. Inhibitors of this pathway have been shown to inhibit endothelial cell motility and angiogenesis. In this study, we investigated the antiangiogenic effect of fasudil, one of the ROCK inhibitors. Fasudil inhibited VEGF-induced endothelial cell migration, viability, and tube formation in vitro in human umbilical vein endothelial cells. VEGF-induced endothelial cell migration was reduced by fasudil associated with loss of stress fiber formation, focal adhesion assembly, and with the suppression of tyrosine phosphorylation of focal adhesion proteins. Furthermore, fasudil inhibited VEGF-induced phosphorylation of myosin light chain, which is one of the main substrates of ROCK. Therefore, the effect of fasudil was suggested to be ROCK dependent. Fasudil not only inhibited VEGF-induced cell proliferation but also reversed the protective effect of VEGF on apoptosis, which resulted in the decrease of cell viability. Moreover, fasudil inhibited VEGF-induced angiogenesis in a directed in vivo angiogenesis assay. These data are the first demonstration that fasudil has antiangiogenic properties. Therefore, fasudil might be useful for the treatment of angiogenesis-related diseases, especially cancer.     

Small interfering RNA targeting of S phase kinase-interacting protein 2 inhibits cell growth of oral cancer cells by inhibiting p27 degradation

S phase kinase-interacting protein 2 (Skp2), an F box protein, is required for the ubiquitination and consequent degradation of p27. It is well known that reduced expression of p27 is frequently observed in various cancers including oral squamous cell carcinoma and is due to an enhancement of its protein degradation. Our previous study showed that overexpression of Skp2 was frequently found in oral squamous cell carcinoma and inversely correlated with p27 expression. Recently, a technique known as RNA interference has been successfully adapted to mammalian cells. In the present study, we investigated if small interfering RNA (siRNA)-mediated gene silencing of Skp2 can be employed in order to inhibit p27 down-regulation in oral squamous cell carcinoma. We used a siRNA plasmid vector, which has an advantage over synthetic siRNAs in determining the effects of decreasing the high constitutive levels of Skp2 protein in oral squamous cell carcinoma. We showed that Skp2 siRNA transfection decreased Skp2 protein and induced the accumulation of p27 protein in oral squamous cell carcinoma cells. Moreover, p27 protein in Skp2 siRNA-transfected cells is more stabilized than that in control siRNA-transfected cells. Interestingly, Skp2 siRNA inhibited the cell proliferation of oral squamous cell carcinoma cells both in vitro and in vivo. Our findings suggest that siRNA-mediated gene silencing of Skp2 can be a novel modality of cancer gene therapy for suppression of p27 down-regulation.     

Interference of lysine-specific demethylase 1 inhibits cellular invasion and proliferation in vivo in gastric cancer MKN-28 cells

BackgroundLysine-specific demethylase 1(LSD1), the first identified histone demethylase, plays an important role in the epigenetic regulation of gene activation and repression. Up-regulated LSD1expression has been reported in several malignant tumors.Our aim, therefore, was to better understand the mechanisms underlying the upregulation of LSD1 in gastric cancer.MethodsWe used lentiviral shRNA to knockdown LSD1 in the gastric cancer MKN-28 cell line. Cell proliferation was measured by MTT assay while cell apoptosis was assessed by Annexin V-FITC/PI double staining flow cytometry. The invasive potential of gastric cancer cells was determined by matrigel invasion assay. Protein expression was detected by Western blot. In vivo, the effect of knocking down LSD1 on tumor growth and protein expression in gastric cancer cells in nude mice was investigated.ResultsLSD1 knockdown in MKN-28 cell lines resulted in increasing the activity of cisplatin in vitro and the inhibition of cancer cell proliferation and invasion, and induced cell apoptosis. The expression of TGF-β1, VEGF, Bcl-2, β-catenin, p-ERK and p-Smad 2/3 proteins was inhibited in LSD1 knockdown cells. Moreover, in an in vivo model of gastric cancer, LSD1 knockdown suppressed tumor growth and protein expression.ConclusionLSD1 knockdown affected the fuction of gastric cancer MKN-28 cell line. LSD1 may be a latent target in the diagnosis and therapy of gastric cancer.     

Knockdown of c-Met by adenovirus-delivered small interfering RNA inhibits hepatocellular carcinoma growth in vitro and in vivo

c-Met is highly expressed and constitutively activated in various human tumors. We employed adenovirus-mediated RNA interference technique to knock down c-Met expression in hepatocellular carcinoma cells and observed its effects on hepatocellular carcinoma cell growth in vitro and in vivo. Among the five hepatocellular carcinoma and one normal human liver cell lines we analyzed, c-Met was highly expressed and constitutively tyrosine phosphorylated in only MHCC97-L and HCCLM3 hepatocellular carcinoma cells. Knockdown of c-Met could inhibit MHCC97-L cells proliferation by arresting cells at G0-G1 phase. Soft agar colony formation assay indicated that the colony forming ability of MHCC97-L cells decreased by approximately 70% after adenovirus AdH1-small interfering RNA (siRNA)/met infection. In vivo experiments showed that adenovirus AdH1-siRNA/met inhibited the tumorigenicity of MHCC97-L cells and significantly suppressed tumor growth when injected directly into tumors. These results suggest that knockdown of c-Met by adenovirus-delivered siRNA may be a potential therapeutic strategy for treatment of hepatocellular carcinoma in which c-Met is overexpressed.