新型基因载体G5-PAMAM-D在前列腺癌HSV-tk/GCV自杀基因系统中的应用

背景与目的:考察纳米级阳离子聚酰胺-胺型树枝状聚合物(polyam idoam ine dendrimers,PAMAM-D)作为基因载体进行前列腺癌自杀基因治疗的可行性,为前列腺癌的基因治疗寻找新的基因载体。方法:以第5代PAMAM-D(G5-PAMAM-D)为载体将含增强型荧光蛋白(EGFP)基因片段的重组质粒pEGFP-C1转染至前列腺癌细胞系PC-3和22Rv1,成功表达EGFP后,再以G5-PAMAM-D为载体将含HSV-tk自杀基因的真核表达重组质粒pcDNA3-tk转染至前列腺癌细胞系PC-3和22Rv1,转染48h后,对转染的两种细胞给予浓度为0、10、100、1000、10000μmol/L前体药更昔洛韦(Ganciclovir,GCV),24h后采用MTT比色法测定药物对细胞增殖的影响。制作前列腺癌皮下荷瘤小鼠,将G5-PAMAM-D/pcDNA3-tk复合物瘤内注射,24h后腹腔注射GCV,观察这一复合物体内对肿瘤生长的抑制作用。结果:荧光观察及FCM结果证明G5-PAMAM-D可将pEGFP-C1转入两种前列腺癌细胞并表达EGFP。采用G5-PAMAM-D将pcDNA3-tk重组质粒转染PC-3和22Rv1细胞,给不同浓度的前体药GCV后,实验组细胞与对照组相比有明显浓度依赖性生长抑制。荷瘤小鼠在给予瘤内注射G5-PAMAM-D/pcDNA3-tk复合物后,行腹腔注射GCV,治疗后第40天,实验组肿瘤体积(1135±245mm3)与对照组裸质粒组(9965±2109mm3)和PAMAM-D组(8357±1956mm3)相比肿瘤生长明显被抑制(P<0.01),生存时间延长。结论:G5-PAMAM-D可作为前列腺腺癌自杀基因治疗的基因载体,有良好的应用前景。     

PSMAe/p特异性驱动shRNA靶向干扰前列腺癌LNCap细胞的研究

探讨前列腺特异性膜抗原增强子/启动子（PSMAe/p）驱动短发夹RNA（shRNA）靶向干扰前列腺癌细胞的特异性。方法:运用RNA干扰技术,以转铁蛋白-聚乙二醇-聚乙烯亚胺（Tf-PEG-PEI）作为基因转移载体,以前列腺癌LNCap细胞为研究对象,并以前列腺癌PC-3细胞、膀胱癌T24细胞及人胚肾HEK293细胞做为对照,将外源增强绿色荧光蛋白（EGFP）基因及以PSMAe/p为启动序列靶向EGFP的干扰质粒转入培养细胞,以实时定量PCR（qRT-PCR）及Western blot分别检测EGFP基因及蛋白在4种细胞各组中的表达情况,研究PSMAe/p驱动shRNA靶向干扰前列腺癌细胞的特异性。结果:在前列腺癌LNCap细胞组中,与单独转入EGFP基因（pEGFP-C1）的细胞组（A组）及转入EGFP基因（pEGFP-C1）+空载体质粒组（pPSMAe/p-UPRT）（C组）相比,转入EGFP基因（pEGFP-C1）及干扰质粒［pPSMAe/p-shEGFP-poly（A）］组（B组）,EGFP基因表达及蛋白表达水平下调,分别与对照组（A组、C组）相比差异具有统计学差异（P<0.05）,而转入EGFP基因（pEGFP-C1）的细胞组（A组）和转入EGFP基因（pEGFP-C1）+空载体质粒组pPSMAe/p-UPRT）（C组）间比较差异无统计学意义（P>0.05）;而在前列腺癌PC-3细胞、膀胱癌T24细胞及人胚肾HEK293细胞中EGFP基因及蛋白表达水平在实验组与对照组、对照组间比较差异无统计学意义（P>0.05）。结论:PSMAe/p驱动shRNA具有细胞特异性,其可驱动特异基因片段在前列腺癌LNCap细胞中表达,从而实现基因治疗的细胞特异性,可作为前列腺癌基因治疗的靶向策略之一。      

RNA干扰技术抑制smad4基因表达对宫颈癌细胞增殖的影响

目的：探讨用RNA干扰技术下调smad4基因表达对宫颈癌细胞增殖的影响。方法：用DNA重组技术将针对人smad4基因不同部位所设计的三对短发夹状RNA（shorthairpinRNA，shRNA）序列克隆到真核表达质粒pGenesil-1中，构建smad4shRNA表达载体pGenesil—smad4一shRNA1、2、3。脂质体介导转染人宫颈癌细胞株HeLa，经G418筛选抑制smad4表达的稳定细胞克隆。MTT法、克隆形成实验、流式细胞术检测抑制smad4基因对宫颈癌细胞增殖的影响。结果：成功构建分别携带三段shRNA及空载体对照的重组质粒pGenesil—smad4-shRNA1、2、3和pGenesil-con，三种shRNA重组质粒中pGenesil—smad4-shRNA2可明显降低细胞内smad4mRNA及smad4蛋白表达，筛选出的HeLa／shRNA2细胞增殖能力明显增强。结论：应用RNA干扰技术能筛选出特异而高效阻断smad4基因表达及功能的shRNA，smad4基因表达下调能明显促进宫颈癌细胞生长。     

RNA干扰对MDA-MB-231乳腺癌细胞株骨保护素基因表达的抑制作用

目的 构建针对骨保护素（OPG）基因的一个载体编码三条短发夹RNA（shRNA）的真核表达质粒,观察其对MDA—MB-231乳腺癌细胞株OPG表达的抑制作用。方法选择3个针对OPG基因的RNA干扰（RNAi）位点,分别设计合成3对编码相应shRNA的DNA单链,每对单链连接形成双链后分别与线性化载体pGenesil-1．1、pGenesil-1．2、pGenesil-1．3连接形成pGenesil-1．1-shRNAl、pGenesil-1．2-shRNA2、pGenesil-1．3-shRNA3,对以上重组载体反复酶切连接,构建成重组质pGenesil—1．1—1．2-1．3-shRNA1-shRNA2-shRNA3,酶切鉴定和测序无误后,将该质粒转染MDA—MB一231细胞,以G418加压筛选,对转染细胞行单克隆化,稳定转染细胞行RT—PCR和Westernblot检测,确定其对OPG基因表达抑制作用。统计分析采用单因素方差分析和SLD分析法。结果成功构建了pGenesil-1．1—1．2-1．3-shRNA1-shRNA2-shRNA3重组质粒;以该质粒稳定转染MDA—MB-231细胞后其OPGmRNA和蛋白表达均较对照差异有统计学意义（P〈0．05）,RNAi对OPGmRNA和蛋白的表达抑制率分别为91％和73％。结论本研究构建了针对OPG的一个载体编码3条shRNA的真核表达质粒,通过RNAi抑制了MDA—MB-231细胞OPG基因表达,为进一步深入探讨肿瘤细胞自身OPG表达在乳腺癌骨转移发生发展中的作用提供了相关实验基础。     

壳聚糖纳米粒介导的CD基因对前列腺癌的体外杀伤作用

目的考察壳聚糖(Chitosan)纳米粒作为自杀基因载体进行前列腺癌自杀基因治疗的可行性,为前列腺癌的基因治疗寻找新的基因载体。方法将壳聚糖纳米粒包裹含报告基因质粒pEGFP-C1转染至前列腺癌细胞系PC-3和22Rvl,观察EGFP的表达情况,再将含胞嘧啶脱氨酶(Cytosinedeamiase,CD)自杀基因的真核表达质粒pcDNA3-CD转染至该2种前列腺癌细胞,转染48h后,以RT-PCR检测CD基因的表达。对转染的两种细胞给予不同浓度的前体药5-氟胞嘧啶(5-fluorocytosine,5-FC)24h后,采用MTT比色法测定药物对细胞增殖的影响,并进行统计学处理。结果表明壳聚糖纳米粒能够将质粒pEGFP-C1转入2种前列腺癌细胞,并表达EGFP,在PC-3细胞其转染效率高于脂质体转染试剂。采用壳聚糖纳米粒转染pcDNA3-CD于2种前列腺癌细胞后,RT-PCR证明在癌细胞中有CD基因的表达。给予前体药物5-FC后,实验组细胞生长抑制率与只给壳聚糖或裸质粒的对照组相比明显增高,说明壳聚糖纳米粒可将CD自杀基因递送至前列腺癌细胞中,并在细胞中进行表达,从而使CD/5-FC自杀基因系统发挥杀伤细胞的作用。结论壳聚糖纳米粒能将质粒基因转入前列腺癌细胞并表达,可做为前列腺癌自杀基因治疗的基因载体,有良好的应用前景。     

叶酸修饰的G5-PAMAM-D介导HSV-TK/GCV自杀基因系统治疗前列腺癌的体外实验研究*

目的:探讨叶酸修饰的第五代聚酰胺- 胺型树枝状聚合物（G5-PAMAM-D-fol ）作为基因载体进行前列腺癌自杀基因治疗的可行性。方法:以G5-PAMAM-D-fol 为基因载体将含有自杀基因HSV-TK 的重组质粒pcDNA 3-tk转染至前列腺癌细胞系PC- 3 和LNCaP ,24h 后以RT-PCR 法检测HSV-TK 基因在两种前列腺癌细胞系中的mRNA 表达;再次转染后24h,对转染的两种细胞分别给予不同浓度的前体药物更昔洛韦（GCV ）,48h 后采用四甲基偶氮唑盐比色法（MTT 法）检测细胞抑制率。结果:RT-PCR结果证明G5-PAMAM-D-fol 可将重组质粒pcDNA 3-tk转染2 种前列腺癌细胞并转录HSV-TK 基因。MTT 实验反映出A 组（G5-PAMAM-D-fol/pcDNA 3-tk）对PC- 3 和LNCaP 细胞有明显的杀伤作用,并且比B 组（G5-PAMAM-D/pcDNA3-tk）的细胞抑制率更高。C 组（G5-PAMAM-D-fol ）和D 组（G5-PAMAM-D）随着GCV 浓度的增加细胞生长并未受到明显的抑制,而且前者并未表现出比后者更明显的细胞杀伤作用。结论:G5-PAMAM-D-fol 能够在体外将自杀基因重组质粒成功转入前列腺癌细胞并表达HSV-TK 基因,其靶向性强且细胞毒性低。      

PCNA的shRNA的真核表达载体的构建及对子宫颈癌细胞的干预性研究

目的构建PCNA的小发夹结构RNA(shRNA)的真核表达载体并在Hela细胞表达,同时观察PCNA的shRNA对人Hela细胞株体外增殖的影响及生物学特性的改变。方法将PCNA的cD-NA的shRNA引物插入真核表达载体pGenesil-1,构建真核表达质粒pGenesil-1-PCNA1-4,并通过酶切和测序等方法进行鉴定,将真核表达质粒pGenesil-1-PCNA1-4转染子宫颈癌细胞,运用Westernblot检测PCNA的蛋白表达,流式细胞仪分析细胞周期变化。结果PCNA的shRNA能显著抑制人Hela细胞的生长,阻滞细胞G0/G1—S期,PCNA的蛋白表达同时受到抑制。结论PCNA的shRNA能显著抑制人Hela细胞的生长,其抑制作用可能通过阻断PCNA蛋白表达实现,实验结果为进一步研究PC-NA的shRNA对子宫颈癌的基因治疗奠定了基础。     

bi-1短发夹状RNA重组真核表达质粒对鼻咽癌和卵巢癌细胞增殖的影响

目的 以真核表达质粒pmU6为基础,针对bi 1基因构建在细胞内表达短发夹状 RNA (shRNA) 的质粒载体,并观察它们对CNE 2Z、CNE 1、HO8910PM、HO8910细胞株生长增殖的影响。方法 人工合成bi 1寡核苷酸链,退火、定向克隆入pmU6载体中产生重组质粒,将重组质粒转染到细胞中,MTT比色法观察转染试剂及质粒载体对细胞生长增殖的影响。结果 与未处理组相比,bi 1shRNA对CNE 2Z、CNE 1、HO8910PM细胞株的生长增殖有明显的抑制作用,而对HO8910细胞株的生长增殖无明显抑制作用。结论 重组质粒能在细胞内表达shRNA,产生RNA干扰(RNA interference, RNAi)效应并特异性抑制靶细胞的生长增殖,为质粒介导的RNAi技术应用于鼻咽癌和卵巢癌的基因治疗提供一定的理论依据。     

RNAi抑制STAT3基因表达对人宫颈癌SiHa细胞化疗敏感性的影响

目的:探讨信号转导因子与转录激活因子3(STAT3)短发夹RNA(short hairpin RNA, shRNA)真核表达载体对宫颈癌SiHa细胞化疗敏感性的作用.方法:构建STAT3基因shRNA真核表达质粒并转染宫颈癌SiHa细胞,RT-PCR及免疫蛋白印迹分别检测STAT3基因的mRNA及蛋白表达水平.细胞经不同浓度顺铂(DDP)作用后,流式细胞术(FCS)检测细胞凋亡率,四甲基偶氮唑蓝比色(MTT)法检测细胞生长抑制率并计算DDP的50%抑制浓度(IC50).结果:与各对照组细胞相比,转染STAT3基因shRNA真核表达载体的SiHa细胞,STAT3基因在mRNA和蛋白水平表达均下降,凋亡率均明显增加,细胞对DDP的IC50值明显下降,差异均有统计学意义.结论:针对STAT3基因的shRNA真核表达载体有效地抑制宫颈癌SiHa细胞STAT3基因表达,增强其对化疗药物DDP的敏感性.     

LRIG1基因特异性RNA干扰表达载体的构建、鉴定和稳定株的筛选

目的构建针对LRIG1（leucine-rich repeats and immunoglobulin-like domains 1,LRIG1）基因的特异性RNA干扰质粒,稳定转染人胶质瘤GL15细胞系,观察其对目的基因LRIG1表达的影响,为探讨LRIG1基因沉默对人脑胶质瘤细胞的生物学行为调控奠定基础。方法根据GenBank提供的LRIG1基因序列设计2条RNA干扰序列,命名为LRIG1-shRNA1、LRIG1-shRNA2,并设计1条非特异性序列作为阴性对照,命名为pGenesil2-negative shRNA。合成各自的寡核苷酸链,退火后与pGenesil2质粒载体连接,转化扩增后测定序列。用不同浓度的G418作用于GL15细胞确定G418对GL15的筛选浓度。将3种重组表达载体转染GL15细胞,G418筛选后挑单克隆并扩增获得稳定株。Western印迹法在蛋白水平上检测LRIG1的表达。结果重组pGenesil2-LRIG1-shRNA质粒经限制性酶切及DNA测序分析证明序列插入正确。G418对GL15细胞的筛选浓度为600mg/L,筛选出稳定转染三种质粒的GL15细胞,转染pGenesil2-LRIG1-shRNA1组细胞LRIG1蛋白表达明显低于转染pGenesil2-negative shRNA组。结论成功构建了针对LRIG1基因的特异性shRNA表达载体（pGenesil2-LRIG1-shRNA1）,转染细胞后可抑制LRIG1基因表达,为下一步研究其功能奠定基础。     

STAT3特异性小干扰RNA表达载体的构建及其对STAT3表达的抑制

目的　构建特异性抑制信号转导与转录激活因子3(STAT3)的小干扰RNA(siRNA)表达载体并检测其对STAT3表达及PC 3细胞增殖的抑制作用。方法　设计、合成STAT3特异性的短链寡核苷酸,经退火形成双链DNA片段,克隆到pSilencerTM2.1 U6载体中,构建STAT3特异siRNA的表达载体,HindIII和BamHI双酶切及测序鉴定重组体,并转染PC 3细胞,G418筛选出稳定表达细胞株,半定量反转录聚合酶链反应(RT PCR)方法和免疫印迹法检测STAT3mRNA和蛋白的表达水平,四甲基偶氮唑蓝(MTT)检测细胞增殖活性。结果　经双酶切与测序鉴定成功构建STAT3 siRNA表达载体,转染前列腺癌细胞株PC 3可显著抑制细胞中STAT3mRNA和蛋白的表达水平(抑制率分别为52.4% 及50.5% ),且细胞增殖活性亦较未转染PC 3细胞显著降低(p<0.05)。结论　运用pSilencerTM2.1 U6载体构建的STAT3 siRNA表达载体可有效抑制STAT3的表达及前列腺癌细胞的增殖。     

肿瘤特异性Survivin、Livin共沉默RNAi载体的构建及其在前列腺癌细胞中的RNA干涉作用

目的:构建含Survivin基因启动子的、肿瘤特异性Survivin、Livin共沉默RNAi载体,研究该载体在前列腺癌细胞中的RNA沉默作用。方法:运用分子克隆技术,选取Survivin、Livin基因RNAi特异性序列,构建以Survivin、Livin基因为靶点的、含Survivin启动子的CGM30 miR-30 shRNA载体。经测序验证,用脂质体法转染PC-3细胞,RT-PCR、免疫印迹实验检测Survivin、Livin的表达变化,并用流式细胞术检测转染后PC-3细胞的凋亡变化。结果:构建的共沉默RNAi重组载体转染PC-3细胞后,Survivin、Livin基因在mRNA和蛋白水平上的表达均明显下调（P＜0.01）,且转染后的细胞凋亡增加了约15％,而正常前列腺上皮BPH-1细胞中则无此作用。结论:成功构建了含Survivin启动子、特异性沉默Survivin、Livin基因表达的RNAi共沉默载体CGM30-SP-svv-liv,转染该重组质粒后可促进肿瘤细胞的凋亡。     

CXCR1/CXCR2受体拮抗剂——G31P抑制前列腺癌转移的体内外实验研究

目的研究CXCR1/CXCR2受体拮抗剂——G31P对人前列腺癌PC-3细胞的体内外转移的抑制作用。方法采用细胞划痕实验研究G31P对PC-3细胞体外转移的抑制作用。通过建立体内的绿色荧光蛋白(GFP)标志人雄激素非依赖性前列腺癌PC-3细胞的裸鼠原位移植瘤模型,观察G31P对裸鼠前列腺癌原位移植瘤体内转移的影响。结果细胞划痕实验结果显示,G31P 100μg/L处理组作用细胞72小时的迁移率为(50.17±22.43)%(P<0.05,与对照组比较)。与对照组(100μL N.S)比较,G31P处理组(0.5 mg/kg)淋巴结(腰淋巴结、肠系膜淋巴结、远处淋巴结)转移略有减少,但差异无统计学意义;胰腺和肝部转移则明显减少(P<0.05)。免疫组织化学结果显示,与对照组比较,G31P处理组MMP-9(P<0.05)的表达明显减少。结论在体内外实验中G31P均能抑制人雄激素非依赖性前列腺癌PC-3细胞系的转移。     

ADAM15 supports prostate cancer metastasis by modulating tumor cell-endothelial cell interaction

Using human tumor and cDNA microarray technology, we have recently shown that the ADAM15 disintegrin is significantly overexpressed during the metastatic progression of human prostate cancer. In the current study, we used lentiviral-based short hairpin RNA (shRNA) technology to down-regulate ADAM15 in the metastatic prostate cancer cell line, PC-3. ADAM15 down-regulation dramatically attenuated many of the malignant characteristics of PC-3 cells in vitro and prevented the s.c. growth of PC-3 cells in severe combined immunodeficient (SCID) mice. By inhibiting the expression of ADAM15 in PC-3 cells, we showed decreased cell migration and adhesion to specific extracellular matrix proteins. This was accompanied by a reduction in the cleavage of N-cadherin by ADAM15 at the cell surface. Fluorescence-activated cell sorting analysis revealed reduced cell surface expression of the metastasis-associated proteins alpha(v) integrin and CD44. Furthermore, matrix metalloproteinase 9 secretion and activity were abrogated in response to ADAM15 reduction. In an in vitro model of vascular invasion, loss of ADAM15 reduced PC-3 adhesion to, and migration through, vascular endothelial cell monolayers. Using an SCID mouse model of human prostate cancer metastasis, we found that the loss of ADAM15 significantly attenuated the metastatic spread of PC-3 cells to bone. Taken together, these data strongly support a functional role for ADAM15 in prostate tumor cell interaction with vascular endothelium and the metastatic progression of human prostate cancer.     

Hypoxia regulates choline kinase expression through hypoxia-inducible factor-1 alpha signaling in a human prostate cancer model

The intensity of the total choline (tCho) signal in spectroscopic images of tumors is spatially heterogeneous. The likewise heterogeneous physiologic tumor microenvironment may contribute to this heterogeneity. We therefore investigated the relationship between hypoxia, choline metabolites, and choline kinase (Chk) in a human prostate cancer model. Human PC-3 prostate cancer cells were engineered to express enhanced green fluorescent protein (EGFP) under hypoxic conditions. These PC-3-5HRE-EGFP cells were characterized in culture and as tumors transplanted in mice using (1)H magnetic resonance spectroscopy (MRS) and MRS imaging (MRSI) combined with EGFP fluorescence microscopy and imaging. Hypoxic EGFP-fluorescing tumor regions colocalized with regions of high tCho in combined MRSI and optical imaging studies. Cellular phosphocholine (PC) and tCho concentrations as well as Chk expression levels significantly increased following exposure of PC-3 cells to hypoxia. A putative promoter region located 5' of the translation start site of the human chk-alpha gene was cloned and luciferase (Luc)-based reporter vector constructs were generated. Luc reporter assays provided evidence that some of the putative hypoxia response elements (HRE) within this putative chk-alpha promoter region functioned in vitro. Chromatin immunoprecipitation assays using an antibody against hypoxia-inducible factor (HIF)-1 alpha showed that HIF-1 can directly bind this region of the endogenous chk-alpha promoter in hypoxic PC-3-5HRE-EGFP cells. These data suggest that HIF-1 activation of HREs within the putative chk-alpha promoter region can increase Chk-alpha expression within hypoxic environments, consequently increasing cellular PC and tCho levels within these environments.     

Heparanase promotes bone destruction and invasiveness in prostate cancer

Heparanase is an endoglycosidase that plays an important role in angiogenesis and metastasis of cancer. Herein we evaluate the effect of heparanase overexpression on invasiveness and bone destruction in prostate cancer bone metastases. The human prostate cancer cell line PC-3 was stably transfected with a plasmid containing the cDNA for human heparanase or with the vector alone as a control. Overexpression of heparanase did not affect the growth of PC-3 cells, but did promote invasiveness of the cells in an in vitro assay. Both cell types were injected into the tibias of nude mice. Four weeks later, the mice were examined radiologically prior to sacrifice and samples of leg tissue were taken to investigate bone destruction and metastasis. Mice injected with PC-3 cells overexpressing heparanase had more severe bone destruction and larger, more invasive, tumors. These results demonstrate that heparanase overexpression can facilitate tumor invasion and accelerate bone destruction caused by prostate cancer bone metastasis.     

壳聚糖纳米粒介导的HSV-tk基因对前列腺癌的体外杀伤作用

目的：考察壳聚糖纳米粒作为自杀基因载体进行前列腺自杀基因治疗的可行性，为前列腺癌的基因治疗寻找新的基因载体。方法：将壳聚糖纳米粒包裹的含报告基因的质粒pEGFP—C1分别转染前列腺癌细胞PC-3和22Rvl，并观察EGFP的表达情况。随后将含HSV-tk自杀基因的真核高效表达质粒pcDNA3-tk经壳聚糖纳米粒包裹后转染上述2种前列腺癌细胞，转染48h后，以RT-PCR检测HSV-tk基因的表达情况。最后对转染的2种细胞给予不同浓度的前体药更昔洛韦（ganci—clovir，GCV）作用24h后，采用MTT比色法测定药物对细胞增殖的影响，并进行统计学分析。结果：壳聚糖纳米粒能够将质粒pEGFP-C1转入2种前列腺癌细胞，并表达EGFP，在PC-3细胞中壳聚糖纳米介导的转染效率高于脂质体。采用壳聚糖纳米粒转染pcDNA3-tk于2种前列腺癌细胞后，RT-PCR证明在癌细胞中有HSV-tk基因的表达。给予前体药物GCV后，实验组细胞生长抑制率与只给壳聚糖或裸质粒的对照组相比明显增高，说明HSV-tk自杀基因纳米粒在细胞中表达，从而使HSV-tk／GCV自杀基因系统发挥杀伤细胞的作用。结论：壳聚糖纳米粒能将质粒基因pcDNA3-tk转入前列腺癌细胞并表达胸苷激酶发挥作用，可做为前列腺癌自杀基因治疗的基因载体。     

shRNA-targeted hTERT suppress cell proliferation of bladder cancer by inhibiting telomerase activity

RNA interference (RNAi) has demonstrated profound prospect in human gene research. hTERT, the rate-limiting component of telomerase activity, is highly expressed in bladder cancer cells. Here, we investigated the anti-proliferation effects of small hairpin interfering RNA (shRNA)-targeted hTERT gene on bladder cancer in vitro and in vivo. The results showed that ph2-shRNA, the most-effective vector carrying shRNA-targeted hTERT, could significantly inhibit the cell proliferation by down-regulating hTERT expression, decreasing telomerase activity, decreasing cell number of S phase, increasing the cell number of G0/G1 phase in T24 cells and xenograft tumor tissues, and attenuate the tumor growth of xenograft mice model compared with controls. Our results demonstrate that hTERT-directed shRNAs are potent inhibitors of bladder cancer. Lin Zou and Penghui Zhang were equally contributed to this paper.