Downregulation of bcl-2 expression in lymphoma cells by bcl-2 ARE-targeted modified,synthetic ribozyme

Synthetic ribozymes are catalytic RNA molecules designed to inhibit gene expression by cleaving specific mRNA sequences. We investigated the potential of synthetic ribozymes to inhibit bcl-2 expression in apoptosis defective bcl-2 overexpressing tumors. A chemically stabilized hammerhead ribozyme has been targeted to the A+U-rich regulative element of bcl-2 mRNA that is involved in bcl-2 gene switch-off during apoptosis. The design of the ribozyme was based on the results of probing accessibility of the RNA target in cellular extracts with antisense DNA. The ribozyme was lipotransfected to a bcl-2 overexpressing human lymphoma cell line (Raji). The cellular uptake of this ribozyme resulted in a marked reduction of both bcl-2 mRNA and BCL-2 protein levels and dramatically increased cellular death by apoptosis. Our results suggest a potential therapeutic application of such ribozyme for the treatment of bcl-2 overexpressing tumors.     

靶向survivin基因siRNA对宫颈癌细胞放射敏感性影响的实验 

 目的 探讨survivin表达对宫颈癌放射敏感性的影响。 方法 RT-PCR和Western blot法分析survivin基因mRNA和蛋白的表达, 平板集落形成实验检测细胞 增殖,流式细胞术检测细胞凋亡率。 survivin siRNA转染HeLa细胞,比较survivin siRNA对细胞凋亡、增殖以及放射敏感性的 影响。 结果 在宫颈癌HeLa细胞中存在survivin的表达,survivin siRNA可诱导HeLa细胞凋亡并抑制增殖 。 结论 survivin siRNA可通过影响细胞凋亡和增殖来增加HeLa细胞的放射敏感性     

Impaired expression of a human septin family gene Bradeion inhibits the growth and tumorigenesis of colorectal cancer in vitro and in vivo

We have identified a novel human septin family gene Bradeion, which is specifically expressed in human colorectal cancer and malignant melanoma. In order to analyze the implications of tumor-specific gene expression, ribozymes and its derivatives were specifically designed and transfected into various colorectal adenocarcinoma cell lines for Bradeion inactivation. We constructed ribozyme expression plasmids controlled by a human tRNA(Val) promoter, and both hammerhead ribozyme and its allosteric derivative maxizyme were used for two different forms of Bradeion mRNA. The sequence-specific cleavage of Bradeion mRNA resulted in significant growth inhibition and G2 arrest in human cancer cell lines, detected by flow cytometry analysis. In addition, in vivo mice studies demonstrated marked tumor growth suppression by the Bradeion-specific ribozymes. Thus, the tumor-specific and selective marker Bradeion also provides valuable tools as a potential target for colorectal cancer therapy.     

RNA干扰survivin基因对卵巢癌细胞生长凋亡及药物敏感性的影响

目的 探讨下调survivin基因对卵巢癌顺铂(DDP)耐药细胞株SKOV3/DDP细胞生长、凋亡及药物敏感性的影响.方法 构建survivin基因的小干扰RNA(siRNA)表达载体pSilencer-survivin,用脂质体方法转染SKOV3/DDP细胞株,另设未转染组和转染pSilencer-control组作为对照.显微镜下观察转染前后细胞的变化,逆转录聚合酶链反应(RT-PCR)和Western blot分别检测survivinmRNA及蛋白的表达,二苯基溴化四氮唑蓝(MTT)法检测细胞生长情况,流式细胞仪检测细胞凋亡及周期的变化.各组细胞加入DDP,检测其对DDP药物敏感性的影响.结果 survivin siRNA可明显下调SKOV3/DDP细胞中survivin mRNA及蛋白表达水平.SKOV3/DDP细胞生长受到明显抑制,生长曲线低平.转染48 h后,转染pSilencer-survivin组细胞凋亡率为19.1%,明显高于末转染组(2.6%)和转染pSilencer-control组(3.5%),G1/G0期细胞所占的比例增高,而G2/M期细胞所占的比例降低.转染pSilencer-survivin组细胞的IC50明显降低,为1.16 μg/mi.结论 survivin siRNA可下调SKOV3/DDP细胞中survivin的表达,使细胞生长减慢,凋亡增加,对DDP的药物敏感性增强.     

Survivin基因对食管癌细胞系KYSE30增殖和侵袭性的影响

目的：研究survivin基因转染对人食管癌细胞系KYSE30增殖和侵袭能力的影响。方法：以脂质体将survivineDNA真核表达载体转染人食管癌KYSE30细胞。应用克隆形成实验检测细胞增殖活性的变化,流式细胞仪检测转染前后细胞周期的变化,Transwell小室侵袭实验检测细胞侵袭能力的变化,MTT检测转染前后细胞对各种化疗药物的敏感性。结果：Survivin基因能促进人食管癌KYSE30细胞增殖,流式细胞仪分析结果显示转染后细胞抗凋亡能力增强,侵袭实验显示转染后细胞侵袭能力较转染前有显著增强,MTF实验显示转染后细胞对化疗药物敏感性下降。结论：Survivin基因能够促进人食管癌细胞系KYSE30的体外增殖和侵袭能力,导致对化疗药敏感性下降,提示其对肿瘤的发生发展和细胞耐药起促进作用,可作为食管癌基因治疗的靶标。     

A novel antisense oligonucleotide targeting survivin expression induces apoptosis and sensitizes lung cancer cells to chemotherapy

Survivin, an inhibitor of apoptosis protein, deserves attention as a selective target for cancer therapy because it lacks expression in differentiated adult tissues but is expressed in a variety of human tumors. We designed 20-mer phosphorothioate antisense oligonucleotides targeting different regions of survivin mRNA and investigated their ability to down-regulate survivin mRNA and induce apoptosis in the lung adenocarcinoma cell line A549. Oligonucleotide 4003, which targets nucleotides 23-251 of survivin mRNA, was identified as the most potent compound. As measured by real-time PCR, 4003 down-regulated survivin mRNA in a dose-dependent manner with an IC50 of 200 nM. Its maximum effect was achieved at a concentration of 400 nM, at which mRNA was down-regulated by 70%. As revealed by increased caspase-3-like protease activity, nuclear condensation and fragmentation, and trypan blue uptake, treatment with 4003 induced apoptosis and sensitized tumor cells to the chemotherapeutic agent etoposide. Oligonucleotide 4003 did not reduce the viability of normal blood leukocytes with marginal levels of survivin mRNA.     

RNA干扰survivin对口腔表皮样癌细胞株 KB生长的抑制作用

 摘要：目的探讨RNA干扰技术沉默survivin基因对口腔表皮样癌细胞株KB增殖和凋亡的影响。方法化学合 成靶向survivin基因的小干扰RNA片段（siRNA）,脂质体法转染KB细胞。通过RT-PCR、蛋白印迹法分别检 测survivin mRNA和蛋白表达变化,流式细胞仪检测细胞生长周期和凋亡,MTT法检测转染后KB细胞增殖情 况。结果siRNA可以有效地降低KB细胞survivin mRNA和蛋白的表达;转染后KB细胞阻滞于G2/M期,凋亡增 加,增殖受抑制。结论应用RNA干扰技术沉默survivin 在KB细胞中的表达可以有效抑制该细胞增殖、促进 癌细胞凋亡,将survivin作为口腔癌基因治疗的靶点,通过RNA干扰技术对口腔癌进行基因治疗具有一定 的可行性。     

Induction of apoptosis in human ovarian epithelial cancer cells by antisurvivin oligonucleotides

Survivin, an anti-apoptosis gene that is abnormally overexpressed in a variety of human tumors, may play an important role in the carcinogenesis and drug resistance of cancer. This study was designed to explore the effects of liposome-survivin antisense oligonucleotide (Lip-ASODN) on the growth and apoptosis of human ovarian cancer cell lines, A2780 and SKOV3. To investigate the use of survivin as a therapeutic target on ovarian cancer, we carried out transfections with Lip-ASODN to induce apoptosis in ovarian cancer cell lines, A2780 and SKOV3. The expression of survivin mRNA and relative protein were evaluated separately by quantitative real-time RT-PCR and Western blot analysis. Cell proliferation inhibition was determined by methyl thiazolyl tetrazolium (MTT) assay, and the induced cell apoptosis was examined using flow cytometry (FCM) after Lip-ASODN transfection. Our results showed that the overexpression of survivin led to infinite carcino-proliferation, and survivin expression in the survivin-positive ovarian cancer cell line A2780 and SKOV3 cells was significantly and gradually reduced when transfected with Lip-ASODN at concentrations of 200, 400 and 600 nM by degrees. Lip-ASODN transfection induced greater apoptosis rates in the human ovarian cancer cell lines A2780 and SKOV3 (p<0.05). The growth inhibition and apoptotic rates of tumor cells change when treated with different concentrations of Lip-ASODN. The cell growth inhibition peak rate was reached when increasing Lip-ASODN concentration to 600 nM. Furthermore, time course evaluation showed that survivin protein expression was inhibited by Lip-ASODN within 12 h after transfection. We concluded that down-regulation of survivin by a targeted antisense oligonucleotide appears to be an effective gene therapy approach in the treatment of ovarian cancer.     

survivin 反义核酸诱导HeLa 细胞凋亡的实验

 目的　探讨survivin 反义核酸诱导HeLa 细胞发生凋亡的可能性,揭示该凋亡发生与Bcl22 和 Bax 之间的关系。方法　采用TUNEL 染色法,研究survivin 反义核酸与HeLa 细胞凋亡的关系;通过免 疫组织化学法检测凋亡相关基因Bcl22 和Bax 的表达。结果　survivin 反义核酸在体外能诱导HeLa 细 胞凋亡,下调Bcl22 的表达,增强Bax 的表达。结论　诱导HeLa 细胞凋亡是survivin 反义核酸抗HeLa 作用的机制之一,survivin 反义核酸可能通过下调Bcl22 的表达,增强Bax 的表达,诱导HeLa 细胞发生 凋亡。     

Survivin小分子干扰RNA对膀胱癌T24细胞生物学行为的影响

背景与目的:膀胱癌是泌尿系统最常见的恶性肿瘤,通常与体内某些基因突变导致细胞周期改变和抗凋亡机制有关。凋亡抑制蛋白家族(IAPs)的新成员Survivin,通过诱导细胞增殖和抑制细胞凋亡参与了人类多种肿瘤的发生,其中包括膀胱癌。本研究探讨转染靶向survivin的小分子干扰RNA(smallinterfering RNA,siRNA)对膀胱癌生物学行为的影响。方法:设计、合成一对survivin编码基因序列特异的siRNA,用脂质体包裹转染T24膀胱癌细胞,分成不同的浓度组(50～200nmol/L);鼠异位膀胱肿瘤模型中瘤内注射不同剂量的siRNA(5!g和50!g)。分别于体外和体内观察siRNA对膀胱癌生物学行为的影响。结果:survivin编码基因序列特异性siRNA能有效下调survivin基因表达水平,最大效应浓度为100nmol/L,此时survivin表达水平下调了75.91%,并显著的抑制了细胞增殖,抑制率达55.29%,与对照组相比差异有统计学意义(P<0.01)。同时,细胞凋亡率亦由2.8%增加至45.70%,与对照组相比差异有统计学意义(P<0.01)。裸鼠移植瘤内重复多次注射50!g survivin-siRNA能够明显抑制肿瘤生长,与对照相比差异有统计学意义(P<0.01)。结论:survivin-siRNA能显著下调膀胱癌细胞survivin基因表达水平,促进细胞凋亡,抑制肿瘤细胞增殖。     

靶向survivin的shRNA对人胆管癌QBC939细胞体外增殖及凋亡的影响

目的:探讨靶向survivin的shRNA对人胆管癌QBC939细胞体外增殖及凋亡的影响。方法:合成具有互补序列的能够编码短发卡RNA(shRNA)的双链寡核苷酸并构建pSilence2.1真核表达质粒,经稳定转染QBC939细胞后对转基因后胆管癌细胞的生物学行为进行观察。结果:neo基因稳定表达于转染阳性质粒及阴性对照质粒的QBC939细胞中。RT-PCR及Western blot检测证实shRNA在mRNA及蛋白质水平抑制survivin表达,抑制率分别为66.2%和61.8%。细胞计数及平板克隆形成实验显示转染细胞生长速度及细胞克隆形成率明显减低(P<0.01)。流式细胞术测定细胞周期显示转染细胞G0/G1期细胞明显增多,S期细胞明显减少。DNA ladder、透射电镜观察及流式细胞术定量研究显示转染细胞凋亡比明显增多。结论:靶向sur-vivin的shRNA能有效抑制目的基因的表达,通过增加细胞凋亡在体外抑制人胆管癌细胞的生长。     

超声靶向微泡破裂法介导RNA干扰沉默survivin基因对宫颈癌HeLa细胞凋亡的影响

目的:通过超声靶向微泡破裂(ultrasound targeted microbubble destruction ,UTMD)法介导基因转染,观察RNA干扰对宫颈癌HeLa细胞survivin基因的沉默效应和诱导细胞凋亡的作用.方法:构建靶向survivin基因的短发夹状RNA(short hairpin RNA,shRNA)表达质粒,将其和微泡共同处理后加入HeLa细胞并予以超声辐照,以空白细胞、单纯质粒、单纯超声辐照、质粒+超声辐照和质粒+Lipofectamine转染作为对照;采用荧光显微镜和FCM法评估基因转染效率,同时对超声辐照参数进行优化;应用FCM法和Hoechst染色及DNA ladder分析法检测细胞凋亡,应用RT-PCR检测survivin mRNA水平的变化.结果:经酶切鉴定及测序分析证实重组质粒构建成功.HeLa细胞采用UTMD处理后,基因转染率显著增加(P＜0.01),并在超声强度为1.0 W/cm2、辐照3 min的条件下最为显著(P＜0.01).PT-PCR检测结果显示,质粒与微泡共同作用加超声辐照组的survivin mRNA抑制率为(83.33±2.73)%,均显著高于各对照组(P＜0.01);FCM法检测结果显示,该组细胞的凋亡率同样显著高于各对照组(P＜0.01);Hoechst染色及DNA ladder检测结果显示,只有经脂质体或UTMD转染处理后的细胞中可检测到明显的细胞凋亡形态及凋亡梯带,其余各组均未检测到明显凋亡.结论:UTMD可有效增强表达质粒的转染效率,是一种新的、有应用前景的非病毒基因转移体系.UTMD介导RNA干扰沉默survivin基因可诱导明显的细胞凋亡,为肿瘤基因治疗及研究提供了新的方法.     

HSP 90与survivin在人乳腺癌细胞中的作用及机制探讨

目的:研究热激蛋白90(heat shock protein 90,HSP 90)和survivin在人乳腺癌细胞中的相互作用以及对肿瘤细胞增殖、凋亡的影响。方法:以人乳腺癌细胞MDA-MB-231为研究对象,用HSP 90抑制剂格尔德霉素(geldanamycin,GA)处理后,Western印迹法检测细胞中P-STAT3、survivin蛋白表达的变化;RT-PCR检测survivin mRNA的变化;MTT法检测其对细胞的增殖活性影响;流式细胞术检测凋亡的变化。结果:HSP90抑制剂GA可使人乳腺癌细胞MDA-MB-231中P-STAT3减少、survivin的蛋白表达减少,细胞的增殖活性降低,凋亡增多。结论:在MDA-MB-231细胞中,HSP 90可能通过激活JAK-STAT3信号通路,影响survivin转录表达,共同促进该肿瘤细胞的高增殖、低凋亡的恶性行为。     

生存蛋白Survivin在人胃癌组织中的表达

目的 :探讨生存蛋白Survivin在胃癌组织中的表达 ,进一步研究化疗前后Sur vivinmRNA表达的差异。方法 :用RT PCR法研究体外培养的胃癌细胞株及人胃癌组织化疗前后SurvivinmRNA表达的变化 ,采用Western blot检测Survivin蛋白的表达。结果 :体外培养的胃癌细胞株化疗药物处理前SurvivinmRNA的表达率为 2 5 % ,接受化疗的细胞株表达率为 70 % ;术前接受化疗的胃癌患者SurvivinmRNA表达率为 60 % ,术前未接受化疗的表达率为 3 5 % ,差异显着。正常胃黏膜细胞未检测到SurvivinmRNA表达。Western blot检测Survivin蛋白的表达具有相同的结果。结论 :Survivin在胃癌细胞中高表达并且接受化疗后Survivin表达率增高 ,提示Survivin表达可能与肿瘤化疗耐受有关     

利用siRNA阻抑survivin基因表达诱导乳腺癌细胞系MCF-7细胞凋亡的研究

背景与目的:survivin属IAP基因家族成员,表达于多种肿瘤组织中,能促使细胞逃避凋亡,并促进细胞的异常有丝分裂。本研究旨在探讨敲除survivin基因后,癌细胞的增殖和凋亡情况。方法:利用RNAi阻抑人乳腺癌细胞内survivin基因的表达,RT-PCR和Westernblot法分析survivin基因mRNA和蛋白的表达,MTT法检测细胞生长增殖抑制率,流式细胞术检测细胞凋亡率。结果:survivinsiRNA转染组,survivin基因表达水平与未转染组比较下调了64%。随着siRNA浓度的增加,细胞增殖抑制率逐渐增高,200nmol/L剂量组细胞增殖抑制率最高,可达60.9%。不同浓度的siRNA可不同程度地诱导细胞凋亡,200nmol/L剂量组凋亡率最高,可达29.0%。结论:survivinsiRNA有可能成为治疗乳腺癌的新药物。     

Survivin 靶向治疗在肿瘤中的研究进展

Survivin（BIRC5）是凋亡抑制蛋白（Inhibitors of apoptosis proteins,IAPs）家族的成员之一,除了表达于胚胎组织、少量表达于能自我更新的正常组织之外,在大多数恶性肿瘤组织中高表达。 Survivin不仅参与了某些正常组织细胞的生理进程,而且也可调节各种肿瘤细胞的增殖和凋亡过程。由于结构和功能的特异性,Survivin作为一种潜在的肿瘤分子靶向治疗的理想靶点受到越来越广泛的关注。针对Survivin的靶向治疗主要包括反义寡核苷酸、核酶、siRNA、显性负性突变体、小分子拮抗剂和免疫治疗。