肺腺癌细胞S100A6基因siRNA慢病毒载体构建及鉴定

目的:构建肺腺癌细胞S100A6基因siRNA慢病毒载体并鉴定。方法:慢病毒载体（pLenR-GPH）构建靶向S100A6基因的RNA干扰重组体,用以建立S100A6基因稳定沉默的肺腺癌A549细胞株。结果:PCR鉴定结果显示3个扩增的阳性片段均已插入pLenR-GPH载体,测序结果证实三个重组慢病毒载体SH1、SH2、SH3的插入序列完全正确,重组慢病毒载体转染到293T细胞中包装成病毒颗粒,将其感染肺癌A549细胞后,RT-PCR和Western blot检测结果均证实三组重组体感染的细胞内S100A6 mRNA和蛋白的表达受到不同程度的抑制,沉默效率分别为98.29%、84.05%及78.24%。结论:成功构建了S100A6基因RNAi慢病毒重组载体,并建立了其稳定表达的肺腺癌A549细胞株,为探讨S100A6基因对肺腺癌生物学行为的影响提供了可靠的细胞模型。     

RNAi-mediated downregulation of <Emphasis Type="Italic">NOB1</Emphasis> suppresses the growth and colony-formation ability of human ovarian cancer cells

Nin one binding protein (NOB1p), encoded by the NOB1 gene, is a crucial molecule in the maturation of the 20S proteasome and protein degradation. The present study evaluates whether NOB1 is an appropriate molecular target for cancer gene therapy. In two ovarian cancer cell lines, SKOV3 and HEY, NOB1 expression was knocked down by a lentiviral short hairpin RNA (shRNA) delivery system. The RNA interference (RNAi)-mediated the downregulation of NOB1 expression markedly reduced the proliferative and colony-formation ability of ovarian cancer cells. Additionally, NOB1 shRNA-expressing lentivirus-treated ovarian cancer cells tended to arrest in the G0/G1 phase. These results suggested that NOB1 may act as an oncogenic factor in ovarian cancer and could be a potential molecular target for ovarian cancer gene therapy.     

RNAi-mediated inhibition of cathepsin B and uPAR leads to decreased cell invasion, angiogenesis and tumor growth in gliomas

RNA interference (RNAi) provides a powerful method for gene silencing in eukaryotic cells, including proliferating mammalian cells. Here, we determined whether RNAi could be utilized to inhibit the expression of proteases implicated in the extracellular matrix degradation, which is characteristic of tumor progression. We have previously shown that antisense stable clones of uPAR and cathepsin B were less invasive and did not form tumors when injected intracranially ex vivo. Since antisense-mediated gene silencing does not completely inhibit the translation of target mRNA and high molar concentrations of antisense molecules are required to achieve gene silencing, we used the RNAi approach to silence uPAR and cathepsin B in this study. We found that the expression of double-stranded RNA leads to the efficient and specific inhibition of endogenous uPAR and cathepsin B protein expression in glioma cell lines as determined by Western blotting. We also found the RNAi of uPAR and cathepsin B reduces glioma cell invasion and angiogenesis in in vitro and in vivo models. Intratumoral injections of plasmid vectors expressing hpRNA for uPAR and cathepsin B resulted in the regression of pre-established intracranial tumors. Further, RNAi for uPAR and cathepsin B inhibited cell proliferation and reduced the levels of pERK and pFAK compared to controls. Taken together, our findings indicate for the first time that RNAi operates in human glioma cells with potential application for cancer gene therapy.     

Inhibition of cervical cancer cell growth in vitro and in vivo with dual shRNAs

RNA interference (RNAi)-based gene silencing is widely used in laboratories for gene function studies and also holds a great promise for developing treatments for diseases. However, in vivo delivery of RNAi therapy remains a key issue. Lentiviral vectors have been employed for stable gene transfer and gene therapy and therefore are expected to deliver a stable and durable RNAi therapy. But this does not seem to be true in some disease models. Here, we showed that lentivirus delivered short-hairpin RNA (shRNA) against human papillomavirus (HPV) E6/E7 oncogenes were effective for only 2 weeks in a cervical cancer model. However, using this vector to carry two copies of the same shRNA or two shRNAs targeting at two different but closely related genes (HPV E6 and vascular endothelial growth factor) was more effective at silencing the gene targets and inhibiting cell or even tumor growth than their single shRNA counterparts. The cancer cells treated with dual shRNA were also more sensitive to chemotherapeutic drugs than single shRNA-treated cells. These results suggest that a multi-shRNA strategy may be a more attractive approach for developing an RNAi therapy for this cancer.     

shRNA沉默多药耐药胃癌细胞7901/VCR MDR1基因表达的实验研究

目的利用RNAi技术干扰胃癌多药耐药细胞（7901/VCR）多药耐药基因（MDR1）的表达,为临床肿瘤的基因治疗奠定基础。方法根据MDR1的碱基序列设计并合成两对短发夹RNA,构建重组载体,用阳离子脂质体法体外转染7901/VCR;采用MTT法检测转染胃癌细胞的生长抑制率,流式细胞术检测细胞周期变化,RT-PCR和Western blot检测转染前后MDR1 mRNA和蛋白表达的变化。结果成功构建RNAi质粒载体,转染后细胞IC50明显降低（P〈0.05）;G1期细胞增加,S期细胞减少（P〈0.05）;MDR1 mRNA转录水平下降（P〈0.05）,P-糖蛋白（P-gp）的表达水平降低（P〈0.05）。结论RNAi能明显抑制胃癌细胞7901/VCR MDR1mRNA和P-gp蛋白的表达,进而对肿瘤细胞多药耐药性有明显的逆转作用,为基因治疗提供了一种新的手段。     

An attenuated Salmonella oral DNA vaccine prevents the growth of hepatocellular carcinoma and colon cancer that express alpha-fetoprotein

Antitumor vaccination therapies using attenuated Salmonella typhimurium carrying plasmid DNA encoding tumor-associated antigens are currently under preclinical development. In the present study, we first established a useful method to facilitate in vivo monitoring of attenuated S. typhimurium uptake using a bioluminescent lux gene operon plasmid. Following transformation with the lux gene operon construct, mice were fed with various amounts of attenuated S. typhimurium-lux to monitor in vivo clearance over a period of 24 h. We found that the ingested attenuated S. typhimurium-lux cells were almost cleared out 9 h postfeeding, as judged by a significant decrease in bioluminescence. We further examined the therapeutic efficacy of vaccination using attenuated S. typhimurium carrying the mouse alpha-fetoprotein (AFP) gene against a cancer line CT26-murine alpha-feto protein (mAFP) that stably expresses AFP and mouse hepatocellular carcinoma (HCC) Hepa1-6. Attenuated S. typhimurium oral DNA vaccine was found to promote protective immunity against both CT26-mAFP and Hepa1-6 tumor cells growth. The oral DNA vaccine significantly increased the life span of tumor-challenged mice in both tumor models. Together, these results suggest that vaccination with the attenuated S. typhimurium oral DNA vaccine that carries the AFP gene could be a promising strategy to prevent HCC development.     

Fundamental study of small interfering RNAs for ganglioside GD3 synthase gene as a therapeutic target of lung cancers

Gangliosides GD3 and GD2 are specifically expressed in neuro-ectoderm-derived tumors, and are considered to play roles in the malignant properties of those cells. We analysed effects of small interfering (si) RNAs against GD3 synthase gene on the expression of ganglioside GD2 and biological phenotypes of human lung cancer cells expressing GD2. An siRNA could suppress the mRNA level of GD3 synthase gene even by single transfection, whereas repeated transfection was required to suppress GD2 expression on the cell surface. Significant reduction in the cell growth and invasion activity was observed in both lung cancer cell lines examined, when repeatedly transfected with the siRNA twice a week. DNA ladder formation was observed after third transfection, indicating the potent induction of apoptosis. Stable transfection of an RNAi expression vector with H1 RNA promoter was also examined. Transfectant cells with the RNAi expression vector showed almost equivalent suppression of GD2 expression and tumor properties in vitro. Furthermore, the stable transfectant cells showed slower cell growth than the control cells in severe combined immunodeficiency mice. These results suggested that siRNAs and/or RNAi expression vectors to generate siRNAs are promising approach to overcome human lung cancers.     

Selective cyclooxygenase-2 silencing mediated by engineered E. coli and RNA interference induces anti-tumour effects in human colon cancer cells

Background:

Cyclooxygenase-2 (COX-2) overexpression is strongly associated with colorectal tumourigenesis. It has been demonstrated that the chronic use of non-steroidal anti-inflammatory drugs (COX inhibitors) partially protects patients from colorectal cancer (CRC) development and progression but induces severe cardiovascular side effects. New strategies for selective COX-2 blockade are required.

Methods:

We developed an improved technique, based on RNA interference (RNAi), to gain a selective COX-2 silencing in CRC cells by a tumour-dependent expression of anti-COX-2 short-hairpin RNA (shCOX-2). Anti-COX-2 shRNA-expressing vectors were delivered in CRC cells (in vitro) and in colon tissues (ex vivo) using engineered Escherichia coli strains, capable of invading tumour cells (InvColi).

Results:

A highly tumour-dependent shCOX-2 expression and a significant COX-2 silencing were observed in CRC cells following InvColi strain infection. Cyclooxygenase-2 silencing was associated with a strong reduction in both proliferative and invasive behaviour of tumour cells. We also demonstrated a pivotal role of COX-2 overexpression for the survival of CRC cells after bacterial infection. Moreover, COX-2 silencing was achieved ex vivo by infecting colon tissue samples with InvColi strains, leading to anti-inflammatory and anti-tumour effects.

Conclusion:

Our RNAi/InvColi-mediated approach offers a promising tool for a highly selective COX-2 blockade in vitro and in vivo.     

S100A6基因干扰对A549肺腺癌细胞生物学行为的影响

目的 探讨S100A6基因干扰对A549肺腺癌细胞生物学行为的影响.方法 构建S100A6基因RNAi载体,慢病毒转染A549肺腺癌细胞,共分为3组,①空载体对照组:转染不携带S100A6基因RNAi的空载质粒;②阴性对照组:未转染任何质粒;③S100A6 RNA干扰组:携带S100A6基因RNAi的质粒.应用实时聚合酶链反应(RT-PCR)和Western blotting鉴定S100A6基因和蛋白表达;采用四甲基偶氮唑蓝、迁移试验和流式细胞仪分别检测细胞增殖、浸润、细胞周期及细胞凋亡等生物学特性.结果 S100A6基因干扰后A549肺腺癌细胞S100A6 mRNA的表达(0.009±0.001)较阴性对照组(0.049 ±0.005)和空载体对照组(0.030 ±0.006)均有明显下降,差异均有统计学意义(=57.56,P=0.000;t=48.21,P=0.000).S100A6基因干扰后A549肺腺癌细胞S100A6蛋白的表达(0.107±0.002)较阴性对照组(0.341 ±0.005)和空载体对照组(0.311 ±0.006)均有明显下降,差异均有统计学意义(t=37.34,P=0.000;=27.51,P=0.001).48 h细胞增殖能力:RNA干扰组(0.230 ±0.008)较阴性对照组(0.292 ±0.038)和空载体对照组(0.307 ±0.013)降低,差异均有统计学意义(t=25.31,P=0.003;t=29.42,P=0.001).细胞浸润能力:RNA干扰组细胞的穿膜细胞数(11.40个±1.36个)较阴性对照组(26.80个±1.83个)和空载体对照组(25.80个±1.93个)降低,差异有统计学意义(t=29.44,P=0.001;t=23.17,P=0.005).细胞周期:RNA干扰组的S期细胞比例(28.26％±0.38％)显著低于空载体对照组(44.73％±0.66％)和阴性对照组(45.15％±1.69％),差异有统计学意义(t=63.69,P=0.000;t =71.55,P=0.000),RNA干扰组的G2-M期细胞比例(26.99％±0.29％)显著高于阴性对照组(13.26％ ±0.49％)和空载体对照组(12.41％±0.46％),差异有统计学意义(t=56.31,P=0.000;t =51.39,P=0.000).RNA干扰组细胞凋亡率(8.90％±0.48％)与阴性对照组(5.84％±0.21％)和空载体对照组(5.99％±0.37％)比较,差异均有统计学意义(t=51.34,P=0.000;t=47.27,P=0.000).结论 S100A6基因参与肺腺癌细胞的增殖、浸润、细胞周期、细胞凋亡等生物学过程,与肿瘤发生、发展、转移等密切相关,有望作为肺腺癌诊断和治疗新的分子靶标.     

Therapeutic potential of RNA interference against cancer

One of the most dramatic events of the past 5 years in the field of molecular biology has been the discovery of RNA interference (RNAi). Although RNAi is an evolutionarily conserved phenomenon for sequence-specific gene silencing in mammalian cells, exogenous small interfering RNA (siRNA) and vector-based short hairpin RNA (shRNA) can also invoke RNAi responses. Both are now not only experimental tools for analyzing gene function but are expected to be excellent avenues for drug target discovery and the emerging class of gene medicine for targeting incurable diseases such as cancer. The success of cancer therapeutic use of RNAi relies on the development of safe and efficacious delivery systems that introduce siRNA and shRNA expression vectors into target tumor cells. For their delivery, a variety of strategies have been used, most of them based on traditional gene therapy delivery systems. In this review, we present siRNA delivery method strategies and discuss the potential of RNAi-based gene therapy in cancer treatment. ( Cancer Sci 2006; 97: 689–696)     

干扰COX-2基因表达对人肺腺癌A2细胞体外恶性增殖的影响

背景与目的 COX-2在多种肿瘤组织中高表达,参与了肿瘤的发生发展,RNA干扰(RNAi)技术是一种经济、快捷、高效的抑制特异基因表达的技术手段.本研究应用RNAi技术研究干扰COX-2基因表达的抑制效果及抑制COX-2基因表达对A2细胞体外恶性增殖的影响.方法 以COX-2为靶点,构建3个带有人U6启动子的COX-2小干扰RNA(short interfering RNA,siRNA)表达载体.用脂质体lipofectamine介导,分别将3个siRNA表达载体及空载体转染到COX-2表达阳性的A2细胞,建立转染细胞株.采用反转录聚合酶链反应(RT-PCR)和Western blot方法检测COX-2表达水平的变化.通过细胞生长曲线、集落形成试验研究干扰COX-2基因表达对A2细胞体外增殖的影响.结果 经过PCR扩增、内切酶鉴定、DNA测序和BLAST比对证实3个siRNA和U6启动子序列正确并准确克隆到pEGFP载体.转染细胞株分别命名为A2-3、A2-7、A2-10和A2-P.转染后24 h、48 h、72 h,A2-P细胞均有绿色荧光表达,而A2-3、A2-7、A2-10细胞均未观察到绿色荧光.RT-PCR和Western blot结果显示,3个siRNA表达载体均发挥作用,COX-2表达受到抑制.与A2细胞比较,A2-3、A2-7、A2-10细胞的COX-2 mRNA表达量分别降低15.6%、20.4%和64.2%,COX-2蛋白表达量分别降低23.7%、36.7%和60.2%.细胞生长曲线、集落形成试验的结果显示,A2-10细胞生长减慢,集落形成率减少,而A2-3和A2-7细胞生长没有明显的改变.结论 应用RNAi技术熄灭COX-2基因表达对A2细胞的体外恶性增殖有明显的抑制作用.     

RNA干扰技术靶向hTERT基因治疗肝癌的实验研究

背景与目的：RNA干扰(RNA interference,RNAi)是由双链RNA介导的、在转录后mRNA水平关闭相应基因表达的新基因阻断技术,在基因功能研究、基因治疗方面已显示出巨大的前景。目前,利用RNAi已抑制了包括cyclophilin、GAPDH、p53、c-myc在内的多个内源基因的表达。同时在艾滋病、病毒性肝炎等的治疗研究中也已取得一定进展。但对肝癌等恶性肿瘤中高表达的hTERT基因,国内外还未见相关研究报道。本研究利用RNAi技术,在体内外抑制hTERT基因表达,探讨RNAi对肝癌治疗的可行性。方法：设计干扰hTERT基因的小片段RNA,构建重组表达质粒pTzu6 1-shRNA-hTERT并导入肝癌SMMC7721细胞株和裸鼠移植瘤,在体内外诱导RNAi,采用流式细胞检测技术、RT—PCR法、免疫组化等同时检测RNAi治疗组和对照组hTERT基因表达及细胞增殖变化,结果：体外细胞实验显示,重组质粒pTZU6 1—shRNA—hTERT导入肝癌SMMC7721细胞株3～7天后,肝癌细胞生长抑制率达37．5％;细胞周期相分布发生显著变化,S期细胞明显减少,G1／G0期细胞显著增加;hTERT的mRNA表达由99．4％下调到53．1％,hTERT蛋白表达由86．3％下调到46．6％。裸鼠体内实验结果显示,质粒pTZu6 1-shRNA-hTERT注射裸鼠皮下移植瘤7天后,瘤体积明显缩小,hTERT的mRNA表达由99．1％下调到76．2％,hT．ERT蛋白表达由87．2％下调到61．8％。体内、外对照组各指标均无变化。结论：RNAi明显抑制了靶基因hTERT的表达及肝癌细胞增殖,是潜在的肿瘤治疗新方法。     

RNA干扰技术逆转神经胶质瘤细胞多药耐药性

目的 探讨利用RNA干扰（RNAi）技术逆转神经胶质瘤细胞多药耐药性。方法 根据多药耐药基因1（MDR1）的碱基序列设计并合成短发夹RNA（shRNA）,构建逆转录病毒质粒载体,用阳离子脂质体法体外转染BT325细胞株,以增强型绿色荧光蛋白（EGFP）表达作为对照。采用定量PCR、Northern blot检测转染前后MDR1 mRNA的表达,Western blot检测蛋白表达;使用CCK-8试剂盒对转染后的细胞进行化疗药物敏感性试验,评价RNAi对多药耐药性的逆转作用。结果 成功构建RNAi质粒载体。共转染实验组RT-PCR定量MDR1 mRNA相对表达水平均有所下降（P〈0．05）;Northern blot表明,转染48h细胞干扰最强;Western blot显示,siRNA各转染组P-糖蛋白（P-gp）的表达分别降低12．9％、30．3％和4．8％,在48h抑制最强;而药物敏感试验显示,转染siRNA后细胞对药物的敏感性明显增强。结论 RNAi能够明显抑制神经胶质瘤细胞系MDR1 mRNA和P-gp蛋白的表达,进而对多药耐药性发挥明显的逆转作用,为基因治疗提供了一种新的手段。     

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.     

慢病毒介导ＲＮＡ干扰抑制脑胶质瘤细胞ＨＭＧＡ１表达对洛铂化疗敏感性的影响

目的：建立逆转录病毒介导入高迁移率族蛋白组Ａ１（ＨＭＧＡ１）基因ＲＮＡ干扰体外表达体系,并观察洛铂对脑胶质瘤细胞株ＳＨＧ-４４化疗敏感性的影响。方法：制备ＨＭＧＡ１ｓｉＲＮＡ慢病毒载体,ＰＣＲ筛选阳性克隆,测序鉴定;转染脑胶质瘤细胞株ＳＨＧ-４４后,ＲＴ-ＰＣＲ检测其对细胞ＨＭＧＡ１基因表达的影响;ＭＴＴ和克隆集落实验检测洛铂对阳性组和阴性组细胞生长、增殖的影响。结果：ＰＣＲ和测序证实,成功构建ＨＭＧＡ１ｓｉＲＮＡ的慢病毒载体;ＲＴ-ＰＣＲ证实阳性组细胞存在ＨＭＧＡ１基因表达下调;分别用不同浓度的洛铂处理两组细胞２４ｈ后,ＭＴＴ法和克隆集落实验显示阳性组细胞生长抑制率较阴性组细胞明显降低。结论：ＨＭＧＡ１ｓｉＲＮＡ能特异性下调脑胶质瘤细胞ＳＨＧ-４４中ＨＭＧＡ１的表达并抑制了其对洛铂的敏感性。     

表达的siRNA对人乳腺癌SKBr-3细胞中survivin基因的干涉作用

目的:构建针对人survivin基因的siRNA真核表达载体,检测其对人乳腺癌SKBr-3细胞中survivin基因表达的干涉作用.方法:将合成的寡核苷酸链退火形成双链,连接入经HindⅢ和BglⅡ双酶切后的pSUPER真核表达载体.对重组质粒进行酶切分析和测序鉴定.通过脂质体介导,把重组质粒稳定转染入SKBr-3细胞,RT-PCR、Western blot印记杂交和细胞免疫化学法检测其对mRNA和蛋白表达的干涉效果.结果:经酶切鉴定及基因测序证实,重组质粒中已插入目的基因片段.RT-PCR显示两个干涉载体均抑制了目的基因的转录;Western blot印记杂交和细胞免疫化学检测结果显示pSUPER-S1对蛋白表达的抑制作用更强.结论:成功地构建了针对人survivin基因的RNA干涉真核表达载体pSUPER-S1和pSUPER-S2,并在人乳腺癌细胞株SKBR-3中有效发挥了对survivin基因的干涉作用.