RNA干涉在肿瘤研究中的应用进展

RNA干涉(RNA interference，RNAi)是生物体内的一种通过双链RNA(double-stranded RNA，dsRNA)来抵抗病毒入侵和抑制转座子活动的自然机制，近来研究发现，21～25nt大小的小干涉RNA(small interfering RNA，siRNA)可在哺乳动物细胞中介导高度序列特异性的基因沉默，并且具有高效性和序列特异性，已经成为功能基因组研究的有力工具，并且有可能在某些疾病如肿瘤等的基因治疗中发挥重要作用。     

RNA干扰技术在肿瘤基因治疗研究中的应用

RNA干扰(RNAi)属于转录后基因沉默(PTGS).其机制为利用双链RNA(dsRNA)特异性地降解相应序列的mRNA成为短干扰RNA,从而特异性地阻断靶基因的表达,现介绍RNA干扰的分子机制、技术应用及在肿瘤基因治疗方面的进展和广阔的发展前景.     

RNA干扰技术在肿瘤基因治疗研究中的应用

RNA干扰(RNAi)属于转录后基因沉默(PTGS)。其机制为利用双链RNA(dsRNA)特异性地降解相应序列的mRNA成为短干扰RNA，从而特异性地阻断靶基因的表达，现介绍RNA干扰的分子机制、技术应用及在肿瘤基因治疗方面的进展和广阔的发展前景。     

shRNA技术在肿瘤发生发展及耐药性研究中的应用

小发夹RNA（short hairpin RNA，shRNA）是近年来兴起的一种序列特异性诱导细胞内目的基因表达沉默的RNA干涉（RNA interference，RNAi）技术，shRNA作用特点主要是高效、持久以及多重抑制性，可广泛应用于基因功能、抗病毒、肿瘤基因治疗研究。本文对shRNA技术在肿瘤发生发展及其耐药性方面的实验研究作一综述。     

RNA干扰技术在胃癌治疗中的研究进展

RNA干扰(RNA interference,RNAi)主要通过导入双链RNA(double strands RNA,dsRNA)进而靶向抑制转录后基因的沉默,小分子干扰RNA(short interfering RNA,siRNA),是RNA干涉的调控的关键.由于RNAi对基因沉默的高效、特异性,在胃癌治疗中,可明显影响胃癌细胞的生物学特性,故这一技术目前已被广泛应用于与胃癌发生相关的基因、蛋白、通路和酶等研究,为胃癌的治疗提供了一种新的途径.     

小干扰RNA的合理设计

引言大量实验表明,针对同一靶基因的不同si RNA序列具有不同的沉默效率,为了能够运用RNAi技术更有效地沉默靶基因,需要在RNAi的第一步,也是其成功与否的关键环节?si RNA序列的设计方面进行更多的改进。本文将在以下几个方面探讨si RNA序列设计方面的最新研究进展,作为应用RNAi技术时的参考。1SiRNA序列的设计RNAi最终要通过si RNA片段与靶基因结合并使之降解,因此,确保高度同源于靶基因而绝无与其他基因同源的si RNA序列,是决定RNAi特异性的关键所在,也是si RNA设计的基本原则。从具有不同沉默效率的si RNA序列中筛选出高效的si R-NA序列,需要经过严密的设计和不断的实验检验[1]。1.1si RNA序列在靶基因中的位置从靶基因起始密码子AUG下游50~100个核苷酸开始搜寻理想的si RNA序列,越靠近靶基因的3′端,其基因沉默效果可能越好[2]。以前的研究表明:不要以5′非翻译区(5′UTR)和3′非翻译区(3′UTR)及起始密码子附近序列作为设计si RNA的模板,这些区域含有调节蛋白结合位点(如翻译起始复合物),调节蛋白可与RISC竞争结合si RNA序列,降低R...     

短发夹状RNA使骨肉瘤细胞缺氧诱导因子HIF-1α基因沉默

目的构建低氧诱导因子HIF-1α发夹状小干涉RNA真核表达载体，观察发夹状双链小干涉RNA对骨肉瘤细胞HIF-1的转录后的基因沉默效果?方法体外合成两对互补的寡核苷酸链，分别针对缺氧诱导因子HIF-1αmRNA序列的两个靶位点。退火形成双链，插入pSilencer^TM neoU62．1真核表达载体。将构建好的载体经脂质体介导转染骨肉瘤SaOS-2细胞，观察HIF-lα基因的沉默效果。半定量RT—PCR检测HIF-1αmRNA的抑制程度，免疫荧光和免疫印迹(Western Blot)检测HIF-1α蛋白表达情况。结果测序证实成功构建HIF-1α发夹状小干涉RNA真核表达载体。转录生成的发夹状小干涉能够特异抑制HIF-1α表达。RT—PCR结果显示针对HIF-1αmRNA序列的两个靶位点的沉默效果分别为69％和92％，免疫荧光显示转染后荧光强度显著降低，免疫印迹显示两个靶位点的发夹状小干涉RNA分别使HIF-1α蛋白表达下降66％和90％。结论通过体内转录生成的发夹状小干涉RNA能够使骨肉瘤细胞缺氧诱导因子HIF-1α基因沉默。     

RNA干扰技术的应用

RNA干扰(RNAi)能产生序列特异性的基因沉默.RNAi的研究与应用在短时间内得到了迅速发展,其应用主要围绕特异性降解同源mRNA的作用方面.现对其应用进展进行综述.     

RNA干扰技术的应用

RNA干扰(RNAi)能产生序列特异性的基因沉默。RNAi的研究与应用在短时间内得到了迅速发展，其应用主要围绕特异性降解同源mRNA的作用方面。现对其应用进展进行综述。     

Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference (RNAi)

Short 21-mer double-stranded RNA (dsRNA) molecules have recently been employed for the sequence-specific silencing of endogenous human genes. This mechanism, called RNA interference (RNAi), is extremely potent and requires only a few dsRNA molecules per cell to silence homologous gene mRNA expression. We used dsRNA targeting the M-BCR/ABL fusion site to kill leukemic cells with such a rearrangement. Transfection of dsRNA specific for the M-BCR/ABL fusion mRNA into K562 cells depleted the corresponding mRNA and the M-BCR/ABL oncoprotein. This was demonstrated by real-time quantitative PCR and Western blots. The BCR/ABL knockdown was accompanied by strong induction of apoptotic cell death. Leukemic cells without BCR/ABL rearrangement were not killed by M-BCR/ABL-dsRNA. In addition, to corroborate the extraordinary sequence specificity of RNAi, we designed another RNA oligo matching the M-BCR/ABL fusion site but having two point mutations within its central region. We show that these two point mutations abolished both p210 reduction and induction of apoptosis in K562 cells. Finally, we compared leukemic cell killing by RNAi to that caused by the ABL kinase tyrosine inhibitor, STI 571, Imatinib. For full induction of apoptosis, dsRNA targeting M-BCR/ABL required 24 h more than Imatinib. This may be caused by the relatively long half-life of the BCR/ABL oncoprotein, which is not targeted by the RNAi mechanism, but is affected by STI 571. When we applied ds M-BCR/ABL RNA and STI 571 in combination, we did not observe a further increase in the induction of apoptosis. Nevertheless, these data may open a field for further studies towards gene-therapeutic approaches using RNA interference to kill tumor cells with specific genetic abnormalities.     

RNAi在胃癌基因治疗中的研究

RNA干扰(RNAi)又称转录后基因沉默,是由外源或内源性的双链RNA(dsRNA)导入细胞而引起同源的mRNA降解,进而抑制其相应的基因表达。作为一项新技术,目前广泛地应用于生物体基因功能研究和多种肿瘤的研究。在胃癌方面,现针对胃癌相关基因治疗进行了积极的探索,并取得了一些突破性的进展。     

RNAi在胃癌基因治疗中的研究

RNA干扰（RNAi）又称转录后基因沉默,是由外源或内源性的双链RNA（dsRNA）导入细胞而引起同源的mRNA降解,进而抑制其相应的基因表达。作为一项新技术,目前广泛地应用于生物体基因功能研究和多种肿瘤的研究。在胃癌方面,现针对胃癌相关基因治疗进行了积极的探索,并取得了一些突破性的进展。     

RNA干扰p53对肝细胞癌SK-Hep-1细胞细胞周期的影响

背景与目的: 肝细胞癌是严重威胁人类健康的恶性肿瘤之一, p53又是重要的抑癌基因,而RNA干扰(RNAinterference, RNAi) 不仅是一种经济、快捷、高效的抑制基因表达的技术手段, 而且有可能在肿瘤基因治疗方面提供新的途径。因此, 我们将外源性p53双链小RNA(smalldoublestrandedRNA, dsRNA) 瞬时转染肝癌SK- Hep -1细胞系, 观察其对肝癌细胞P53和P21蛋白表达及细胞周期的影响。方法: 合成的p53dsRNA和EGFPdsRNA, 分别以200ng和400ng的p53dsRNA用脂质体转染法转染SK- Hep- 1细胞, 同时设0. 9%NaCl空白对照和EG- FP及EGFP+EGFPdsRNA阳性对照组, 每组3个复孔, 分别在转染24h和48h时用流式细胞仪进行细胞周期检测, 对转染p53dsRNA48h后应用WesternBlot检测P53和P21蛋白的表达,初步探讨p53的dsRNA干扰对肝癌细胞影响的机制。结果: SK- Hep -1细胞在转染200ngp53dsRNA后G0 -G1 期细胞明显减少, 24h时与空白对照相比减少了52 .53%, 与转染同剂量EG FP+EGFPdsRNA的阳性对照组相比也减少了50 .29% (P<0. 05); S期细胞明显增多, 24h时与空白对照相比增加了146 8%, 与转染同剂量EGFP+EGFPdsRNA的阳性对照组相比增加了128. 62% (P<0 .05); G2-M期细胞也明显增多, 24h时与空白对照相比增加了30. 56% (P<0. 05),     

RNA interference is a functional pathway with therapeutic potential in human myeloid leukemia cell lines

BACKGROUND: RNA interference (RNAi) is a cellular pathway of gene silencing in a sequence-specific manner at the messenger RNA level. The basic mechanism behind RNAi is the breaking of a double-stranded RNA (dsRNA) matching a specific gene sequence into short pieces called short interfering RNA, which trigger the degradation of mRNA that matches its sequence. In this study, we explored the effects of RNAi in reducing the target gene expression in human myeloid leukemia cell lines. METHODS: Four myeloid leukemia cell lines (HL-60, U937, THP-1, and K562) were transfected with dsRNA duplexes corresponding to the endogenous c-raf and bcl-2 genes and the gene expression inhibition was assessed. The effect of RNAi on cell differentiation was studied; the apoptosis induction and the sensitization of the leukemia cell lines to etoposide and daunorubicin were quantified by flowcytometric methods. RESULTS: Transfection of the myeloid leukemia cell lines with dsRNA corresponding to c-raf and bcl-2 genes decreased the expression of Raf-1 and Bcl-2 proteins. RNAi for c-raf gene blocked the appearance of the monocytic differentiation induced by treatment with TPA. Combined RNAi for c-raf and bcl-2 induced apoptosis in HL-60, U937, and THP-1 cells and increased chemosensitivity to etoposide and daunorubicin. CONCLUSIONS: RNAi is a functional pathway in human myeloid leukemia cell lines and combined RNAi of c-raf and bcl-2 genes may represent a novel approach to leukemia, providing a means to overcome the resistance to chemotherapeutic agents and ultimately to augment the efficacy of chemotherapy in myeloid leukemia.     

Cationized gelatin delivery of a plasmid DNA expressing small interference RNA for VEGF inhibits murine squamous cell carcinoma

Double-stranded RNA (dsRNA) plays a major role in RNA interference (RNAi), a process in which segments of dsRNA are initially cleaved by the Dicer into shorter segments (21-23 nt) called small interfering RNA (siRNA). These siRNA then specifically target homologous mRNA molecules causing them to be degraded by cellular ribonucleases. RNAi down regulates endogenous gene expression in mammalian cells. Vascular endothelial growth factor (VEGF) is a key molecule in vasculogenesis as well as in angiogenesis. Tumor growth is an angiogenesis-dependent process, and therapeutic strategies aimed at inhibiting angiogenesis are theoretically attractive. To investigate the feasibility of using siRNA for VEGF in the specific knockdown of VEGF mRNA, thereby inhibiting angiogenesis, we have performed experiments with a DNA vector based on a siRNA system that targets VEGF (siVEGF). It almost completely inhibited the expression of three different isoforms (VEGF120, VEGF164 and VEGF188) of VEGF mRNA and the secretion of VEGF protein in mouse squamous cell carcinoma NRS-1 cells. The siVEGF released from cationized gelatin microspheres suppressed tumor growth in vivo. A marked reduction in vascularity accompanied the inhibition of a siVEGF-transfected tumor. Fluorescent microscopic study showed that the complex of siVEGF with cationized gelatin microspheres was still present around the tumor 10 days after injection, while free siVEGF had vanished by that time. siVEGF gene therapy increased the fraction of vessels covered by pericytes and induced expression of angiopoietin-1 by pericytes. These data suggest that cationized-gelatin microspheres containing siVEGF can be used to normalize tumor vasculature and inhibit tumor growth in a NRS-1 squamous cell carcinoma xenograft model.     

Studies on the inhibition of feline EGFR in squamous cell carcinoma: enhancement of radiosensitivity and rescue of resistance to small molecule inhibitors

This study investigated different methods of EGFR (Epithelial Growth Factor Receptor) targeting in feline squamous cell carcinoma with the ultimate aim of establishing a large animal model of human head and neck cancer. Both small molecule receptor tyrosine kinase inhibitor (TKI) and RNA interference (RNAi) techniques were employed to target the feline EGFR. We demonstrated that the human drug gefitinib caused a reduction in cell proliferation and migration in a feline cell line. However, we also document the development of resistance that was not associated with mutation in the kinase domain. RNAi caused a potent reduction in EGFR activity and was able to overcome acquired gefitinib resistance. In addition, RNAi targeting of EGFR, but not gefitinib, caused an additive effect on cell killing when combined with radiation. These results support the use of feline SCC as a model of head and neck cancer in man in the search for novel and effective treatments for both tumors.     

Inhibition of CD147 expression reduces tumor cell invasion in human prostate cancer cell line via RNA interference

CD147, also named extracelluar matrix metalloproteinase inducer (EMMPRIN), has been proved to be involved in the invasion and metastasis processes of tumor cells in many types of cancers. To determine the role of CD147 in the invasiveness properties of prostate cancer, we successfully downregulated CD147 by RNA interference (RNAi) technology, in PC-3 cell line at high level of CD147 expression. PC-3 cells were transfected with a pSilencer 4.1-CMV neo Vector coding for an RNA composed of two identical 19-nucleotide sequence motifs in an inverted orientation, separated by a 9-bp spacer to form a hairpin dsRNA capable of mediating target CD147 inhibition. Gelatin zymography was employed to determine the effect on reducing secretions of MMP-2 and MMP-9 of the transfected cells. Matrigel invasion assay was performed to evaluate the invasion ability of PC-3 cells in vitro. Our results showed that CD147 expression was significantly inhibited by small interfering RNAs (siRNA) transfectants in PC-3 cells at mRNA and protein levels, which resulted in dramatic reduction of invasion ability in tumor cells. Moreover, downregulation of CD147 resulted in reducing secretions of MMP-2, MMP-9. Taken together, CD147 downregulation by RNAi technology decreases the invasive capability of prostate cancer cells, demonstrating that stable expression of siRNA CD147 could potentially be an experimental approach for prostate cancer gene therapy.