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CN1434054A - Dowble-stranded RNA and use thereof - Google Patents

Dowble-stranded RNA and use thereof Download PDF

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CN1434054A
CN1434054A CN 03115489 CN03115489A CN1434054A CN 1434054 A CN1434054 A CN 1434054A CN 03115489 CN03115489 CN 03115489 CN 03115489 A CN03115489 A CN 03115489A CN 1434054 A CN1434054 A CN 1434054A
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tumor
rna
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CN1176937C (en )
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白春学
张新
张敏
陈杰
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复旦大学附属中山医院
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Abstract

The present invention relates to a double-stranded RNA and its application for inhibiting mammalian tumor cell. Said invention adopts the RNA interference technique to introduce the externally-synthetic double-stranded RNA into mammalian tumor cell by means of combination of non-viral carrier and/or viral carrier, and can inhibit the expression of epidermal growth factor receptor so as to change NSCLC cell biological character and raise the sensitivity of mammalian tumor cell to radiation therapy and chemical therapy. Said invented double-strand RNA can be used for preparing antitumor biological preparation and antitumor medicine to make direct injection in tumor body in travenous medication for effectively inhibiting tumor growth.

Description

一种双链RNA及其用途 A double-stranded RNA and its use

技术领域 FIELD

本发明属生物技术领域,涉及一种双链RNA及其抗肿瘤用途。 The present invention belongs to the field of biotechnology, and relates to a double-stranded RNA antitumor use. 具体涉及一种21-nt双链RNA及其在抑制非小细胞肺癌细胞株表皮生长因子受体表达的用途。 In particular to a 21-nt double-stranded RNA and its inhibition of non-small cell lung cancer cell line of epidermal growth factor receptor expression.

目前,抗肿瘤药物的研制主要针对治疗靶标的高通量药物筛选和针对治疗靶标的理性化药物设计。 At present, the development of anticancer drugs mainly screening and rational drug design for a therapeutic target for the treatment of high-throughput drug target. 近年来受到广泛重视的反义核酸技术是后者的一个重要代表,通过设计和靶标蛋白基因的反义核酸,导入到细胞中,对靶标蛋白的表达进行抑制,达到杀死和治疗肿瘤的目的。 In recent years, extensive attention antisense nucleic acid technology is an important representative of the latter, by designing antisense nucleic acids and the target gene is introduced into a cell, target protein expression can be suppressed, and the purpose of treating a tumor kill . 反义核酸由于其作用靶点序列清楚、易设计制备,被认为是极具潜力的肿瘤治疗药物,但在实际应用中,存在抑制效率低,专一性差,较大的毒性以及用药量较大和成本高等缺陷。 The antisense nucleic acid sequence due to its targets clear, easily prepared, is considered to be great potential cancer therapy, but in practical applications, the presence of low suppression efficiency, poor specificity, toxicity, and larger and larger dosage and high costs.

RNA干扰(RNA interference,RNAi)是由双链RNA(double-stranded RNA,dsRNA)引发的序列依赖性的转录后基因沉默机制(posttranscriptional genesilencing,PTGS)。 RNA interference (RNA interference, RNAi) is a post of a double-stranded sequence-dependent RNA (double-stranded RNA, dsRNA) transcriptional gene silencing mechanism initiated (posttranscriptional genesilencing, PTGS). 在哺乳动物细胞中,21-23nt dsRNA进入细胞后,能以其为模板,降解与之序列相应的mRNA,从而达到专一性抑制特定基因的蛋白生成。 In mammalian cells, 21-23nt dsRNA into the cell, can be used as a template, mRNA degradation sequence corresponding thereto, so as to achieve specific suppression of gene specific protein production. RNAi是真核生物中普遍存在的抵抗病毒入侵、抑制转座子活动、调控基因表达的监控机制。 RNAi is ubiquitous in eukaryotes against viruses, suppress transposon activity, regulatory monitoring mechanism of gene expression. 在药物开发方面,RNAi除了具有和反义核酸相同的优点外,还具有针对靶点抑制的专一性高,用量少,药物毒性小的特点。 In drug development, RNAi and antisense nucleic acids in addition to having the same advantages, but also has a high specificity for target inhibition, with less drug toxicity small features. 目前将RNAi技术用于抗病毒(如HIV病毒)和细胞信号传递系统的研究方兴未艾,但用于肿瘤治疗未见报道。 The RNAi technology currently used anti-viral (e.g., HIV virus) studies and cell signaling systems in the ascendant, but have not been reported for the treatment of tumors.

本发明的进一步目的是提供新的抗肿瘤生物制剂和抗肿瘤药物。 A further object of the present invention is to provide a novel antitumor biological agents and an antitumor agent.

本发明选定哺乳动物肿瘤细胞表皮生长因子受体(EGFR)作为肿瘤治疗靶点,采用RNA干扰(RNAi)技术,体外合成与EGFR cDNA序列互补的21nt双链RNA,与非病毒载体和/或病毒载体结合导入哺乳动物肿瘤细胞,抑制表皮生长因子受体的表达,从而改变NSCLC细胞生物学特性,增加放化疗敏感性。 The present invention is selected mammalian tumor epidermal growth factor receptor (EGFR) as a target for cancer therapy, use of RNA interference (RNAi) technology, synthesized in vitro with EGFR cDNA sequence complementary 21nt double-stranded RNA, and non-viral vectors, and / or viral vectors introduced into mammalian tumor cell binding, inhibit the expression of epidermal growth factor receptors, thereby altering the biological characteristics of NSCLC cells, increased sensitivity to chemotherapy.

本发明所述哺乳动物肿瘤细胞表皮生长因子受体(epidermal growthfactor receptor,EGFR)是一分子量为170KD的跨膜糖蛋白,可调控细胞周期,调节细胞增生、存活、粘附、迁移和分化,促进损伤修复。 The present invention is a mammalian tumor cell epidermal growth factor receptor (epidermal growthfactor receptor, EGFR) is a transmembrane glycoprotein of a molecular weight of 170KD, can regulate the cell cycle, regulate cell proliferation, survival, adhesion, migration and differentiation and promote damage repair. EGFR在包括非小细胞肺癌(non-small-cell-lung cancer,NSCLC)在内的多种上皮源性肿瘤中过度表达,与肿瘤增生、转移、放化疗敏感性下降关系密切。 EGFR in a variety of epithelial tumors including non-small cell lung cancer (non-small-cell-lung cancer, NSCLC) including overexpressed, tumor proliferation, metastasis, decreased sensitivity to chemotherapy closely.

本发明按下述方法和步骤进行,1、测定EGFR受体(1)选用EGFR阳性的细胞株A549、SPC-A-1,通过免疫组化法和免疫荧光法对EGFR定性、定位。 The present invention as described below and steps, 1, EGFR receptor assay (1) selection of EGFR-positive cell lines of A549, SPC-A-1, by immunohistochemistry and immunofluorescence to EGFR qualitative positioning.

免疫组化设低倍镜、高倍镜阴性对照和阳性对照。 Immunohistochemistry set low magnification, high magnification, the negative and positive controls. 免疫组化结果阳性对照在细胞膜,细胞浆和细胞核显示棕色颗粒,阴性对照未见特异性着色。 Immunohistochemistry results positive control in the cell membrane, cytoplasm and nucleus show brown particles, a negative control and no specific coloration.

上述方法所用一抗为鼠抗人EGFR单克隆抗体,由中科院细胞生物所提供,二抗为SABC标记的兔抗鼠IgG。 The above-described method using a murine anti-human anti-EGFR monoclonal antibodies, biological cells provided by the Chinese Academy of Sciences, rabbit anti-mouse IgG secondary antibody is labeled SABC.

所述细胞株由中科院上海细胞生物学研究所细胞库提供。 The cell line provided by the Chinese Academy of Sciences Shanghai Institute of Cell Biology, Cell Bank.

免疫荧光法设阴性和阳性对照荧光照片。 Immunofluorescence set negative and positive controls fluorescence photograph. 试验结果同免疫组化染色。 The test results with immunohistochemical staining. 所述二抗采用FITC标记的兔抗鼠IgG。 The use of a secondary antibody rabbit anti-mouse IgG FITC-labeled.

(2).测定受体数量采用流式细胞仪测定受体数量,结果为A549:%gated:78.58%;mean:54.51,SPC-A-1:%gated:98.89%mean:103.79,H69:%gated:0.67%mean:67.54。 (2) determining the amount of receptor receptor number was measured by flow cytometry and found to A549:% gated:. 78.58%; mean: 54.51, SPC-A-1:% gated: 98.89% mean: 103.79, H69:% gated: 0.67% mean: 67.54.

2、筛选EGFR高表达细胞株取细胞株A549,SPC-A-1,ECV,HFL,H7402,BEL7402、SKOV3和H69进行筛选,结果表明A549,SPC-A-1为EGFR高表达细胞株,ECV、HFL、H7402、BEL7402、SKOV3细胞株EGFR表达较低。 2, screening and EGFR-expressing cell lines taken cell line A549, SPC-A-1, ECV, HFL, H7402, BEL7402, SKOV3 and H69 screening results show that A549, SPC-A-1 is a high expression of EGFR cell line, the ECV , HFL, H7402, BEL7402, low EGFR expression SKOV3 cell line. H69细胞株无EGFR表达。 H69 cell line no EGFR expression. 设H69细胞株为阴性对照。 H69 cell line is provided as a negative control.

所述细胞株由中科院上海细胞生物学研究所细胞库提供。 The cell line provided by the Chinese Academy of Sciences Shanghai Institute of Cell Biology, Cell Bank.

3、按下述原则设计ssRNA,1)EGFR的cDNA启动子AUG下游75碱基处,2)找到第一个AA二聚体,3)记录AA下游19个核苷酸(siRNA-EGFR),其核苷酸序列为:5'AAGGAGCUGCCCAUGAGAAAU……mRNA4)计算嘌呤和嘧啶的百分比(G/C),G/C比值为30%至70%,50%最佳。 3, according to the following design principles ssRNA, 1) EGFR cDNA was sub-AUG starts 75 bases downstream, 2) to find the first AA dimer, 3) 19 nucleotides downstream of the recording AA (siRNA-EGFR), nucleotide sequence: percentage (G / C) 5'AAGGAGCUGCCCAUGAGAAAU ...... mRNA4) calculating the purine and pyrimidine, G / C ratio of 30% to 70%, best 50%.

4、按已知的体外化学合成方法合成siRNA-EGFR,其核苷酸序列为:GGAGCUGCCCAUGAGAAAUdTdT……siRNAAUUUCUCAUGGGCAGCUCCdTdT……siRNA complement5、拟和(annealing)siRNA双链1)独立分装上述化学合成的RNA单链,用无RNase水稀释,2)正义、反义RNA混合,加入拟和缓冲液, 4, according to the known chemical synthetic methods in vitro synthesized siRNA-EGFR, which nucleotide sequence: GGAGCUGCCCAUGAGAAAUdTdT ...... siRNAAUUUCUCAUGGGCAGCUCCdTdT ...... siRNA complement5, fit (annealing) siRNA duplexes 1) above independently dispensing chemically synthesized RNA single chain, diluted with RNase-free water, 2) sense and antisense RNA were mixed, and intended buffer,

3)90℃孵育,离心,收集管底液体。 3) 90 ℃ incubation, centrifuge, a liquid collection tube bottom.

4)37℃孵育1小时。 4) 37 ℃ for 1 hour.

5)拟和的双链RNA的终浓度为20uM。 5) the final concentration of double-stranded RNA, and proposed for 20uM. 形成的双链具有如下序列。 Double strand has the following sequence. GGAGCUGCCCAUGAGAAAUdTdT……siRNAAUUUCUCAUGGGCAGCUCCdTdT……siRNA complement6、sRNA-unrelated的设计、合成以及拟和同dsRNA-EGFR。 GGAGCUGCCCAUGAGAAAUdTdT ...... siRNAAUUUCUCAUGGGCAGCUCCdTdT ...... siRNA complement6, sRNA-unrelated design, synthesis and fitting with the dsRNA-EGFR. 序列为:5'-GAACUUCAGGGUCAGCUUGCCUU-3'——dsRNA-unrelated5'-GGCAAGCUGACCCUGAAGUUCUU-3'——unrelated completement7、15%PAGE-SDS电泳鉴定拟和的双链,拟和完全,双链片段大小正确。 Sequence: 5'-GAACUUCAGGGUCAGCUUGCCUU-3 '- dsRNA-unrelated5'-GGCAAGCUGACCCUGAAGUUCUU-3' - unrelated completement7,15% PAGE-SDS electrophoresis and identification of the double stranded proposed, and intends to complete, double-stranded fragments of the correct size.

8、筛选阳离子脂质体取阳离子脂质体TKO,Oligofectamine,LipofectamineTM2000进行筛选,采用高转导效率的LipofectamineTM2000用于dsRNA的转导。 8, taken screened cationic liposomes cationic liposomes TKO, Oligofectamine, LipofectamineTM2000 screening using high transduction efficiency LipofectamineTM2000 used to transduce the dsRNA.

9、RNA干扰技术用于肿瘤细胞:1)制备贴壁细胞,2)制备dsRNA-Lipofectamine复合物,3)流式细胞仪检测EGFR受体数目。 9, RNA interference on tumor cells: 1) Preparation of adherent cells, 2) the preparation of dsRNA-Lipofectamine complex, 3) EGFR receptor number of flow cytometry.

实验结果证实,A549细胞株EGFR表达降低了73.23%,SPC-A-1细胞株受体表达降低了76.01%。 Experimental results show that, EGFR expressing A549 cells reduced 73.23%, receptor expression SPC-A-1 cell line reduced 76.01%. 本发明将体外合成的dsRNA借助阳离子脂质体导入哺乳动物肿瘤细胞,能抑制表皮生长因子受体的表达,从而改变NSCLC细胞生物学特性,增加哺乳动物肿瘤细胞对放化疗的敏感性,获得肯定的RNAi效果。 The present invention is synthesized in vitro by means of cationic liposomes dsRNA into mammalian tumor cells, can inhibit the expression of epidermal growth factor receptors, thereby altering the biological characteristics of NSCLC cells, mammalian increased sensitivity of tumor cells to chemotherapy, obtaining a positive the RNAi effect.

本发明双链RNA可与非病毒载体和/或病毒载体结合,制备新的抗肿瘤生物制剂和抗肿瘤药物,行瘤体内直接注射或静脉给药有效抑制肿瘤生长。 Double-stranded RNA of the invention may be combined with non-viral vectors and / or viral vectors, the preparation of novel antitumor and antineoplastic biological agents, direct intratumoral injection or intravenous administration line effective to inhibit tumor growth.

其中1:分子量标准,2:正义RNA-EGFR,3:反义RNA-EGFR, Wherein 1: molecular weight standards, 2: sense RNA-EGFR, 3: antisense RNA-EGFR,

4:双链RNA-EGFR,5:非特异双链RNA图2是RNAi对SPC-A-1细胞EGFR的抑制作用。 4: double-stranded RNA-EGFR, 5: non-specific double-stranded RNA inhibition of FIG. 2 is an RNAi SPC-A-1 cells EGFR.

按已知体外化学合成方法合成siRNA-EGFR,其序列为:5'AAGGAGCUGCCCAUGAGAAAU……mRNA,GGAGCUGCCCAUGAGAAAUdTdT……siRNA,长度:21,分子量:6732.9(g/mole),ODU260:31.9(ug/ODU260),数量:50nmol/管×2管,AUUUCUCAUGGGCAGCUCCdTdT……siRNA complement长度:21,分子量:6583.7(g/mole),ODU260:33.4(ug/ODU260),数量:50nmol/管×2管,上述合成的每条RNA单链独立分装,RNase灭活的水稀释成50uM,正义RNA30ul与反义RNA30ul混合,加入15ul 5×拟和缓冲液,终体积为75ul,90℃孵育1分钟,离心15秒,收集管底液体,37℃孵育1小时,dsRNA双链终浓度为20uM。 Synthesized by known chemical synthetic methods in vitro siRNA-EGFR, having the sequence: 5'AAGGAGCUGCCCAUGAGAAAU ...... mRNA, GGAGCUGCCCAUGAGAAAUdTdT ...... siRNA, length: 21, molecular weight: 6732.9 (g / mole), ODU260: 31.9 (ug / ODU260), number: 50nmol / tube pipe × 2, AUUUCUCAUGGGCAGCUCCdTdT ...... siRNA complement length: 21, molecular weight: 6583.7 (g / mole), ODU260: 33.4 (ug / ODU260), number: 50nmol / pipe × 2 tubes, each of the above-synthesized dispensing separate single strand RNA, RNase inactivation diluted aqueous 50 uM, the antisense justice RNA30ul RNA30ul mixed, added to 15ul 5 × buffer and intended final volume of 1 minute incubation 75ul, 90 ℃, centrifuged for 15 seconds, the collection tube bottom liquid, 37 ℃ incubated for 1 hour, dsRNA duplex final concentration of 20uM. 拟和缓冲液的组成为:100mMKOAc,30mMHEPES-KOHpH7.4,2mM MgOAc。 And buffer composition is proposed: 100mMKOAc, 30mMHEPES-KOHpH7.4,2mM MgOAc. 经15%PAGE-SDS电泳,结果表明拟和的双链拟和完全,双链片段大小正确。 By electrophoresis 15% PAGE-SDS, and the results show that the proposed fitting fully double stranded, double-stranded fragments of the correct size.

实施例2 RNA干扰技术用于非小细胞肺癌细胞制备贴壁细胞:转染前一天接种A549或SPC-A-1细胞于12孔培养板,使得次日转染的时候细胞密度达40-50%(0.5-2×105细胞/孔/12孔培养板),更换培养液(DMEM+10%FBS),体积1ml。 Example 2 RNA interference techniques for the preparation of non-small cell lung cancer cells adherent cells: A549 were seeded one day or SPC-A-1 cells before transfection in 12-well culture plates, so that the next day transfected when cell density reached 40-50 % (0.5-2 × 105 cells / well / 12 well plate), replacing the culture medium (DMEM + 10% FBS), the volume of 1ml.

制备dsRNA-Lipofectamine复合物:1)将dsRNA稀释于100ul无血清DMEM,混匀。 DsRNA-Lipofectamine complex was prepared: 1) the dsRNA diluted in 100ul serum free DMEM, and mix.

2)Lipofectamine使用前混匀,然后4ul Lipofectamine与100ul无血清DMEM混匀。 2) Lipofectamine mix before use, and 4ul Lipofectamine mix with 100ul serum free DMEM. 室温孵育5min。 5min incubation at room temperature.

3)将dsRNA稀释液与Lipofectamine稀释液混匀,总体积200ul,室温孵育20min,室温保存。 3) The dsRNA Lipofectamine dilution with Diluent, 200 ul of total volume, incubated at room temperature 20min, stored at room temperature.

4)加入12孔培养板,混匀,培养箱内孵育24-48小时。 4) 12-well culture plate was added, mixed and incubated for 24-48 hours incubator.

流式细胞仪检测受体数目,结果为:A549细胞株EGFR表达降低了73.23%(dsRNA-EGFR16ul/lipofectamine2000 4ul/12孔培养板)。 Receptor number detected by flow cytometry, the result is: EGFR expressing A549 cells reduced 73.23% (dsRNA-EGFR16ul / lipofectamine2000 4ul / 12 well plate). SPC-A-1细胞株受体表达降低了76.01%(dsRNA-EGFR16 ul/lipofectamine 2000 4ul/12孔培养板)。 Receptor expression SPC-A-1 cell line decreased 76.01% (dsRNA-EGFR16 ul / lipofectamine 2000 4ul / 12 well plate).

结果证实,本发明dsRNA能抑制表皮生长因子受体的表达,改变NSCLC细胞生物学特性,增加哺乳动物肿瘤细胞对放化疗的敏感性,获得肯定的RNAi效果。 The results confirmed that, according to the present invention is a dsRNA can inhibit the expression of epidermal growth factor receptor, alter the biological properties of NSCLC cells, mammalian tumor cells to increased sensitivity to chemotherapy, an affirmative RNAi effect.

Claims (7)

1.一种双链RNA,其特征是具有如下序列的21-nt双链RNA。 A double-stranded RNA, wherein the 21-nt double-stranded RNA having the sequence. GGAGCUGCCCAUGAGAAAUdTdT……siRNAAUUUCUCAUGGGCAGCUCCdTdT……siRNA complement GGAGCUGCCCAUGAGAAAUdTdT ...... siRNAAUUUCUCAUGGGCAGCUCCdTdT ...... siRNA complement
2.按权利要求1所述的双链RNA,其特征是所述的双链RNA是体外合成。 2. The double-stranded RNA according to claim 1, wherein the double stranded RNA is synthesized in vitro.
3.按权利要求1和2所述的双链RNA,其特征是所述双链RNA与非病毒载体和/或病毒载体结合导入肿瘤细胞,制备抗肿瘤生物制剂和抗肿瘤药物。 3. The double-stranded RNA according to claim 1 and claim 2, wherein said double-stranded RNA and non-viral vectors and / or viral vector into the tumor cells in combination, the antitumor preparation of biological agents and an antitumor agent.
4.按权利要求3所述的双链RNA,其特征是所述非病毒载体是阳离子脂质体。 4. The double-stranded RNA according to claim 3, wherein said non-viral vectors to cationic liposomes.
5.按权利要求4所述的双链RNA,其特征是所述阳离子脂质体是Lipofecta-mineTM2000。 As claimed in claim 5. said double stranded RNA 4, wherein said cationic liposome is Lipofecta-mineTM2000.
6.按权利要求3所述的双链RNA,其特征是所述肿瘤细胞是哺乳动物肿瘤细胞。 6. The double-stranded RNA according to claim 3, wherein said tumor cell is a mammalian tumor cell.
7.按权利要求3所述的双链RNA,其特征是所述肿瘤细胞是非小细胞肺癌细胞。 7. The double-stranded RNA according to claim 3, wherein the tumor cells are non-small cell lung cancer cells.
CN 03115489 2003-02-21 2003-02-21 Double-stranded RNA and use thereof CN1176937C (en)

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