CN105063048A - SiRNA (small interfering ribonucleic acid) capable of inhibiting expression of Survivin genes and application of siRNA - Google Patents

SiRNA (small interfering ribonucleic acid) capable of inhibiting expression of Survivin genes and application of siRNA Download PDF

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CN105063048A
CN105063048A CN201510497040.6A CN201510497040A CN105063048A CN 105063048 A CN105063048 A CN 105063048A CN 201510497040 A CN201510497040 A CN 201510497040A CN 105063048 A CN105063048 A CN 105063048A
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sirna
cancer
survivin
expression
application
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谢晶
邢高扬
李剑光
滕乐生
孟庆繁
逯家辉
刘艳
权宇彤
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吉林大学
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Abstract

The invention discloses siRNA (small interfering ribonucleic acid) capable of inhibiting expression of Survivin genes and an application of siRNA and particularly relates to an application of the efficient siRNA in preparation of medicines for controlling the tumor cell cycle. Efficient siRNA double-strand sequence can specifically inhibit expression of protein related to a disease after entering an organism in a certain way, so that related disease genes are silenced, and the purpose of treatment is achieved. SiRNA molecules have spectral anti-tumor activity, have a remarkable inhibition effect on diseases such as leukemia, gastric cancer, prostate, bladder cancer, cervical cancer, breast cancer, lung cancer, liver cancer and the like and can be used for preparing medicines for inhibiting proliferation and transfer of tumor cells.

Description

-种抑制Survivin基因表达的siRNA及其应用 - Species and Its Application siRNA inhibiting expression of Survivin

技术领域 FIELD

[0001] 本发明公开了一种抑制survivin基因表达的siRNA分子及其在制备抗肿瘤药物的应用,属于生物医药技术领域。 [0001] The present invention discloses a method of inhibiting the expression of survivin siRNA molecule and its application in the manufacture of anticancer drugs, the field of biotechnology medicine.

背景技术 Background technique

[0002] 目前,恶性肿瘤临床进展快、生存期短以及传统临床治疗的中远期疗效均不理想, 化疗药物一方面起到治疗作用,同时另一方面,化疗药物的毒副作用对患者的身体起到破坏作用。 [0002] Currently, cancer clinical progress fast, short life as well as traditional clinical treatment of the long-term effects are not ideal, chemotherapy drugs play a therapeutic effect on the one hand, while on the other hand, side effects of chemotherapy drugs on the patient's body play a destructive role. 因此,因而寻找新的肿瘤治疗策略是全球临床及科研工作者所面临的重大问题。 So, therefore the search for new therapeutic strategies is a major global problem and clinical researchers face. 随着分子生物学的发展,针对肿瘤基因进行靶向治疗成为癌症治疗研究的方向。 With the development of molecular biology, has become the direction targeted therapy for tumor therapy for cancer gene. 药物作用于肿瘤某分子、某基因等靶向部位,一方面提高了疗效,另一方面减少了药物的用量。 Drug molecules for targeting a tumor site, a gene or the like, on the one hand to improve the effect, on the other hand reducing the amount of the drug. 寻找能成为肿瘤治疗的标靶已成为治疗肿瘤的前提之一。 Looking to become a target for cancer treatment has become a prerequisite for tumor therapy. 可能成为药物作用的标靶包括:癌细胞具有的特殊的抗原、特殊生长因子受体、肿瘤血管生长相关因子、癌细胞特殊信息传递途径中的各种分子、癌细胞内细胞周期和细胞凋亡的调控分子。 The drug may become targets include: cancer cells have special antigens, specific growth factor receptor, tumor angiogenesis related growth factors, various cancer specific information transfer pathway molecule, cycle and apoptosis in cancer cells Cells the regulatory molecules.

[0003] 人类survivin基因是1997年美国耶鲁大学的Ambrosini等利用效应细胞蛋白酶受体-1(effectorcellproteasereceptor_l,EPR-l)cDNA在人类基因组的杂交筛选中分离并克隆出来的,与EPR-1同位于染色体17q25,全长14. 7kb,由3个内含子和4个外显子组成,其mRNA长约1.9kb,其编码产生的蛋白由142个氨基酸组成,相对分子量为16. 5X103, 是近年来发现的凋亡抑制蛋白(inhibitorofapoptosisprotein,IAP)家族中的一个新成员,具有抑制细胞凋亡和维持细胞有丝分裂的双重功能,在正常的终末分化组织中几乎不表达。 [0003] In human survivin gene is 1997 Ambrosini et Yale University using the effector cell protease receptor -1 (effectorcellproteasereceptor_l, EPR-l) cDNA isolated in the hybridization screening of the human genome and cloned, with the EPR-1 located at the same chromosome 17q25, a full-length 14. 7kb, by three introns and 4 exons, which mRNA is about 1.9 kb, encoding a protein produced by the 142 amino acids, relative molecular weight of 16. 5X103, in recent years inhibition of apoptosis to find a new member of the family of proteins (inhibitorofapoptosisprotein, IAP), and inhibition of apoptosis has a dual function of maintaining cell mitosis, almost no expression in normal terminally differentiated tissues. 在生理状态下,survivin仅表达于成人分泌期子宫内膜、胎盘、睾丸、胸腺及胚胎中,但在病理状态下,survivin广泛表达于人类各种恶性肿瘤组织中,如胃癌、食管癌、结直肠癌、肝癌、乳腺癌、宫颈癌、肺癌、白血病、黑色素瘤、淋巴瘤、III~IV期神经母细胞瘤等。 In physiological conditions, Survivin expression only in adult secretory endometrium, placenta, testis, thymus and embryos, but in pathological conditions, Survivin widely expressed in various human malignant tumors, such as gastric cancer, esophageal cancer, colorectal colorectal cancer, liver cancer, breast cancer, cervical cancer, lung cancer, leukemia, melanoma, lymphoma, III ~ IV stage neuroblastoma like. survivin表达增高可抑制肿瘤细胞凋亡而促进其增殖,而且其表达与病程进展明显相关。 inhibit survivin expression increased tumor cell apoptosis and promoting proliferation, and its expression was significantly related to progression. 因此,survivin可能作为一种新的肿瘤标志物,成为检测恶性肿瘤的指标。 Accordingly, Survivin may serve as a novel tumor marker, an index detection of malignant tumors.

[0004] RNA干扰survivin表达技术,RNA干扰(RNAinterference,RNAi)是指在生物体细胞内,外源性或内源性的双链RNA(double-strandedRNA)引起与其同源的mRNA特异性的降解,从而抑制survivin基因的转录和表达,具有高度特异性、高效性的特点。 [0004] RNA interference survivin expression technology, RNA interference (RNAinterference, RNAi) refers to a cell in an organism, exogenous or endogenous double-stranded RNA (double-strandedRNA) induced specific degradation of homologous mRNA , thereby inhibiting transcription and expression of survivin, having a high specificity, efficiency characteristics. 总结近年来的研究成果,其在临床应用中具有较广阔的前景。 Summarize recent research results, which has a broader prospect in clinical application.

发明内容 SUMMARY

[0005] 本发明公开一种抑制survivin基因表达的双链siRNA分子其在抗肿瘤药物的应用,是一种新的siRNA序列,可以抑制肿瘤细胞的增殖和转移。 [0005] The present invention discloses a method of inhibiting the application of double-stranded siRNA molecule survivin gene expression in anticancer drugs, is a new siRNA sequences may inhibit the proliferation and metastasis of tumor cells.

[0006] 本发明所述的双链SiRNA分子,其特征在于,该双链SiRNA分子是一种高效靶向survivin基因的广谱双链的小分子干扰RNA (siRNA),具有如下序列结构: [0006] The present invention is double-stranded SiRNA molecule, wherein the double-stranded molecule is an efficient SiRNA targeting survivin gene broad spectrum duplex small interfering RNA (siRNA), the structure has the following sequence:

[0007] 正义链5 ' -GAGACAGAAUAGAGUGAUA-3 'SEQIDN0:1 [0007] The sense strand 5 '-GAGACAGAAUAGAGUGAUA-3' SEQIDN0: 1

[0008] 反义链5 ' -UAUCACUCUAUUCU⑶CUC-3 'SEQIDN0:2 [0008] The antisense strand 5 '-UAUCACUCUAUUCU⑶CUC-3' SEQIDN0: 2

[0009] 本发明所述的双链siRNA分子的主干序列即为SEQIDNO:1和SEQIDNO:2所示的核苷酸序列,即正义链和反义链分别具有SEQIDN0:1和2所示的核苷酸序列的双链分子。 [0009] The double stranded siRNA molecule backbone sequence according to the present invention is the SEQIDNO: 1 and SEQIDNO: 2 nucleotide sequence, i.e., the sense strand and antisense strand each having SEQIDN0: 1 and a core 2 shown in FIG. nucleotide sequence of the double-stranded molecule.

[0010] 优选的,所述双链SiRNA分子,具有如下的序列结构: [0010] Preferably, the double stranded SiRNA molecule, having the sequence structure:

[0011]正义链:5 '-GAGACAGAAUAGAGUGAUAtt-3 'SEQIDN0:3 [0011] sense strand: 5 '-GAGACAGAAUAGAGUGAUAtt-3' SEQIDN0: 3

[0012] 反义链:5 ' -UAUCACUCUAUUCU⑶CUCtt-3 'SEQIDN0:4。 [0012] Antisense strand: 5 '-UAUCACUCUAUUCU⑶CUCtt-3' SEQIDN0: 4.

[0013] 本发明的siRNA分子来源于针对survivin基因开放阅读框的功能保守区而制备的siRNA分子库,该序列的正义链与survivin基因的mRNA结合,干扰survivin基因mRNA 的翻译,从而抑制肿瘤细胞的分裂,使肿瘤细胞阻滞在G2/M期。 [0013] siRNA molecules siRNA molecules of the invention library prepared from survivin gene open reading frame for the function of the conserved regions, and survivin mRNA sense strand of the gene sequence binding interference survivin gene mRNA translation, thereby inhibiting tumor cell division, tumor cells arrested in G2 / M phase.

[0014] 本发明的siRNA分子的优点在于,所制备的siRNA随机分布于survivin基因开放阅读框区段,长度可控性分布于19-23bp,可以提高有效靶点的命中率。 [0014] siRNA molecules advantages of the present invention, siRNA prepared randomly distributed in the survivin open reading frame gene segments, the length of the controlled distribution in 19-23bp, can effectively improve the hit rate targets.

[0015] 本发明还提出了一种siRNA重组质粒,所述的siRNA重组质粒包含前面所述的任意一种双链siRNA分子的序列。 [0015] The present invention further provides a recombinant plasmid siRNA, siRNA according to any one recombinant plasmid comprising the sequence of a double stranded siRNA molecule previously described. 由此,利用siRNA重组质粒转染动物细胞,能够有效的沉默surivivin基因,进而诱导细胞凋亡、抑制肿瘤细胞分裂、抑制肿瘤血管的生成,从而能够有效的治疗肿瘤。 Thus, using siRNA plasmids were transfected into an animal cell, it can effectively silence gene surivivin, thereby inducing apoptosis, inhibition of tumor cell division, inhibition of tumor angiogenesis, thereby enabling effective treatment of tumors. 进一步,该siRNA分子与siRNA重组质粒能够有效的用于抗肿瘤药物的制备,从而利用制备的抗肿瘤药物对患者进行给药,有效的治疗肿瘤。 Further, the recombinant plasmid siRNA siRNA molecule can be effectively used for the preparation of antineoplastic, anti-tumor drugs using the thus prepared administered to a patient, an effective treatment of tumors.

[0016] 根据本发明还提出了一种survivin基因沉默试剂盒,所述试剂盒包含前面所述的任意一种双链siRNA分子或siRNA重组质粒。 [0016] The present invention further provides a kit survivin gene silencing, said kit comprising a double stranded siRNA molecule of any one recombinant plasmids or siRNA as previously described. 利用本发明的试剂盒能够有效的将肿瘤患者的细胞进行survivin基因沉默,从而能够有效的治疗肿瘤。 Using the kit of the present invention can be effective in patients with cancer cells survivin gene silencing, thereby enabling effective treatment of tumors.

[0017] 本发明的siRNA分子可以作为抗肿瘤药物的有效成分,肿瘤疾病包括如白血病、 胃癌、前列腺癌、膀胱癌、宫颈癌,乳腺癌,肺癌,肝癌等。 [0017] siRNA molecules of the invention may be used as an active ingredient of antitumor drugs, including neoplastic diseases such as leukemia, gastric cancer, prostate cancer, bladder cancer, cervical cancer, breast cancer, lung cancer, liver cancer and the like.

[0018] 因而,本发明还提出了前面所述的任意一种双链siRNA分子、或者siRNA重组质粒在制备抗肿瘤药物中的应用。 [0018] Accordingly, the present invention also provides a double stranded siRNA molecule of any one of the foregoing, or in the preparation of the recombinant plasmid by siRNA antineoplastic.

[0019] 本发明提出一种抗肿瘤药物,该药物含有有效量的siRNA分子或siRNA重组质粒, 该抗肿瘤药物可以采用常规方法制备成相应制剂,如可被制成针剂形式,例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。 [0019] The present invention provides an antitumor agent, the drug molecule containing an effective amount of a siRNA or siRNA recombinant plasmids, the respective antineoplastic formulations prepared by conventional methods can be employed as such can be made in the form of injection, for example, with physiological saline or an aqueous solution containing glucose and other adjuvants were prepared by conventional methods. 针剂、溶液等宜在无菌条件下制造。 Should injections, solutions and the like produced under sterile conditions.

[0020] 本发明的积极效果在于:提供了一种新的高效革E1向survivin基因的广谱双链的小分子干扰RNA(siRNA),同时还公开了其在制备抗肿瘤药物中的应用。 [0020] The positive effect of the present invention is: to provide a new efficient leather E1 to a small molecule interfering double-stranded broad spectrum of survivin gene RNA (siRNA), also discloses their use in the preparation of anti-tumor drugs.

附图说明 BRIEF DESCRIPTION

[0021] 图1是化学合成的siRNA转染两种细胞后实时定量PCR检测survivinmRNA表达量柱形图,纵坐标表示survivin相对于GAPDH的mRNA表达水平;横坐标表示四个处理实验组,分别为未转染的细胞组,转染实验包括三个siRNA浓度,分别为10nM、20nM和30nM。 [0021] FIG. 1 is a real-time quantitative PCR detection of the expression of the bar graph survivinmRNA chemically synthesized siRNA transfection in both cell ordinate indicates survivin relative to GAPDH mRNA expression; the abscissa represents four experimental treatment groups, respectively, untransfected cell group, three transfection experiments including siRNA concentration, respectively, 10nM, 20nM and 30nM.

[0022]图2是WesternBlot检测siRNA转染人前列腺癌细胞PC-3、肝癌细胞IfepG2、乳腺癌细胞MCF-7和宫颈癌细胞Hela后的survivin蛋白表达量,NC所对应的是阴性对照组, EG所对应的是加入20nM/孔的siRNA组。 [0022] FIG. 2 is a siRNA transfection WesternBlot detection of human prostate cancer PC-3, hepatoma cells IfepG2, MCF-7 cancer cells and the expression of the breast cancer cells Hela survivin protein, NC is the corresponding negative control group, EG is added to the corresponding 20nM / well siRNA group.

具体实施方式 Detailed ways

[0023] 为使本发明更加容易理解,下面结合具体实施例,进一步阐述本发明。 [0023] In order that the invention may be more readily understood, specific embodiments in conjunction with the following examples further illustrate the present invention. 这些实施例只用于说明本发明而不用于限制本发明的范围。 These embodiments of the present invention is illustrative only and not intended to limit the scope of the invention.

[0024] 实施例1 :siRNA沉默效率验证 [0024] Example 1: siRNA silencing efficiency verification

[0025] 1.主要仪器、试剂和材料. [0025] 1. The main instruments, reagents and materials.

[0026] 1. 1仪器:核酸合成仪(GE公司)、PCR仪(ABI公司);实时定量PCR(Bio-Rad);细胞培养箱(Thermo)等。 [0026] 1.1 Instrument: nucleic acid synthesizer (GE Company), PCR instrument (ABI Co.); quantitative real-time PCR (Bio-Rad); cell incubator (Thermo) and the like.

[0027]1. 2 材料和试剂:RNAiMAX™ (invitrogen),DMEM培养基(Gibco), [0027] 12 Materials and reagents:. RNAiMAX ™ (invitrogen), DMEM medium (Gibco),

[0028]TurboCapturemRNAkit(QIAGEN),SensiMix™one-StepKit(Quantace)等。 [0028] TurboCapturemRNAkit (QIAGEN), SensiMix ™ one-StepKit (Quantace) and the like. 其他生化试剂均购于上海生工生物工程技术服务有限公司。 Other biochemical reagents were purchased from Shanghai Biological Engineering Technology Services Limited.

[0029] 1. 3PCR引物(百奥迈科生物技术有限公司合成): [0029] 1. 3PCR primers (one hundred Synthesis Aomai Ke Biotechnology Co., Ltd.):

[0030]survivin正向引物:5 ' -AGAACTGGCCCTTCTTGGAGG-3 ' [0030] survivin forward primer: 5 '-AGAACTGGCCCTTCTTGGAGG-3'

[0031]survivin反向引物:5 ' -CTTTTTATGTTCCTCTATGGGGTC-3 ' [0031] survivin reverse primer: 5 '-CTTTTTATGTTCCTCTATGGGGTC-3'

[0032]GAPDH正向引物;5 ' -GAAGGTGAAGGTCGGAGTC-3 ' [0032] GAPDH forward primer; 5 '-GAAGGTGAAGGTCGGAGTC-3'

[0033]GAPDH反向引物:5 ' -GAAGATGGTGATGGGAITTC-3 ' [0033] GAPDH reverse primer: 5 '-GAAGATGGTGATGGGAITTC-3'

[0034] 2•化学合成siRNA [0034] 2 • chemically synthesized siRNA

[0035] 根据survivin基因的核苷酸序列,设计小分子干扰siRNA: [0035] The nucleotide sequence of the survivin gene, the design of small interfering siRNA:

[0036]正义链:5,-GAGACAGAAUAGAGUGAUAtt-3, [0036] sense strand: 5, -GAGACAGAAUAGAGUGAUAtt-3,

[0037]反义链:5,-UAUCACUCUAUUCU⑶CUCtt-3, [0037] Antisense strand: 5, -UAUCACUCUAUUCU⑶CUCtt-3,

[0038] 将该序列在人类EST数据库中比对,确定没有与它相同的其它基因序列。 [0038] The sequence alignments of human EST database, identify other gene sequence is not identical with it. siRNA由广州锐博生物科技有限公司合成。 siRNA synthesis from Guangzhou Rui Bo Biological Technology Co., Ltd.

[0039]3.RT-PCR检测survivin基因mRNA水平 [0039] 3.RT-PCR detection of survivin mRNA levels

[0040] 3. 1细胞培养:宫颈癌细胞Hela和人肺癌细胞A549分别在含10%FBS的DMEM培养基中,37°C、5 %C02培养箱培养。 [0040] 3.1 Cell culture: Hela cervical cancer cells and A549 cells were in DMEM medium containing 10% FBS in, 37 ° C, 5% C02 incubator.

[0041] 3. 2细胞铺板并转染:将宫颈癌细胞Hela和人肺癌细胞A549按1X105个/孔接种到96孔细胞培养板中,两种细胞分别设置未转染组、siRNA10nM组、siRNA20nM组和siRNA30nM组,共8组。 [0041] 3.2 cells were plated and transfected: Hela cervical cancer cells and A549 cells by 1X105 cells / well into 96-well cell culture plate, the two cells are disposed untransfected, siRNA10nM group, siRNA20nM siRNA30nM group and group 8 group. 在37°C、5%C02培养箱细胞培养过夜,在无双抗含10%FBS的DMEM 培养基中,转染按照RNAiMAX™的说明书转染,将实施例1步骤2中合成的siRNA分别按10nM/孔、20nM/孔和30nM/孔加入两组细胞对应的分组。 At 37 ° C, 5% C02 incubator cells were cultured overnight in DMEM medium containing 10% FBS anti unparalleled in transfected according to the transfection RNAiMAX ™ specification, Example 1, Step 2 in the embodiment respectively synthesized siRNA 10nM / well, 20 nM / well and 30 nM / well of cell packets corresponding to the two groups.

[0042] 用基因特异性引物检测样本中survivinmRNA表达水平,同时扩增看家基因GAPDH作为内参对照。 [0042] with gene-specific primers the expression level of a test sample survivinmRNA, simultaneous amplification of the housekeeping gene GAPDH was used as loading control. 每个反应做3个平行。 Each reaction was done three parallel. 建立如下25yL反应体系:4yL模板RNA, 12. 5yL2XSensiMixone-step, 1yL5 '正向引物(6yM),1yL3 '反向引物(6yM)0.5yL50XSYBRGreenI,用无RNase水补足体系至25yL。 25yL reaction system established as follows: 4yL template RNA, 12. 5yL2XSensiMixone-step, 1yL5 'forward primer (6yM), 1yL3' reverse primer (6yM) 0.5yL50XSYBRGreenI, made up with RNase-free water to the system 25yL. 反应条件:40°C反转录30min,95°C预变性7min,95°C变性20sec,60°C退火30sec,72°C延伸30sec,循环45 次。 Reaction conditions: 40 ° C reverse transcriptase 30min, 95 ° C denaturation 7min, 95 ° C denaturation 20sec, 60 ° C annealing 30sec, 72 ° C extend 30sec, 45 cycles.

[0043] 3. 4结果分析:用实时定量PCR分析实验结果,并作出柱状图,如图1所示,实验分为四组,其中"未转染"为未经任何处理的细胞样品,其他三组为本发明siRNA,浓度分别为10nM、20nM、30nM。 [0043] 3.4 Analysis of results: The results by real-time PCR, and to make a histogram, as shown in FIG. 1 were divided into four groups, wherein "non transfection" is a sample of cells without any treatment, other three groups of the present invention, siRNA, concentrations of 10nM, 20nM, 30nM. 结果显示革E1向survivin基因的siRNA沉默效果显著,具有一定的浓度依赖性。 The results show that the siRNA leather E1 survivin gene silencing effect significantly, with a certain concentration dependent. 在siRNA为30nM时,survivin基因的沉默效率即可达到80%以上。 When siRNA was 30nM, survivin gene silencing efficiency can reach 80% or more.

[0044] 实施例2 :survivin基因沉默对肿瘤细胞增殖影响 [0044] Example 2: survivin gene silencing on proliferation of tumor cells

[0045]将宫颈癌细胞Hela、肝癌IfepG2和人肺癌细胞A549按5X103个/孔分别接种到96孔细胞培养板中,三种细胞分别培养,分别进行试验。 [0045] The cervical cancer cell Hela, and human hepatocellular carcinoma A549 cells IfepG2 Press 5X103 cells / well were seeded into 96-well cell culture plate, three types of cells were cultured, were tested. 在37 °C、5 % 0)2培养箱细胞培养24 小时,在无双抗含10%FBS的DMEM培养基中,按照RNAiMAX™的说明书转染,实施例1步骤2中合成的siRNA按10、20、40nM/孔加入,37°C孵育48小时后,每孔加入20yLMTT(5mg/ mL),37°C继续孵育4h,吸除培养基,加入200yLDMSO溶解甲瓒结晶,于490nm处测定吸光度值。 At 37 ° C, 5% 0) 2 incubator cells were cultured for 24 hours in DMEM medium containing 10% FBS anti unparalleled in accordance with instructions RNAiMAX ™ transfection, 2 synthesized in Example 1, step siRNA according to embodiment 10, after 20,40nM / well was added, 37 ° C for 48 hours, and added to each well 20yLMTT (5mg / mL), 37 ° C incubation continued for 4h, the medium removed by suction, was added to dissolve formazan crystals 200yLDMSO, absorbance measured at 490nm in .

[0046]MTT法检测siRNA对肿瘤细胞增殖的抑制结果,siRNA在20nM至50nM对肿瘤肺癌A549、肝癌IfepG2、宫颈癌Hela和乳腺癌MCF-7的抑制作用逐渐增强,抑制率结果见表1。 [0046] MTT assay siRNA on tumor cell proliferation inhibition results, siRNA A549, liver IfepG2, inhibition of Hela and MCF-7 breast tumor gradually increased in lung 20nM to 50nM, inhibition rates (Table 1). 用临床上常用的抗肿瘤的顺铂作为siRNA的药效对照药物,顺铂在10yM至40yM对肿瘤肺癌A549、肝癌IfepG2、宫颈癌Hela和乳腺癌MCF-7的抑制作用逐渐增强,作用48小时后, MTT法检测的对肿瘤细胞的抑制率结果,见表2。 By conventional cisplatin antitumor efficacy of siRNA clinically as a control drug, cisplatin 10yM to 40yM A549, liver IfepG2, inhibition of Hela and MCF-7 breast cancer gradually increased lung tumor, for 48 hours after inhibition rate detection result of MTT assay on tumor cells (Table 2).

[0047] 表1siRNA作用48小时对肿瘤细胞生长的抑制率(% ) [0047] TABLE 48 hours 1siRNA inhibition of tumor cell growth rate (%)

Figure CN105063048AD00061

[0050] 表2顺铂作用48小时对肿瘤细胞生长的抑制率(% )[0051] [0050] Table 2 cisplatin 48 hours inhibition of tumor cell growth rate (%) [0051]

Figure CN105063048AD00062

[0048] [0048]

[0049] [0049]

[0052] 由于细胞株A549、细胞株IfepG2、细胞株Hela和细胞株MCF-7分别为肺癌细胞株、 肝癌细胞株、宫颈癌细胞株和乳腺癌细胞株,所以本发明所述的siRNA可以用于肝癌、宫颈癌和乳腺癌的治疗。 [0052] Since the cell lines A549, cell lines IfepG2, carcinoma Hela cell line MCF-7 and lung cancer cell lines, respectively, hepatocellular carcinoma cell line, cervical cancer cell lines and breast cancer cell lines, the siRNA of the present invention can be used in the treatment of liver cancer, cervical cancer and breast cancer.

[0053] 实施例3:WesternBlot检测siRNA对survivin表达的抑制情况 [0053] Example 3: Detection WesternBlot siRNA inhibition of survivin expression in

[0054] 将人前列腺癌细胞PC-3、肝癌细胞IfepG2、乳腺癌细胞MCF-7细胞和宫颈癌细胞Hela分别按1. 5X105个/孔接种到6孔细胞培养板中,四种细胞分别培养,分别进行试验,在37°C、5% 0)2培养箱细胞培养24小时,在无双抗含10%FBS的DMEM培养基中,按照RNAiMAX™的说明书转染,siRNA按20nM/孔加入,37°C孵育48小时后,吸除培养基,用PBS洗两遍,加入100iiL细胞裂解液,在冰浴上裂解lOmin,用细胞刮将细胞刮下,转移至1. 5ml离心管中,lOOOOrpm离心5min后,取上清液测定蛋白浓度。 [0054] Human prostate cancer PC-3, hepatoma cells IfepG2, breast cancer cells MCF-7 cells and cervical carcinoma Hela cells were press 1. 5X105 cells / well were seeded into 6-well cell culture plates were four cell culture were tested at 37 ° C, 5% 0) 2 incubator cells were cultured for 24 hours in DMEM medium containing 10% FBS anti unparalleled in accordance with instructions RNAiMAX ™ transfection, siRNA was added at 20nM / hole, after 37 ° C for 48 hours, the medium was removed by suction, washed twice with PBS, added 100iiL cell lysate, lOmin lysed in an ice bath, the cells were scraped using a cell scraper and transferred to a 1. 5ml centrifuge tube, lOOOOrpm after centrifugation 5min, the supernatant protein concentration was determined. 每组取30yg蛋白进行SDS-PAGE, 电泳完成后转至PVDF膜上,封闭后进行survivin-抗及羊抗兔二抗处理,然后进行化学发光反应,显影。 Protein from each group 30yg SDS-PAGE, transferred to PVDF membrane after the electrophoresis was completed, for survivin- anti-rabbit secondary antibody and goat anti-blocking treatment, and then the chemiluminescent reaction developed. 结果见图2,加入siRNA后细胞的suivivin蛋白表达量明显低于不经过siRNA处理的阴性对照组。 The results shown in Figure 2, protein expression after addition of siRNA suivivin cells without significantly lower than the negative control siRNA-treated group.

[0055]在四种细胞系中,siRNA都能显著的降低survivin蛋白的表达,所以本发明所述的siRNA可以用于前列腺癌、肝癌、乳腺癌和宫颈癌的治疗。 [0055] In the four cell lines, siRNA can significantly reduce the expression of the survivin protein, the siRNA of the present invention can be used to treat prostate cancer, liver cancer, breast cancer and cervical cancer.

Claims (7)

1. 一种抑制survivin基因表达的双链siRNA分子,其特征在于,具有如下序列结构: 正义链5 ' -GAGACAGAAUAGAGUGAUA-3 ' SEQ ID N0:1 反义链5 ' -UAUCACUCUAUUCU⑶CUC-3 ' SEQ ID N0:2。 A double stranded siRNA molecule inhibiting the expression of survivin, characterized in that the structure has the following sequence: sense strand 5 '-GAGACAGAAUAGAGUGAUA-3' SEQ ID N0: 1 antisense strand 5 '-UAUCACUCUAUUCU⑶CUC-3' SEQ ID N0 :2.
2. -种抑制survivin基因表达的双链siRNA分子,其特征在于,具有如下序列结构: 正义链:5 ' -GAGACAGAAUAGAGUGAUA tt-3 ' SEQ ID N0:3 反义链:5 ' -UAUCACUCUAUUCU⑶CUC tt-3 ' SEQ ID N0:4。 2. - kind of double-stranded siRNA molecules inhibiting the expression of survivin, characterized in that the structure has the following sequence: sense strand: 5 '-GAGACAGAAUAGAGUGAUA tt-3' SEQ ID N0: 3 antisense strand: 5 '-UAUCACUCUAUUCU⑶CUC tt-3 'SEQ ID N0: 4.
3. -种siRNA重组质粒,其特征在于,所述siRNA重组质粒包含权利要求1或2中所述的双链siRNA分子。 3. - siRNA species recombinant plasmid, wherein said recombinant plasmid comprising siRNA siRNA duplex molecule as claimed in claim 1 or claim 2.
4. 一种Survivin基因沉默试剂盒,其特征在于,所述试剂盒包含权利要求1或2中所述的双链siRNA分子或权利要求2所述的siRNA重组质粒。 Survivin gene silencing A kit, wherein said kit comprises a siRNA as claimed in claim 2, the recombinant plasmid in double stranded siRNA molecule of claim 1 or claim 2 or claim.
5. 权利要求1中或2所述的双链siRNA分子或权利3所述的siRNA重组质粒在制备抗肿瘤药物或抗肿瘤药物组合物中的应用。 Application of siRNA recombinant plasmid of claim 3 or claim double stranded siRNA molecule of claim 1 or 2 in the preparation of the antitumor agent or antitumor agent composition.
6. -种抗肿瘤药物,其特征在于,含有有效量的如权利要求1或2中所述的双链siRNA 分子或权利要求3所述的siRNA重组质粒。 6 - Antitumor, characterized in that, in a double stranded siRNA molecule of claim 1 or claim 2 or comprising an effective amount of a siRNA as claimed in claim in claim 3, the recombinant plasmid.
7. 根据权利6中所述的抗肿瘤药物,其中肿瘤是白血病、胃癌、前列腺癌、膀胱癌、宫颈癌,乳腺癌,肺癌,肝癌中至少一种。 The antitumor agent according to claim 6, wherein the tumor is leukemia, gastric cancer, prostate cancer, bladder cancer, cervical cancer, breast cancer, lung cancer, liver cancer at least one.
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