CN112442500A - siRNA for inhibiting MCM7, composition and application thereof - Google Patents
siRNA for inhibiting MCM7, composition and application thereofDownload PDFInfo
- Publication number
- CN112442500A CN112442500ACN201910818294.1ACN201910818294ACN112442500ACN 112442500 ACN112442500 ACN 112442500ACN 201910818294 ACN201910818294 ACN 201910818294ACN 112442500 ACN112442500 ACN 112442500A
- Authority
- CN
- China
- Prior art keywords
- cancer
- sirna
- composition
- cells
- mcm7
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y306/00—Hydrolases acting on acid anhydrides (3.6)
- C12Y306/04—Hydrolases acting on acid anhydrides (3.6) acting on acid anhydrides; involved in cellular and subcellular movement (3.6.4)
- C12Y306/04012—DNA helicase (3.6.4.12)
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Animal Behavior & Ethology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Virology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Epidemiology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Translated fromChineseDescription
Translated fromChinese技术领域technical field
本发明属于生物医药领域，特别涉及抑制MCM7的siRNA、组合物及其应用。The invention belongs to the field of biomedicine, and particularly relates to siRNA for inhibiting MCM7, compositions and applications thereof.
背景技术Background technique
MCM复合体由MCM2～MCM7亚基组成，在细胞内具有解旋酶活性，在DNA复制前打开DNA双链，参与到DNA复制起始(Bik Tye,Annual Review of Biochemistry,1999)。而且MCM复合体还对细胞增殖、DNA损伤修复和细胞周期起到重要的调控作用。The MCM complex is composed of MCM2-MCM7 subunits, which have helicase activity in cells, open DNA double strands before DNA replication, and participate in the initiation of DNA replication (Bik Tye, Annual Review of Biochemistry, 1999). Moreover, the MCM complex also plays an important role in the regulation of cell proliferation, DNA damage repair and cell cycle.
小干扰RNA(Small interfering RNA，siRNA)是一种长20～25个核苷酸的双链RNA，最早在植物的转录后基因沉默现象中发现，公开报道了人工合成的siRNA可沉默哺乳动物细胞的特定基因表达(Thomas Tuschl et al.,Nature,2001；Thomas Tuschl et al.,Science,2001；Thomas Tuschl et al.,Cell,2002)。由于siRNA可以在基因水平靶向干扰，不需要依赖靶点蛋白的晶体结构。因此科学家们研究了一系列利用siRNA进行RNA干扰(RNAinterference，RNAi)来抑制靶标基因的表达的方法，从而进行基因功能研究与疾病治疗。Small interfering RNA (siRNA) is a double-stranded RNA with a length of 20-25 nucleotides. It was first discovered in the phenomenon of post-transcriptional gene silencing in plants. It has been publicly reported that artificially synthesized siRNA can silence mammalian cells. specific gene expression (Thomas Tuschl et al., Nature, 2001; Thomas Tuschl et al., Science, 2001; Thomas Tuschl et al., Cell, 2002). Since siRNA can target interference at the gene level, there is no need to rely on the crystal structure of the target protein. Therefore, scientists have studied a series of methods to use siRNA to conduct RNA interference (RNA interference, RNAi) to inhibit the expression of target genes, so as to conduct gene function research and disease treatment.
由于靶标基因上的不同位点的序列不同而具有不同的二级结构和不同的热动力学特性，因此，不同位点被siRNA干扰的可能性和程度会有很大差异。另外，相同siRNA在不同种类的细胞中的活性也可能不同。因此，对于任何一个细胞中的任何靶标基因，高活性siRNA的设计、测试和获得是一个创造性发明的过程。Due to the different sequences of different sites on the target gene with different secondary structures and different thermodynamic properties, the possibility and degree of interference by siRNA at different sites will vary greatly. In addition, the activity of the same siRNA in different kinds of cells may also be different. Therefore, for any target gene in any one cell, the design, testing and acquisition of highly active siRNA is a process of creative invention.
目前，尚未见有关用于抑制肝癌、胃癌、前列腺癌等癌细胞的MCM7基因的siRNA的报道。At present, there is no report on siRNA for suppressing the MCM7 gene of cancer cells such as liver cancer, gastric cancer, and prostate cancer.
发明内容SUMMARY OF THE INVENTION
本发明的首要目的在于提供一种抑制MCM7的siRNA。The primary object of the present invention is to provide an siRNA that inhibits MCM7.
本发明的另一目的在于提供上述siRNA的应用。Another object of the present invention is to provide the application of the above-mentioned siRNA.
本发明的再一目的在于提供一种siRNA特异性靶向MCM7基因而预防或治疗肿瘤/癌症的方法。Another object of the present invention is to provide a method for preventing or treating tumor/cancer by specifically targeting MCM7 gene with siRNA.
发明人针对MCM7基因设计和测试了许多RNA干扰片段，但是大部分siRNAs的干扰效率低，无法有效进行后期肿瘤治疗研究。发明人经过探索研究，创造性发明了一些高效的siRNA序列，用于干扰MCM7基因。这对于RNA干扰的应用至关重要，即靶基因不同位点的siRNA作用效果差异很大，可能与siRNA二级(元)结构和热动力学性质、碱基分布等因素有关。The inventors have designed and tested many RNA interference fragments for the MCM7 gene, but most of the siRNAs have low interference efficiency and cannot effectively carry out later tumor treatment research. After exploration and research, the inventor creatively invented some efficient siRNA sequences for interfering with the MCM7 gene. This is very important for the application of RNA interference, that is, the effect of siRNA at different sites of the target gene is very different, which may be related to the secondary (meta) structure of siRNA, thermodynamic properties, base distribution and other factors.
发明人进一步探索发现，通过改变siRNA正义链的一个或多个碱基，使得正义链和反义链构成不完全互补配对，因此改变整个双链RNA热动力学性质，提高反义链进入RNA干扰复合体蛋白的效率，从而提高了siRNA抑制靶标MCM7基因的效率。The inventors further explored and found that by changing one or more bases of the siRNA sense strand, the sense strand and the antisense strand form an incomplete complementary pairing, thus changing the thermodynamic properties of the entire double-stranded RNA and improving the entry of the antisense strand into RNA interference. The efficiency of the complex protein, thereby improving the efficiency of siRNA inhibition of the target MCM7 gene.
为达到上述目的，本发明所采取的技术方案是：In order to achieve the above object, the technical scheme adopted by the present invention is:
发明人以MCM7蛋白为靶点设计了siRNA。该siRNA通过抑制人MCM7基因的表达从而抑制MCM7蛋白的合成，从而阻扰了整个MCM复合体(MCM2-MCM7)的形成，从而抑制DNA复制和细胞增殖来达到肿瘤/癌症预防或治疗的目的。The inventors designed siRNA targeting MCM7 protein. The siRNA inhibits the synthesis of MCM7 protein by inhibiting the expression of human MCM7 gene, thereby blocking the formation of the entire MCM complex (MCM2-MCM7), thereby inhibiting DNA replication and cell proliferation to achieve the purpose of tumor/cancer prevention or treatment.
通过siRNA对MCM复合体任何一个亚基的表达抑制，可以抑制整个复合体的形成，进而抑制细胞增殖产生抗肿瘤作用。By inhibiting the expression of any subunit of the MCM complex by siRNA, the formation of the entire complex can be inhibited, thereby inhibiting cell proliferation and producing anti-tumor effects.
本发明一个方面，提出了siRNA，所述siRNA可抑制MCM7基因表达，由正义链和反义链组成，其中，所述siRNA选自：In one aspect of the present invention, an siRNA is proposed, which can inhibit the expression of MCM7 gene, and consists of a sense strand and an antisense strand, wherein the siRNA is selected from:
siRNA-1：正义链：5'-GGACUUAAUUUGUGAGAAU-3'；siRNA-1: sense strand: 5'-GGACUUAAUUUGUGAGAAU-3';
反义链：5'-AUUCUCACAAAUUGAGUCC-3'；Antisense strand: 5'-AUUCUCACAAAUUGAGUCC-3';
或or
siRNA-2：正义链：5'-GGAAGUGGUAAAUAAAGAU-3'；siRNA-2: sense strand: 5'-GGAAGUGGUAAAUAAAGAU-3';
反义链：5'-AUCUUUAUUUACCACUUCC-3'；Antisense strand: 5'-AUCUUUAUUUUACCACUUCC-3';
或与所述siRNA-1或siRNA-2的正义链或反义链序列具有80％以上，或更好地，90％以上同源性，或其碱基可以修饰成为核酸的衍生物，且具有相同功能。Or it has more than 80%, or better, more than 90% homology with the sense strand or antisense strand sequence of said siRNA-1 or siRNA-2, or its base can be modified into a derivative of nucleic acid, and has Same function.
进一步的，所述siRNA正义链和反义链的3'端需添加两个呈单链悬挂结构的脱氧核糖核苷酸dT或dN。Further, two deoxyribonucleotides dT or dN in a single-stranded pendant structure need to be added to the 3' ends of the sense strand and antisense strand of the siRNA.
进一步的，所述siRNA通过抑制MCM7基因表达而预防或治疗肿瘤/癌症。Further, the siRNA prevents or treats tumor/cancer by inhibiting MCM7 gene expression.
进一步的，所述肿瘤/癌症选自肝癌、胃癌、前列腺癌、乳腺癌、肺癌、胰腺癌、宫颈癌、子宫内膜癌、大肠癌、肺癌、鼻咽癌、卵巢癌、皮肤癌、食管癌或脑瘤。Further, the tumor/cancer is selected from liver cancer, gastric cancer, prostate cancer, breast cancer, lung cancer, pancreatic cancer, cervical cancer, endometrial cancer, colorectal cancer, lung cancer, nasopharyngeal cancer, ovarian cancer, skin cancer, esophageal cancer or brain tumor.
进一步的，所述肿瘤可因MCM7基因表达被抑制而减慢或停止生长、缩小或消失。Further, the tumor may slow down or stop growing, shrink or disappear due to the inhibition of MCM7 gene expression.
进一步的，所述MCM7基因选自人MCM7基因。Further, the MCM7 gene is selected from human MCM7 gene.
进一步的，所述的siRNA序列，其序列可以在部分位点局部修改，只要不影响其对靶标的结合及抑制。Further, the sequence of the siRNA can be locally modified at some sites, as long as it does not affect the binding and inhibition of the target.
进一步的，所述siRNA序列及其局部修改序列，其碱基可以修饰成为核酸的衍生物，只要不影响其对靶标的结合及抑制。Further, the bases of the siRNA sequence and its locally modified sequence can be modified into nucleic acid derivatives, as long as the binding and inhibition of the target are not affected.
本发明的又一个方面，提出了siRNA在制备预防或治疗肿瘤/癌症药物或组合物中的应用。In yet another aspect of the present invention, the application of siRNA in the preparation of medicaments or compositions for preventing or treating tumors/cancer is proposed.
进一步的，所述肿瘤/癌症选自肝癌、胃癌、前列腺癌、乳腺癌、肺癌、胰腺癌、宫颈癌、子宫内膜癌、大肠癌、肺癌、鼻咽癌、卵巢癌、皮肤癌、食管癌或脑瘤。Further, the tumor/cancer is selected from liver cancer, gastric cancer, prostate cancer, breast cancer, lung cancer, pancreatic cancer, cervical cancer, endometrial cancer, colorectal cancer, lung cancer, nasopharyngeal cancer, ovarian cancer, skin cancer, esophageal cancer or brain tumor.
进一步的，所述肿瘤/癌症可因MCM7基因表达被抑制而减慢或停止生长、缩小或消失。Further, the tumor/cancer may slow down or stop growing, shrink or disappear due to the inhibition of MCM7 gene expression.
进一步的，siRNA的浓度为5～150nM，优选10～100nM，更优选15～60nM，最优选20～40nM。Further, the concentration of siRNA is 5-150 nM, preferably 10-100 nM, more preferably 15-60 nM, and most preferably 20-40 nM.
还可以具有下列附加技术特征：It can also have the following additional technical features:
所述siRNA可抑制MCM7基因表达，由正义链和反义链组成，The siRNA can inhibit MCM7 gene expression, and is composed of a sense strand and an antisense strand,
其中，所述siRNA选自：Wherein, the siRNA is selected from:
siRNA-1：正义链：5'-GGACUUAAUUUGUGAGAAU-3'；siRNA-1: sense strand: 5'-GGACUUAAUUUGUGAGAAU-3';
反义链：5'-AUUCUCACAAAUUGAGUCC-3'；Antisense strand: 5'-AUUCUCACAAAUUGAGUCC-3';
或or
siRNA-2：正义链：5'-GGAAGUGGUAAAUAAAGAU-3'；siRNA-2: sense strand: 5'-GGAAGUGGUAAAUAAAGAU-3';
反义链：5'-AUCUUUAUUUACCACUUCC-3'；Antisense strand: 5'-AUCUUUAUUUUACCACUUCC-3';
或与所述siRNA-1或siRNA-2的正义链或反义链序列具有80％以上，或更好地，90％以上同源性，或其碱基可以修饰成为核酸的衍生物，且具有相同功能。Or it has more than 80%, or better, more than 90% homology with the sense strand or antisense strand sequence of said siRNA-1 or siRNA-2, or its base can be modified into a derivative of nucleic acid, and has Same function.
进一步的，所述siRNA正义链和反义链的3'端需添加两个呈单链悬挂结构的脱氧核糖核苷酸dT或dN。Further, two deoxyribonucleotides dT or dN in a single-stranded pendant structure need to be added to the 3' ends of the sense strand and antisense strand of the siRNA.
进一步的，所述MCM7基因选自人的MCM7基因。Further, the MCM7 gene is selected from human MCM7 gene.
进一步的，siRNA可以在部分位点局部修改，只要不影响其对靶标的结合及抑制。Further, siRNA can be locally modified at some sites, as long as it does not affect the binding and inhibition of its target.
进一步的，siRNA序列及其局部修改序列，其碱基可以修饰成为核酸的衍生物，只要不影响其对靶标的结合及抑制。Further, the bases of the siRNA sequence and its locally modified sequence can be modified into nucleic acid derivatives, as long as the binding and inhibition of the target are not affected.
本发明的又一个方面，提出了一种预防或治疗肿瘤/癌症的药物或组合物，所述药物或组合物包括：In yet another aspect of the present invention, a medicament or composition for preventing or treating tumor/cancer is proposed, and the medicament or composition comprises:
上述任一所述的siRNA；any of the above-mentioned siRNA;
或可表达上述任一所述的siRNA的表达体系。Or an expression system capable of expressing any of the above-mentioned siRNAs.
进一步的，所述肿瘤/癌症选自肝癌、胃癌、前列腺癌、乳腺癌、肺癌、胰腺癌、宫颈癌、子宫内膜癌、大肠癌、肺癌、鼻咽癌、卵巢癌、皮肤癌、食管癌或脑瘤。Further, the tumor/cancer is selected from liver cancer, gastric cancer, prostate cancer, breast cancer, lung cancer, pancreatic cancer, cervical cancer, endometrial cancer, colorectal cancer, lung cancer, nasopharyngeal cancer, ovarian cancer, skin cancer, esophageal cancer or brain tumor.
进一步的siRNA的浓度为5～150nM，优选10～100nM，更优选15～60nM，最优选20～40nM。The concentration of further siRNA is 5-150 nM, preferably 10-100 nM, more preferably 15-60 nM, most preferably 20-40 nM.
进一步的，所述肿瘤可因MCM7基因表达被抑制而减慢或停止生长、缩小或消失。Further, the tumor may slow down or stop growing, shrink or disappear due to the inhibition of MCM7 gene expression.
进一步的，上述所述药物或组合物，还包括：Further, the above-mentioned medicine or composition also includes:
药学上可接受的载体；a pharmaceutically acceptable carrier;
预防或治疗肿瘤的其它活性成分。Other active ingredients for the prevention or treatment of tumors.
进一步的，药学上可接受的载体和/或辅料包括但不限于缓冲剂、乳化剂、悬浮剂、稳定剂、防腐剂、生理食盐、赋形剂、填充剂、凝结剂与调和剂、界面活性剂、扩散剂、消泡剂。Further, pharmaceutically acceptable carriers and/or adjuvants include, but are not limited to, buffers, emulsifiers, suspending agents, stabilizers, preservatives, physiological salts, excipients, fillers, coagulants and blending agents, interfacial active agents agent, diffusing agent, defoamer.
进一步的，所述预防或治疗肿瘤的其他活性成分包括：化疗剂、放疗剂或抗体药物。Further, the other active ingredients for preventing or treating tumors include chemotherapeutic agents, radiotherapy agents or antibody drugs.
进一步的，所述药物或组合物的形式适用于：直接裸RNA注射法、脂质体包裹RNA直接注射法、蛋白或多肽包裹RNA直接注射法、金包被RNA基因枪轰击法、细菌携带质粒表达RNA法或病毒表达RNA法。Further, the form of the medicine or composition is suitable for: direct naked RNA injection method, liposome-encapsulated RNA direct injection method, protein or polypeptide encapsulated RNA direct injection method, gold-coated RNA gene gun bombardment method, bacteria-carried plasmids. Expression RNA method or virus expression RNA method.
进一步的，所述siRNA药物或组合物可以选自固体、液体、凝胶、半流质、气雾任一形式。Further, the siRNA drug or composition can be selected from any form of solid, liquid, gel, semi-liquid and aerosol.
进一步的，所述MCM7基因选自人的MCM7基因。Further, the MCM7 gene is selected from human MCM7 gene.
利用siRNA可以有效抑制MCM7基因表达和蛋白的合成，起到治疗肿瘤/癌症的目的。The use of siRNA can effectively inhibit MCM7 gene expression and protein synthesis for the purpose of treating tumors/cancers.
本发明的有益效果是：The beneficial effects of the present invention are:
本发明通过改变siRNA-1序列正义链的5’端顺数的第6个碱基，使得正义链和反义链构成不完全互补配对，因此改变整个双链RNA热动力学性质，提高反义链进入RNA干扰复合体蛋白的效率，从而提高siRNA-1抑制靶标MCM7基因的效率。In the present invention, by changing the 6th base of the cis-numbered 5' end of the sense strand of the siRNA-1 sequence, the sense strand and the antisense strand form an incomplete complementary pairing, thus changing the thermodynamic properties of the entire double-stranded RNA and improving the antisense The efficiency of strand entry into RNA interference complex proteins, thereby increasing the efficiency of siRNA-1 inhibition of the target MCM7 gene.
本发明通过提供一种靶向MCM7基因的siRNA，与常规基因敲除技术相比，本发明操作简便、试验周期短；在mRNA和蛋白水平上，所述siRNA对MCM7的抑制效果高达90％以上，抑制效率极高、特异性好；同时能够有效抑制癌症细胞的DNA复制、增殖和克隆形成能力，对于开发新的抗癌基因药物和提高癌症的治疗效果有重要的意义，具有显著的临床应用前景和经济价值。The present invention provides a siRNA targeting MCM7 gene. Compared with conventional gene knockout technology, the present invention has simple operation and short test period; at the level of mRNA and protein, the inhibitory effect of the siRNA on MCM7 is as high as more than 90% , the inhibition efficiency is extremely high and the specificity is good; at the same time, it can effectively inhibit the DNA replication, proliferation and clone formation of cancer cells, which is of great significance for the development of new anti-cancer gene drugs and improving the therapeutic effect of cancer, and has significant clinical application prospects and economic value.
附图说明Description of drawings
图1为MCM7特异性的siRNA-1在HepG2和Hep3B肝癌细胞MCM7mRNA水平的沉默效果。Figure 1 shows the silencing effect of MCM7-specific siRNA-1 on the level of MCM7 mRNA in HepG2 and Hep3B liver cancer cells.
图2的A～C图分别为MCM7特异性的siRNA-1在HepG2和Hep3B肝癌细胞和siRNA-2在HepG2肝癌细胞中MCM7蛋白水平的沉默效果，其中β-actin为内部参照蛋白。Panels A to C of Figure 2 show the silencing effect of MCM7-specific siRNA-1 in HepG2 and Hep3B hepatoma cells and siRNA-2 in HepG2 hepatoma cells, respectively, where β-actin is the internal reference protein.
图3为siRNA-1抑制HepG2肝癌细胞DNA复制的EdU显阳性的细胞荧光显微镜图，其中图3A、C和E分别是阴性对照组细胞NC转染HepG2肝癌细胞后的EdU显阳性的细胞荧光显微镜图、Hochst染细胞核DNA图、EdU显阳性的细胞荧光显微镜图和Hochst染细胞核DNA图的重叠图；其中图3B、D和F分别是siRNA-1转染HepG2肝癌细胞后的EdU显阳性的细胞荧光显微镜图、Hochst染细胞核DNA图、EdU显阳性的细胞荧光显微镜图和Hochst染细胞核DNA图的重叠图。Figure 3 is a fluorescent microscope image of EdU positive cells showing DNA replication of HepG2 liver cancer cells inhibited by siRNA-1, in which Figure 3A, C and E are EdU positive cell fluorescence microscope images of negative control cells after NC transfection of HepG2 liver cancer cells, respectively Figure, Hochst-stained nuclear DNA image, EdU-positive cytofluorescence microscopy image and Hochst-stained nuclear DNA image overlay; Figure 3B, D and F are EdU-positive cells after siRNA-1 transfection of HepG2 hepatoma cells, respectively Fluorescence microscopy images, Hochst-stained nuclear DNA images, fluorescence microscopy images of EdU-positive cells, and Hochst-stained nuclear DNA images.
图4为EdU掺入显阳性的HepG2细胞比例统计图。Figure 4 is a statistical graph of the proportion of HepG2 cells with positive EdU incorporation.
图5A～E分别为siRNA-1抑制HepG2肝癌细胞、Hep3B肝癌细胞、SGC-7907胃癌细胞、PC3前列腺癌细胞和MCF7乳腺癌细胞增殖的生长曲线。Figures 5A-E show the growth curves of siRNA-1 inhibiting the proliferation of HepG2 liver cancer cells, Hep3B liver cancer cells, SGC-7907 gastric cancer cells, PC3 prostate cancer cells and MCF7 breast cancer cells, respectively.
图6为siRNA抑制各种癌细胞的克隆生成的结果图，图A～E分别为用siRNA-1或阴性对照NC转染HepG2肝癌细胞、Hep3B肝癌细胞、SGC-7907胃癌细胞、PC3前列腺癌细胞和MCF7乳腺癌细胞后，癌细胞克隆生成的数目对照图。Figure 6 shows the results of siRNA inhibiting the clone formation of various cancer cells. Figures A to E are HepG2 liver cancer cells, Hep3B liver cancer cells, SGC-7907 gastric cancer cells, and PC3 prostate cancer cells transfected with siRNA-1 or negative control NC, respectively. After and MCF7 breast cancer cells, the control chart of the number of cancer cell clones generated.
图7为siRNA抑制各种癌细胞的克隆生成能力，图A～E分别为用siRNA-1或阴性对照NC转染HepG2肝癌细胞、Hep3B肝癌细胞、SGC-7907胃癌细胞、PC3前列腺癌细胞和MCF7乳腺癌细胞后，癌细胞的克隆总面积占孔总面积的比例。Figure 7 shows the ability of siRNA to inhibit the clonogenicity of various cancer cells. Figures A to E are HepG2 liver cancer cells, Hep3B liver cancer cells, SGC-7907 gastric cancer cells, PC3 prostate cancer cells and MCF7 transfected with siRNA-1 or negative control NC, respectively. After breast cancer cells, the proportion of total clonal area of cancer cells to total pore area.
具体实施方式Detailed ways
下面结合实施例对本发明中的技术方案进行清楚、完整的说明，但并不局限于此。The technical solutions in the present invention will be clearly and completely described below with reference to the embodiments, but are not limited thereto.
实施例1 siRNA设计Example 1 siRNA design
根据siRNA靶序列基本原则，设计、合成人MCM7基因转录本(NM_001278595.1)表达的siRNA序列(1条21个核苷酸)，即siRNA的正义链和反义链，其碱基序列为：According to the basic principles of siRNA target sequence, the siRNA sequence (one 21 nucleotides) expressed by the human MCM7 gene transcript (NM_001278595.1) was designed and synthesized, that is, the sense strand and antisense strand of the siRNA. The base sequences are:
siRNA-1正义链：5'-GGACUUAAUUUGUGAGAAUdTdT-3'；(SEQ ID NO.1)siRNA-1 sense strand: 5'-GGACUUAAUUUGUGAGAAUdTdT-3'; (SEQ ID NO. 1)
siRNA-1反义链：5'-AUUCUCACAAAUUGAGUCCdTdT-3'(SEQ ID NO.2)。siRNA-1 antisense strand: 5'-AUUCUCACAAAUUGAGUCCdTdT-3' (SEQ ID NO. 2).
或or
siRNA-2正义链：5'-GGAAGUGGUAAAUAAAGAUdTdT-3'(SEQ ID NO.3)；siRNA-2 sense strand: 5'-GGAAGUGGUAAAUAAAGAUdTdT-3' (SEQ ID NO. 3);
siRNA-2反义链：5'-AUCUUUAUUUACCACUUCCdTdT-3'(SEQ ID NO.4)。siRNA-2 antisense strand: 5'-AUCUUUAUUUUACCACUUCCdTdT-3' (SEQ ID NO. 4).
阴性对照RNA(NC)的碱基序列为：The base sequence of the negative control RNA (NC) is:
正义链：5'-CUCUUAGCCAAUAUUCGCUdTdT-3'(SEQ ID NO.5)；Sense strand: 5'-CUCUUAGCCAAUAUUCGCUdTdT-3' (SEQ ID NO. 5);
反义链：5′-AGCGAAUAUUGGCUAAGAGdTdT-3'(SEQ ID NO.6)。Antisense strand: 5'-AGCGAAUAUUGGCUAAGAGdTdT-3' (SEQ ID NO. 6).
本发明siRNA序列和对照RNA正义链和反义链的3’端，添加了两个呈单链悬挂结构的脱氧核糖核苷酸(dT或dN)，以增强siRNA在体内和体外的稳定性，防止被核酸酶降解。Two deoxyribonucleotides (dT or dN) in a single-stranded suspension structure are added to the 3' ends of the sense and antisense strands of the siRNA sequence of the present invention and the control RNA to enhance the stability of the siRNA in vivo and in vitro, Protected against degradation by nucleases.
本发明siRNA序列可以在部分位点局部修改，只要不影响其对靶标的结合及抑制。The siRNA sequence of the present invention can be locally modified at some sites, as long as it does not affect the binding and inhibition of the target.
本发明siRNA序列及其局部修改序列，其碱基可以修饰成为核酸的衍生物，只要不影响其对靶标的结合及抑制。The bases of the siRNA sequences of the present invention and their locally modified sequences can be modified into nucleic acid derivatives as long as they do not affect the binding and inhibition of the target.
实施例2 siRNA在细胞中的转染Example 2 Transfection of siRNA in cells
采用脂质体Lipofectamine RNAiMax作为转染试剂，步骤按照Thermo FisherScientific公司的操作规程。采用的细胞系为HepG2和Hep3B肝癌细胞系、SGC-7907胃癌细胞系、PC3前列腺癌细胞系和MCF7乳腺癌细胞系。Lipofectamine RNAiMax was used as a transfection reagent, and the steps were in accordance with the operating procedures of Thermo Fisher Scientific. The cell lines used were HepG2 and Hep3B liver cancer cell lines, SGC-7907 gastric cancer cell line, PC3 prostate cancer cell line and MCF7 breast cancer cell line.
本发明未注明具体条件的实验方法，通常按照常规条件如Sambrook等人《分子克隆：实验室指南》(New York：Cold Spring Harbor Laboratory Press，1989)中所述的条件，或按照制造厂商所建议的条件。The present invention does not specify the experimental method of specific conditions, usually according to the conditions described in "Molecular Cloning: Laboratory Guide" (New York: Cold Spring Harbor Laboratory Press, 1989) of people such as Sambrook according to conventional conditions, or according to the manufacturer's instructions recommended conditions.
转染步骤如下：将上述不同的细胞系分别接种于12孔板中，37℃、5％CO2培养过夜，使细胞生长密度达到40～50％。参照Lipofectamine RNAiMax(Thermo FisherScientific)操作规程，将本发明制备的siRNA和阴性对照RNA(NC)分别转染到不同的细胞系。转染后收集细胞，采用qRT-PCR和蛋白印迹(western blotting)进一步检测siRNA的干扰效果。The transfection steps are as follows: the above-mentioned different cell lines are respectively inoculated into 12-well plates, and cultured at 37° C. and 5% CO2 overnight to make the cell growth density reach 40-50%. Referring to the operating procedures of Lipofectamine RNAiMax (Thermo Fisher Scientific), the siRNA prepared in the present invention and the negative control RNA (NC) were respectively transfected into different cell lines. The cells were collected after transfection, and the interference effect of siRNA was further detected by qRT-PCR and western blotting.
实施例3 siRNA抑制MCM7mRNA表达检测Example 3 Detection of siRNA inhibiting MCM7 mRNA expression
方法：转染后收集细胞，提取总RNA，逆转录后进行实时荧光定量PCR，检测siRNA处理癌细胞后MCM7mRNA的表达量。Methods: Cells were collected after transfection, total RNA was extracted, reverse transcribed, and real-time quantitative PCR was performed to detect the expression of MCM7 mRNA after siRNA treatment of cancer cells.
将本发明制备的siRNA和阴性对照RNA(NC)分别转染到肝癌细胞系HepG2，使用同样的方法也转染到肝癌细胞系Hep3B，转染24小时后，收集细胞，取适量细胞重新接种于6孔板中，72小时后收集细胞提取总RNA。逆转录，并进行实时荧光定量PCR检测MCM7mRNA。The siRNA and negative control RNA (NC) prepared by the present invention were respectively transfected into the liver cancer cell line HepG2, and also transfected into the liver cancer cell line Hep3B using the same method. In a 6-well plate, cells were collected after 72 hours to extract total RNA. Reverse transcription was performed, and real-time quantitative PCR was performed to detect MCM7mRNA.
1.总RNA提取1. Total RNA extraction
(1)分别收集肿瘤细胞至离心管，离心800rpm，3min，弃上清，加入PBS洗涤一次并转移至EP管中；(1) Collect tumor cells into centrifuge tubes, centrifuge at 800 rpm for 3 min, discard the supernatant, add PBS to wash once and transfer to EP tubes;
(2)再次离心800rpm，3min，弃上清，加入0.5ml TRIzol，反复吹打使肿瘤细胞溶解，室温静置5～10min；(2) Centrifuge again at 800 rpm for 3 min, discard the supernatant, add 0.5 ml of TRIzol, lyse the tumor cells by repeated pipetting, and let stand for 5-10 min at room temperature;
(3)加入0.2ml氯仿/ml TRIzol，剧烈振荡混匀15sec，室温静置10min；(3) Add 0.2ml chloroform/ml TRIzol, shake vigorously for 15sec, and let stand for 10min at room temperature;
(4)12000rpm，4℃，离心15min；(4) 12000rpm, 4°C, centrifugation for 15min;
(5)离心后液体分为三层，从下至上依次为酚/氯仿层、中间蛋白层、上层无色水相，RNA存于上层水相中；(5) liquid is divided into three layers after centrifugation, from bottom to top are phenol/chloroform layer, intermediate protein layer, upper layer colorless water phase, RNA is stored in upper layer water phase;
(6)吸取上层水相至新的EP管中，注意避免将中间蛋白吸出；(6) Suck the upper aqueous phase into a new EP tube, taking care to avoid sucking out the intermediate protein;
(7)加入预冷的异丙醇0.5ml/ml TRIzol，颠倒混匀，室温静置10min；(7) Add pre-cooled isopropanol 0.5ml/ml TRIzol, invert and mix, and let stand for 10min at room temperature;
(8)12000rpm，4℃，离心10min；(8) 12000rpm, 4°C, centrifugation for 10min;
(9)弃上清，用75％乙醇(750μl无水乙醇，250μl DEPC水现配)洗涤RNA沉淀物，12,000rpm，4℃，离心5min；(9) Discard the supernatant, wash the RNA precipitate with 75% ethanol (750 μl absolute ethanol, 250 μl DEPC water), centrifuge at 12,000 rpm, 4 °C for 5 min;
(10)弃上清，超净台中鼓风干燥3分钟左右，RNA呈半透明状；(10) Discard the supernatant and blow dry for about 3 minutes in ultra-clean Taizhong, and the RNA is translucent;
(11)加入15～20μl 1‰DEPC水溶解RNA沉淀，紫外分光光度计测定浓度及OD值，-70℃保存或直接用于反转录反应。(11) Add 15-20 μl of 1‰ DEPC water to dissolve the RNA precipitate, measure the concentration and OD value with an ultraviolet spectrophotometer, and store at -70°C or use it directly for reverse transcription reaction.
2.反转录成cDNA2. Reverse transcription into cDNA
高温预变形5min后冰上急冻，反转录反应体系：After pre-deformation at high temperature for 5 minutes, it was snap-frozen on ice. The reverse transcription reaction system:
反应条件：37℃15min，50℃5min，95℃5min，4℃保持。合成的cDNA可立即用于下游实验或-20℃冰箱保存。Reaction conditions: 37°C for 15 min, 50°C for 5 min, 95°C for 5 min, hold at 4°C. The synthesized cDNA can be used immediately for downstream experiments or stored in a -20°C freezer.
3.荧光定量实时PCR3. Real-time PCR
将反转录的cDNA样品以适当比例稀释后，使用THUNDERBRID SYBR qPCR Mix配置如下PCR反应体系：After diluting the reverse transcribed cDNA samples in an appropriate ratio, use THUNDERBRID SYBR qPCR Mix to configure the following PCR reaction system:
其中：in:
上游引物序列为5’-GTGAAGGATCCTGCGACACA-3’(SEQ ID NO.7)；The upstream primer sequence is 5'-GTGAAGGATCCTGCGACACA-3' (SEQ ID NO.7);
下游引物序列为5’-ACACGCGTTCTTTTGTTCCG-3’(SEQ ID NO.8)；The downstream primer sequence is 5'-ACACGCGTTCTTTTGTTCCG-3' (SEQ ID NO.8);
内参上游引物序列为5’-ACACGCGTTCTTTTGTTCCG-3’(SEQ ID NO.9)；The upstream primer sequence of the internal reference is 5'-ACACGCGTTCTTTTGTTCCG-3' (SEQ ID NO.9);
内参下游引物序列为5’-GGACTCCATGCCCAGGAAGGAA-3’(SEQ ID NO.10)。The sequence of the primer downstream of the internal reference is 5'-GGACTCCATGCCCAGGAAGGAA-3' (SEQ ID NO. 10).
结果：图1A和B表明，相对于对照组NC，用siRNA-1转染癌细胞后，分别有效抑制HepG2、Hep3B肝癌细胞中的MCM7mRNA表达量，沉默效果达90％以上。Results: Figures 1A and B show that, compared with the control group NC, after transfection of cancer cells with siRNA-1, the expression of MCM7 mRNA in HepG2 and Hep3B liver cancer cells was effectively inhibited, and the silencing effect reached more than 90%.
实施例4 siRNA抑制MCM7蛋白表达检测Example 4 Detection of siRNA inhibition of MCM7 protein expression
方法：将本发明制备的siRNA和阴性对照RNA(NC)分别转染到肝癌细胞系HepG2，使用同样的方法也转染到肝癌细胞系Hep3B，转染24小时后，收集细胞，取适量细胞重新接种于12孔板中，72小时后收集细胞做蛋白印迹实验。Methods: The siRNA and negative control RNA (NC) prepared in the present invention were respectively transfected into the liver cancer cell line HepG2, and also transfected into the liver cancer cell line Hep3B by the same method. The cells were seeded in 12-well plates, and the cells were collected for Western blotting after 72 hours.
1、吸去培养基，加入适量2×laemmli缓冲液，轻轻摇晃12孔板裂解细胞，收集到PE管中，与PE管架的孔摩擦震碎DNA,95℃煮2min；1. Aspirate the medium, add an appropriate amount of 2×laemmli buffer, gently shake the 12-well plate to lyse the cells, collect them into a PE tube, rub against the holes of the PE tube rack to smash the DNA, and cook at 95°C for 2 minutes;
2、将煮沸变性的样品进行聚丙烯酰胺凝胶电泳、Western印迹，之后用5％脱脂牛奶对PVDF膜室温封闭0.5小时；2. The boiled denatured samples were subjected to polyacrylamide gel electrophoresis and Western blotting, and then the PVDF membrane was blocked with 5% skim milk for 0.5 hours at room temperature;
3、选用合适的一抗(鼠抗人MCM7单克隆抗体，Santa Cruz Biotechnology)，以适当的稀释比例，与PVDF膜4℃孵育过夜；3. Select an appropriate primary antibody (mouse anti-human MCM7 monoclonal antibody, Santa Cruz Biotechnology), incubate with PVDF membrane at 4°C overnight at an appropriate dilution ratio;
4、次日，TBST洗3遍，每遍10min；4. The next day, wash 3 times with TBST, 10min each time;
5、选用与一抗宿主种属相对应的HRP标记的抗小鼠IgG二抗，以适当的稀释比例，与PVDF膜室温孵育1小时；5. Select the HRP-labeled anti-mouse IgG secondary antibody corresponding to the host species of the primary antibody, and incubate it with the PVDF membrane at room temperature for 1 hour at an appropriate dilution ratio;
6、TBST洗3遍，每遍10min；6.
7、ECL液显影，检测MCM7蛋白在肿瘤细胞中的表达情况。7. ECL solution was developed to detect the expression of MCM7 protein in tumor cells.
结果：图2A、B和C显示，相对于对照组NC，用siRNA-1转染癌细胞后，可有效抑制HepG2以及Hep3B细胞中MCM7蛋白的表达；用siRNA-2转染癌细胞后，可有效抑制HepG2细胞中MCM7蛋白的表达；siRNA-1和siRNA-2的沉默效果均可达90％以上。Results: Figure 2A, B and C show that, compared with the control group NC, after transfection of cancer cells with siRNA-1, the expression of MCM7 protein in HepG2 and Hep3B cells could be effectively inhibited; It can effectively inhibit the expression of MCM7 protein in HepG2 cells; the silencing effect of siRNA-1 and siRNA-2 can reach more than 90%.
实施例5 siRNA抑制癌细胞DNA复制Example 5 siRNA inhibits DNA replication of cancer cells
方法：将本发明制备的siRNA和阴性对照RNA(NC)分别转染到肝癌细胞系HepG2，使用同样的方法也转染到肝癌细胞系Hep3B，转染24小时后，收集细胞，取适量细胞重新接种于96孔板中。12小时后，将mimosine试剂添加到细胞中孵育24小时，使细胞同步在G1期和S期交界处。Methods: The siRNA and negative control RNA (NC) prepared by the present invention were respectively transfected into the liver cancer cell line HepG2, and also transfected into the liver cancer cell line Hep3B using the same method. seeded in 96-well plates. After 12 hours, mimosine reagent was added to the cells and incubated for 24 hours to synchronize the cells at the junction of G1 and S phases.
使用新鲜培养基清洗细胞三次，每次间隔三分钟，将细胞从mimosine的抑制中释放出来。添加新鲜培养基培养细胞3.5小时，再加入50mmol/L EdU(5-Ethynyl-2'-deoxyuridine，一种胸腺嘧核苷类似物)继续培养0.5小时。固定细胞并染色，在荧光显微镜下观察并统计EdU掺入显阳性的细胞的比例。Cells were released from mimosine inhibition by washing the cells three times with fresh medium at three-minute intervals. Fresh medium was added to culture the cells for 3.5 hours, and then 50 mmol/L EdU (5-Ethynyl-2'-deoxyuridine, a thymidine analog) was added to continue the culture for 0.5 hours. Cells were fixed and stained, and the proportion of cells positive for EdU incorporation was observed under a fluorescence microscope and counted.
结果：图3和图4表明，siRNA-1转染癌细胞后，与阴性对照NC相比，EdU掺入显阳性的细胞的比例明显减少，表明siRNA-1显著抑制了癌细胞的DNA复制。Results: Figures 3 and 4 show that after siRNA-1 was transfected into cancer cells, compared with the negative control NC, the proportion of cells with positive EdU incorporation was significantly reduced, indicating that siRNA-1 significantly inhibited the DNA replication of cancer cells.
所述新鲜培养基采用Gibco RPMI 1640。The fresh medium used Gibco RPMI 1640.
实施例6 siRNA抑制癌细胞增殖Example 6 siRNA inhibits cancer cell proliferation
方法：将本发明制备的siRNA和阴性对照RNA(NC)分别转染不同的癌细胞系，转染24小时后收集细胞。取适量细胞分为五等分，重新接种于12孔板中，连续计数五天，每天选取一个孔的细胞计数。绘制转染后的细胞生长曲线。Methods: The siRNA and negative control RNA (NC) prepared by the present invention were respectively transfected into different cancer cell lines, and the cells were collected 24 hours after transfection. Divide an appropriate amount of cells into five equal parts, re-seek them in a 12-well plate, and count them for five consecutive days. Cells in one well are selected every day for counting. Plot the cell growth curve after transfection.
结果：如图5结果表明，siRNA-1可高效抑制HepG2肝癌细胞、Hep3B肝癌细胞、SGC-7907胃癌细胞、PC3前列腺癌细胞和MCF7乳腺癌细胞的增殖。Results: As shown in Figure 5, siRNA-1 can effectively inhibit the proliferation of HepG2 liver cancer cells, Hep3B liver cancer cells, SGC-7907 gastric cancer cells, PC3 prostate cancer cells and MCF7 breast cancer cells.
同样的，siRNA-2也可高效抑制上述癌细胞的增殖。Similarly, siRNA-2 can also effectively inhibit the proliferation of the above-mentioned cancer cells.
实施例7 siRNA抑制癌细胞克隆生成Example 7 siRNA inhibits cancer cell clone formation
方法：将本发明制备的siRNA和阴性对照RNA(NC)分别转染到上述不同的癌细胞系，转染24小时后收集细胞。将细胞接种到6孔板中，细胞密度为0.4×103个/孔。培养14天后，用甲醇固定，并使用结晶紫染色。Methods: The siRNA and negative control RNA (NC) prepared in the present invention were respectively transfected into the above-mentioned different cancer cell lines, and the cells were collected 24 hours after transfection. Cells were seeded into 6-well plates at a cell density of 0.4×103 cells/well. After 14 days of culture, they were fixed with methanol and stained with crystal violet.
结果：如图6和图7表明，图6A～E中可知，siRNA-1转染癌细胞后，与阴性对照NC相比，癌细胞克隆数目显著减少；图7A～E中可知，siRNA-1转染癌细胞后，与阴性对照NC相比，克隆总面积占孔总面积的比例减少，以上可说明siRNA可高效抑制HepG2肝癌细胞、Hep3B肝癌细胞、SGC-7907胃癌细胞、PC3前列腺癌细胞和MCF7乳腺癌细胞的克隆生成能力。Results: As shown in Figure 6 and Figure 7, it can be seen from Figure 6A to E that after siRNA-1 was transfected into cancer cells, compared with the negative control NC, the number of cancer cell clones was significantly reduced; Figure 7A to E showed that siRNA-1 After transfection of cancer cells, compared with the negative control NC, the proportion of the total clone area to the total pore area decreased. The above shows that siRNA can effectively inhibit HepG2 liver cancer cells, Hep3B liver cancer cells, SGC-7907 gastric cancer cells, PC3 prostate cancer cells and Clonogenic capacity of MCF7 breast cancer cells.
实施例8 MCM7siRNA应用Example 8 Application of MCM7siRNA
本发明MCM7siRNA在制备预防或治疗肿瘤/癌症药物中的应用，所述癌症选自肝癌、胃癌、前列腺癌、乳腺癌、肺癌、胰腺癌、宫颈癌、子宫内膜癌、大肠癌、肺癌、鼻咽癌、卵巢癌、皮肤癌、食管癌或脑瘤。The application of the MCM7siRNA of the present invention in the preparation of a drug for preventing or treating tumor/cancer, the cancer is selected from liver cancer, gastric cancer, prostate cancer, breast cancer, lung cancer, pancreatic cancer, cervical cancer, endometrial cancer, colorectal cancer, lung cancer, nasal cancer Pharyngeal, ovarian, skin, esophageal, or brain tumors.
综上所述，本发明的siRNA有效抑制了MCM7基因表达，从而降低了MCM7蛋白的合成，siRNA的抑制效果高达90％以上，抑制效率极高、特异性好；同时能够有效抑制癌症细胞的DNA复制、增殖和克隆形成能力，对于开发新的抗癌基因药物和提高癌症的治疗效果有重要的意义，具有显著的临床应用前景和经济价值。To sum up, the siRNA of the present invention effectively inhibits the expression of MCM7 gene, thereby reducing the synthesis of MCM7 protein, the inhibition effect of siRNA is as high as more than 90%, the inhibition efficiency is extremely high, and the specificity is good; at the same time, it can effectively inhibit the DNA of cancer cells. The ability of replication, proliferation and clone formation is of great significance for the development of new anti-cancer gene drugs and improving the therapeutic effect of cancer, and has significant clinical application prospects and economic value.
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.
SEQUENCE LISTINGSEQUENCE LISTING
<110> 恩智（广州）医药科技有限公司 恩康药业科技（广州）有限公司<110> Enzhi (Guangzhou) Pharmaceutical Technology Co., Ltd. Enkang Pharmaceutical Technology (Guangzhou) Co., Ltd.
佛山英特医药科技有限公司 广州英特基因科技有限公司Foshan Inte Pharmaceutical Technology Co., Ltd. Guangzhou Inte Gene Technology Co., Ltd.
<120> 抑制MCM7 的siRNA、组合物及其应用<120> MCM7-inhibiting siRNA, composition and application thereof
<130><130>
<160> 10<160> 10
<170> PatentIn version 3.5<170> PatentIn version 3.5
<210> 1<210> 1
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工引物<213> Artificial primers
<400> 1<400> 1
ggacuuaauu ugugagaaut t 21ggacuuaauu ugugagaaut t 21
<210> 2<210> 2
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工引物<213> Artificial primers
<400> 2<400> 2
auucucacaa auugagucct t 21auucucacaa auugagucct t 21
<210> 3<210> 3
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工引物<213> Artificial primers
<400> 3<400> 3
ggaaguggua aauaaagaut t 21ggaaguggua aauaaagaut t 21
<210> 4<210> 4
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工引物<213> Artificial primers
<400> 4<400> 4
aucuuuauuu accacuucct t 21aucuuuauuu accacuucct t 21
<210> 5<210> 5
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工引物<213> Artificial primers
<400> 5<400> 5
cucuuagcca auauucgcut t 21cucuuagcca auauucgcut t 21
<210> 6<210> 6
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工引物<213> Artificial primers
<400> 6<400> 6
agcgaauauu ggcuaagagt t 21agcgaauauu ggcuaagagt t 21
<210> 7<210> 7
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工引物<213> Artificial primers
<400> 7<400> 7
gtgaaggatc ctgcgacaca 20gtgaaggatc ctgcgacaca 20
<210> 8<210> 8
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工引物<213> Artificial primers
<400> 8<400> 8
acacgcgttc ttttgttccg 20acacgcgttc ttttgttccg 20
<210> 9<210> 9
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工引物<213> Artificial primers
<400> 9<400> 9
acacgcgttc ttttgttccg 20acacgcgttc ttttgttccg 20
<210> 10<210> 10
<211> 22<211> 22
<212> DNA<212> DNA
<213> 人工引物<213> Artificial primers
<400> 10<400> 10
ggactccatg cccaggaagg aa 22ggactccatg cccaggaagg aa 22
Claims (10)
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910818294.1ACN112442500A (en) | 2019-08-30 | 2019-08-30 | siRNA for inhibiting MCM7, composition and application thereof |
| PCT/CN2020/112560WO2021037264A1 (en) | 2019-08-30 | 2020-08-31 | Sirna for inhibiting mcm7 gene expression, composition and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910818294.1ACN112442500A (en) | 2019-08-30 | 2019-08-30 | siRNA for inhibiting MCM7, composition and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112442500Atrue CN112442500A (en) | 2021-03-05 |
Family
ID=74684317
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910818294.1APendingCN112442500A (en) | 2019-08-30 | 2019-08-30 | siRNA for inhibiting MCM7, composition and application thereof |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN112442500A (en) |
| WO (1) | WO2021037264A1 (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1867672A (en)* | 2003-08-18 | 2006-11-22 | 财团法人日本健康科学振兴财团 | Improved siRNA molecule and method of inhibiting gene expression with the use of the same |
| CN104561003A (en)* | 2015-01-16 | 2015-04-29 | 上海生博生物医药科技有限公司 | Micromolecule RNA of interference MCM7 gene for inhibiting glioma proliferation as well as preparation method and application thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006006948A2 (en)* | 2002-11-14 | 2006-01-19 | Dharmacon, Inc. | METHODS AND COMPOSITIONS FOR SELECTING siRNA OF IMPROVED FUNCTIONALITY |
| KR101545705B1 (en)* | 2010-09-15 | 2015-08-25 | 중앙대학교 산학협력단 | Marker for diagnosing of clonorchiasis and liver cancer comprising Mcm7 gene |
| CN109288855B (en)* | 2018-09-21 | 2021-08-17 | 中国人民解放军军事科学院军事医学研究院 | Uses of reagents in the preparation of drugs, interference fragments, methods for inhibiting self-renewal of liver cancer stem cells, and drugs for treating liver cancer |
- 2019
- 2019-08-30CNCN201910818294.1Apatent/CN112442500A/enactivePending
- 2020
- 2020-08-31WOPCT/CN2020/112560patent/WO2021037264A1/ennot_activeCeased
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1867672A (en)* | 2003-08-18 | 2006-11-22 | 财团法人日本健康科学振兴财团 | Improved siRNA molecule and method of inhibiting gene expression with the use of the same |
| CN104561003A (en)* | 2015-01-16 | 2015-04-29 | 上海生博生物医药科技有限公司 | Micromolecule RNA of interference MCM7 gene for inhibiting glioma proliferation as well as preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| NCBI: ""Homo sapiens minichromosome maintenance complex component 7 (MCM7), transcript variant 3, mRNA"", 《GENBANK DATABASE》, 5 July 2019 (2019-07-05), pages 1 - 5* |
| 孙雯等: ""RNAi沉默MCM7基因对人肝癌细胞SMMC-7721裸鼠移植瘤影响研究"", 《中华肿瘤防治杂志》, 31 January 2015 (2015-01-31), pages 7 - 12* |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2021037264A1 (en) | 2021-03-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109797151B (en) | Application of Circ-CDH1 inhibitor | |
| CN107661509B (en) | A kind of targeted inhibitor of linc00673 gene and use thereof | |
| CN107541513A (en) | For silence CD317 small molecules interference RNA, recombinant vector, medicine and its application | |
| CN109481685B (en) | Application of CD317 inhibitor in preparation of medicament for treating liver cancer | |
| RU2583290C2 (en) | Preventive or therapeutic agent for treating fibrosis | |
| CN106434864A (en) | Tumor marker LIMK1 and application thereof | |
| CN108004322A (en) | A kind of applications of lncRNA in adenocarcinoma of lung is diagnosed and/or treated | |
| CN109288855B (en) | Uses of reagents in the preparation of drugs, interference fragments, methods for inhibiting self-renewal of liver cancer stem cells, and drugs for treating liver cancer | |
| CN107523566B (en) | Targeting inhibitor of MCM3AP-AS1 gene and application thereof | |
| CN110251529A (en) | Application of miR-124-3p and its analogs in the preparation of anti-breast cancer drugs | |
| CN104328122A (en) | siRNA aiming at human annexin A2 acceptor gene and application thereof | |
| CN116286819B (en) | siRNA targeting PABPC4 and its application in the treatment of liver cancer | |
| CN109988765B (en) | Targeting inhibitor of FENDRR gene and application thereof | |
| CN112442500A (en) | siRNA for inhibiting MCM7, composition and application thereof | |
| CN107541514B (en) | siRNA for specifically inhibiting CO L12A 1 gene expression, recombinant vector and application thereof | |
| CN108251425A (en) | Inhibit the siRNA molecule of RIOK2 genes and its antitumor application | |
| CN105779575B (en) | Uses of human EIF3A gene and related drugs | |
| CN110358765A (en) | Inhibit siRNA and its application of people TNFAIP1 gene expression | |
| CN108192895B (en) | siRNA molecule targeting NOB1 gene and application thereof | |
| WO2021037265A1 (en) | Sirna capable of inhibiting expression of mcm7 gene, composition, and application thereof | |
| CN114606233A (en) | siRNA targeting long non-coding RNA HOXD-AS2 and its application in the treatment of liver cancer | |
| CN110241118B (en) | Targeting inhibitor of ZFAS1 gene and application thereof | |
| CN107988230A (en) | Suppress the siRNA molecule of NOB1 genes | |
| CN119303094B (en) | Application of DKC1 inhibitor in preparation of medicament for treating multiple myeloma | |
| CN116173059B (en) | Use of human AURKB gene in the preparation of anti-tumor drugs |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right | Effective date of registration:20220214 Address after:510700 unit C501, 5th floor, No. 9, helix 4th Road, International Biological Island, Guangzhou, Guangdong Applicant after:Enkang Pharmaceutical Technology (Guangzhou) Co.,Ltd. Applicant after:FOSHAN INTELGEN PHARMACEUTICALS Co.,Ltd. Address before:Unit C501, 5th floor, No.9, helix 4th Road, International Biological Island, Guangzhou, Guangdong 510700 Applicant before:ENZHI (GUANGZHOU) PHARMACEUTICAL TECHNOLOGY Co.,Ltd. Applicant before:Enkang Pharmaceutical Technology (Guangzhou) Co.,Ltd. Applicant before:FOSHAN INTELGEN PHARMACEUTICALS Co.,Ltd. Applicant before:INTELGEN (GUANGZHOU) Ltd. | |
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |