WO2024065900A1 - Sirna for inhibiting tumor growth and use thereof - Google Patents

Sirna for inhibiting tumor growth and use thereof Download PDF

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WO2024065900A1
WO2024065900A1 PCT/CN2022/126237 CN2022126237W WO2024065900A1 WO 2024065900 A1 WO2024065900 A1 WO 2024065900A1 CN 2022126237 W CN2022126237 W CN 2022126237W WO 2024065900 A1 WO2024065900 A1 WO 2024065900A1
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seq
cancer
sirna
group
tumor
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许强
王冠
魏金旺
王堃
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领星生物科技（上海）有限公司
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Definitions

  • the invention relates to a method for inhibiting tumor cell growth by targeting AURKB with allele-specific siRNA, as well as siRNA for inhibiting tumor growth and application thereof.
  • Cancer is one of the leading causes of death in the world. As a populous country in the world, China's cancer data is not optimistic. Whether it is the number of new cases or deaths, China far exceeds other countries and ranks first in the world. In 2022, it is estimated that there will be about 4,820,000 newly diagnosed cancer cases and about 3,210,000 deaths in China. The increasing number of new cases and deaths has prompted my country to need more cancer prevention and treatment interventions to reduce the burden of cancer in the future.
  • Single nucleotide polymorphism refers to a DNA sequence polymorphism caused by a variation in a single nucleotide position on a chromosome sequence, and the frequency of occurrence in the population is greater than 1%.
  • the two genotypes containing SNP are alleles.
  • Tumor cells often have loss of heterozygosity (LOH), which means that tumor cells with LOH can only rely on one of the genotypes. If the gene that has LOH is also an essential gene, we can specifically inhibit the expression of this genotype, thereby killing tumor cells, while normal cells, since LOH does not occur, can still rely on the other genotype to normally express the essential gene without being affected.
  • LOH heterozygosity
  • AURKB is a mitotic serine/threonine protein kinase that belongs to the Aurora kinase family. Aurora kinase was discovered in 1995, but it was not until 1998 that it was first applied to the observation of human cancer tissue expression. During mitosis, Aurora kinase is involved in spindle formation, centrosome maturation, chromosome differentiation and cytokinesis. AURKB is a member of the chromosome passenger protein complex and plays an important role in cell cycle progression. Dysregulation of AURKB has been observed in some tumors. The gene is highly expressed in a variety of tumors and is associated with tumor cell invasion, metastasis and drug resistance.
  • AURKB Aurora kinase B
  • siRNA Small interfering RNA
  • mRNA messenger ribonucleotides
  • RISC RNA-induced silencing complex
  • the present invention provides a siRNA that can specifically target the expression of the AURKB gene of a specific genotype, thereby achieving the purpose of eliminating tumor cells without affecting the function of normal cells.
  • the siRNA designed according to the SNP of the present invention can specifically inhibit and kill tumor cells that have LOH, while the growth of heterozygous cells (representing normal cells) is not significantly affected. Therefore, this area can become a new siRNA drug targeting area for inhibiting tumor growth, thereby achieving the goals of the siRNA drug of the present invention, such as good specificity, high efficiency, easy development, and reusability.
  • the purpose of the present invention is to solve the problem of insufficient specificity of small molecule inhibitors and obvious side effects of medication at this stage.
  • siRNAs By designing different siRNAs to target the two haplotype sequences of the AURKB gene respectively, tumor cells expressing the haplotype can be specifically inhibited, thereby reducing the impact on heterozygous normal cells. This can solve the problem of high toxicity caused by the inability of small molecule AURKB inhibitors to distinguish genotypes.
  • the two SNPs in the two haplotypes H1 (CGTGCCCAT) (SEQ ID NO: 45) and H2 (AGTGCCCAG) (SEQ ID NO: 46) are only 7bp apart. Therefore, this region is very suitable as a siRNA targeting region, which can improve the specificity of siRNA, thereby inhibiting the growth of tumor cells with heterozygous loss.
  • small interfering nucleic acid As used herein, the terms “small interfering nucleic acid,” “siNA or SINA” molecule, “small interfering RNA,” “siRNA,” “small interfering nucleic acid molecule,” “small interfering oligonucleotide molecule” refer to any nucleic acid molecule that is capable of inhibiting or downregulating gene expression through the RNA interference mechanism.
  • target sequence refers to a continuous portion of the nucleotide sequence of an mRNA molecule (including messenger RNA (mRNA), which is a product of RNA processing primary transcript products) formed during transcription of the H1/H2 gene.
  • mRNA messenger RNA
  • the target portion of the sequence will be at least long enough to serve as a substrate for siRNA to guide cutting in this portion or its vicinity.
  • the target sequence will generally be from 9-36 nucleotides in length, for example, 15-30 nucleotides in length, including all subranges therebetween.
  • the target sequence can have from 15-30 nucleotides, 15-26 nucleotides, 15-23 nucleotides, 15-22 nucleotides, 15-21 nucleotides, 15-20 nucleotides, 15-19 nucleotides, 15-18 nucleotides, 15-17 nucleotides, 18-30 nucleotides, 18-26 nucleotides, 18-23 nucleotides, 18-22 nucleotides, 18-21 nucleotides, 18-20 nucleotides, 19-30 nucleotides, 19-26 nucleotides, 19-23 nucleotides, 19-22 nucleotides, 19-21 nucleotides, 19-20 nucleotides, 20-30 nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24 nucleotides, 20-23 nucleotides, 20-22 nucleotides, 20-21 nucleotides, 21-30 nucleotides, 21-
  • RNA refers to a molecule comprising at least one ribonucleotide residue, including double-stranded RNA, single-stranded RNA, isolated RNA, partially purified, pure or synthetic RNA, recombinantly produced RNA, and altered RNA or analogs of naturally occurring RNA.
  • RNA refers to an iRNA comprising an RNA molecule or a molecular complex having a hybrid duplex region comprising two antiparallel and substantially complementary nucleic acid strands, which are referred to as having "sense” and “antisense” orientations relative to the target RNA.
  • the duplex region can have any length that allows for specific degradation of the desired target RNA via the RISC pathway, but will generally range from 9 to 36 base pairs in length, e.g., 15-30 base pairs in length. For a duplex between 9 and 36 base pairs, the length of the duplex region is preferably 15-30 base pairs.
  • the duplex can have any length within this range, for example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 and any subranges therebetween, including but not limited to 15-30 base pairs, 15-26 base pairs, 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 1 22 base pairs, 20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs, 21-23 base pairs, or 21-22 base pairs.
  • dsRNAs produced by processing in cells with Dicer and similar enzymes typically have a base pair length ranging from 19 to 22.
  • One chain of the duplex region of dsDNA contains a sequence that is substantially complementary to the region of the target RNA.
  • the two chains forming the duplex structure can be from a single RNA molecule having at least one self-complementary region, or can be formed by two or more independent RNA molecules.
  • the duplex region is composed of two chains of a single molecule, the molecule can have a duplex region separated by a single nucleotide chain (referred to herein as a "hairpin loop") between the 3'-end of one chain forming the duplex structure and the 5'-end of the corresponding other chain.
  • the hairpin loop can include at least one non-paired nucleotide; in some embodiments, the hairpin loop can include at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 31 or more non-paired nucleotides.
  • the two substantially complementary chains of dsRNA are composed of separate RNA molecules, these molecules do not need to be covalently linked, but can be covalently linked.
  • the connecting structure is referred to as a "joint".
  • siRNA is also used herein to refer to dsRNA as described above.
  • the term "inhibit" means that the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, or the activity of one or more protein subunits is upregulated or downregulated, so that the expression, level or activity is greater or less than that observed when the regulator is not present.
  • the expression of the AURKB gene is inhibited by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% by administering the siRNA characterized in the present invention.
  • the AURKB gene is inhibited by at least about 60%, 70% or 80% by administering the siRNA described herein.
  • the AURKB gene is inhibited by at least about 85%, 90%, 95%, 98%, 99%, or more by administering the siRNA described herein.
  • gene refers to a nucleic acid encoding an RNA sequence, including but not limited to a structural gene encoding a polypeptide.
  • siRNA double-stranded RNA
  • mRNA small RNA
  • DNAi deoxyribonucleic acid interference
  • shRNA small hairpin RNA
  • the present invention relates to a siRNA targeting an mRNA fragment encoded by a sequence shown in SEQ ID NO: 45 or SEQ ID NO: 46.
  • At least one chain consists of a nucleotide chain with a length of 19-30 nucleotides, preferably a nucleotide chain with a length of 19-29, 19-28, 19-27, 19-26, or 19-25 nucleotides.
  • the siRNA comprises at least one modified nucleotide.
  • At least one of the modified nucleotides is selected from the group consisting of a 2'-O-methyl modified nucleotide, a nucleotide comprising a 5'-phosphorothioate group, a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group.
  • At least one of the modified nucleotides is selected from the group consisting of 2'-deoxy modified nucleotides, locked nucleotides, non-basic nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidate nucleotides and nucleotides containing non-natural bases.
  • At least one strand of the siRNA comprises a 3' overhang of at least one nucleotide.
  • each strand of the siRNA is modified with a pendant base.
  • the overhanging bases consist of any one or more deoxyribonucleotides selected from dA, dT, dG, and dC.
  • the overhanging bases consist of any two deoxyribonucleotides selected from dA, dT, dG, and dC.
  • the siRNA has a structure selected from the group consisting of: SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 21 and SEQ ID NO: 22 No.: SEQ ID NO: 39 and SEQ ID NO: 40; SEQ ID NO: 41 and SEQ ID NO: 42; and SEQ ID NO: 43 and SEQ ID NO: 44.
  • the second aspect of the present invention relates to a vector comprising the siRNA as described in the first aspect of the present invention.
  • the third aspect of the present invention relates to a cell comprising the siRNA as described in the first aspect of the present invention.
  • the present invention relates to a pharmaceutical composition for inhibiting tumor growth, comprising the siRNA as described in the first aspect of the present invention and a pharmaceutically acceptable excipient.
  • the tumor is selected from the group consisting of: glioma, leukemia, brain cancer, esophageal cancer, gastric cancer, lung cancer, liver cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, breast cancer, intestinal cancer, nasopharyngeal cancer, endometrial cancer, and prostate cancer.
  • the tumor is selected from the group consisting of brain cancer, lung cancer and liver cancer.
  • the present invention relates to a pharmaceutical composition for inhibiting tumor growth, comprising siRNA for inhibiting AURKB expression and a pharmaceutically acceptable excipient.
  • the tumor is selected from the group consisting of: glioma, leukemia, brain cancer, esophageal cancer, gastric cancer, lung cancer, liver cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, breast cancer, intestinal cancer, nasopharyngeal cancer, endometrial cancer, and prostate cancer.
  • the tumor is selected from the group consisting of brain cancer, lung cancer and liver cancer.
  • the present invention relates to a method for reducing the expression of AURKB in target cells, wherein the method is performed by administering the siRNA described in the first aspect of the present invention.
  • the present invention relates to a method for inhibiting tumor growth, which is carried out by administering siRNA to a subject in need thereof, wherein the siRNA targets an mRNA fragment encoded by a sequence shown in SEQ ID NO: 45 or SEQ ID NO: 46.
  • At least one strand of the siRNA consists of a nucleotide chain of 19-30 nucleotides, preferably a nucleotide chain of 19-29, 19-28, 19-27, 19-26, or 19-25 nucleotides in length.
  • the siRNA comprises at least one modified nucleotide.
  • each strand of the siRNA is modified with a pendant base.
  • the overhanging bases consist of any one or more deoxyribonucleotides selected from dA, dT, dG, and dC.
  • the overhanging bases consist of any two deoxyribonucleotides selected from dA, dT, dG, and dC.
  • the siRNA has a structure selected from the group consisting of: SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 21 and SEQ ID NO: 22 No.: SEQ ID NO: 39 and SEQ ID NO: 40; SEQ ID NO: 41 and SEQ ID NO: 42; and SEQ ID NO: 43 and SEQ ID NO: 44.
  • the tumor is selected from the group consisting of: glioma, leukemia, brain cancer, esophageal cancer, gastric cancer, lung cancer, liver cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, breast cancer, intestinal cancer, nasopharyngeal cancer, endometrial cancer, and prostate cancer.
  • the tumor is selected from the group consisting of brain cancer, lung cancer and liver cancer.
  • the present invention relates to a method for inhibiting tumor growth, comprising administering siRNA that inhibits AURKB expression to a subject in need thereof.
  • the tumor is selected from the group consisting of: glioma, leukemia, brain cancer, esophageal cancer, gastric cancer, lung cancer, liver cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, breast cancer, intestinal cancer, nasopharyngeal cancer, endometrial cancer, and prostate cancer.
  • the tumor is selected from the group consisting of brain cancer, lung cancer and liver cancer.
  • the present invention relates to the use of siRNA in the preparation of a drug for inhibiting tumor growth, wherein the siRNA targets an mRNA fragment encoded by a sequence shown in SEQ ID NO: 45 or SEQ ID NO: 46.
  • At least one strand of the siRNA consists of a nucleotide chain of 19-30 nucleotides, preferably a nucleotide chain of 19-29, 19-28, 19-27, 19-26, or 19-25 nucleotides in length.
  • the siRNA comprises at least one modified nucleotide.
  • each strand of the siRNA is modified with a pendant base.
  • the overhanging bases consist of any one or more deoxyribonucleotides selected from dA, dT, dG, and dC.
  • the overhanging bases are composed of any two deoxyribonucleotides selected from dA, dT, dG, and dC.
  • the siRNA has a structure selected from the group consisting of: SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 21 and SEQ ID NO: 22 No.: SEQ ID NO: 39 and SEQ ID NO: 40; SEQ ID NO: 41 and SEQ ID NO: 42; and SEQ ID NO: 43
  • the tumor is selected from the group consisting of: glioma, leukemia, brain cancer, esophageal cancer, gastric cancer, lung cancer, liver cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, breast cancer, intestinal cancer, nasopharyngeal cancer, endometrial cancer, and prostate cancer.
  • the tumor is selected from the group consisting of brain cancer, lung cancer and liver cancer.
  • AURKB is an essential gene in many tumors.
  • a CAS-9 value less than 0 indicates inhibition of tumor growth, and a value less than -3 indicates that tumor cells die rapidly after gene knockout, indicating that the gene is an essential gene.
  • Figure 2 Compared with the expression in normal tissues, AURKB expression was significantly upregulated in liver cancer, lung cancer, and brain glioma;
  • Figure 3 Patients with high AURKB expression in multiple cancer types have poor overall survival prognosis.
  • Figure 3A shows Lingxing data;
  • Figure 3B shows TCGA data;
  • FIG. 4 The newly discovered siRNA targetable region in the AURKB (NM_004217) gene is located between 8205000-8205040 on chromosome 17 (GRCh38.p14, NC_000017.11). The boxed positions are two SNP sites contained in this region;
  • FIGS 5A-5C siRNA transfection results
  • FIG. 6 Cell viability test results using the Cell Titer-Glo (CTG) method.
  • Example 1 AURKB is an essential gene for multiple tumors
  • AURKB is an essential gene (included in the Achilles_common_essentials essential gene list).
  • the CRISPR scores were all negative and basically less than -2 ( Figure 1). Therefore, the AURKB gene is an essential gene in many tumors (such as hepatocellular carcinoma, lung cancer and brain cancer, etc.).
  • Example 2 In liver cancer, lung cancer, and brain cancer, AURKB expression is upregulated in tumor tissues
  • TCGA data show that the expression of AURKB in liver cancer and lung cancer is higher than that in normal tissues.
  • Select the AURKB gene in the public website GEPIA2 http://gepia2.cancer-pku.cn/#analysis), select the corresponding cancer type, and then select Box Plot.
  • the webpage can display the expression comparison chart of the AURKB gene in tumor and normal tissues. Compared with the expression in normal tissues, the expression of AURKB in liver cancer, lung cancer, and brain glioma is significantly upregulated. This indicates that AURKB is closely related to tumor occurrence ( Figure 2).
  • Example 3 GC and TCGA data show that patients with low AURKB expression in hepatocellular carcinoma, lung cancer and brain cancer have better overall survival
  • siRNA haplotypes were found in the AURKB exons.
  • the newly discovered siRNA targetable region in the AURKB (NM_004217) gene is located between 8205000-8205040 on chromosome 17 (GRCh38.p14, NC_000017.11). This region is a completely new siRNA targeting region (Figure 4). The results showed that two SNPs 7 bp apart can significantly improve the inhibition efficiency of siRNA.
  • siRNAs Based on the targeted region and SNP information, a series of siRNAs that can cover the two genotypes were designed and transfection experiments were carried out to observe their interference efficiency.
  • siRNAs designed for this region were screened to obtain the following siRNAs with good knockdown efficiency, as shown in Table 2, which are a series of siRNA sequences designed for haplotype H1 (CGTGCCCAT) (SEQ ID NO.45); as shown in Table 3, which are a series of siRNA sequences designed for haplotype H2 (AGTGCCCAG) (SEQ ID NO.46).
  • hepatocellular carcinoma cell lines were selected to represent three common genotypes: HepG2 (H1 type), Huh-6 (heterozygous, including two genotypes, H1 and H2) and SNU387 (H2 type).
  • the three cell lines were transfected with siRNAs in Tables 3 and 4, as well as negative control siRNA (Reibo Bio) and positive control siRNA (Reibo Bio).
  • Lipofectamine TM RNAiMAXP Thermo Fisher Scientific
  • siRNAs si-1# and si-2# targeting H1 type have the best interference efficiency in HepG2 cells carrying H1, which can inhibit 60% of gene expression, while the rest, such as si-5#, si-6#, si-7#, si-8# and si-11#, can achieve 40% inhibition efficiency.
  • the inhibition efficiency of si-1# and si-2# in the heterozygous Huh-6 carrying H1 and H2 was 50%, and it could only inhibit about 20% in SNU387 carrying H2.
  • si-A6# could inhibit about 40% of the gene expression in SNU387, while the inhibition rate became worse in HepG2 and Huh6.
  • the experimental results confirmed the specific inhibition of siRNA on the expression of different genotypes.
  • siRNA targeting this region can effectively inhibit the expression of AURKB gene in tumor cells and inhibit tumor growth, but has no effect on the growth of hybrid cells
  • siRNAs were selected for the CellTiter-Glo (CTG) growth experiment to observe the effect of AURKB interference on the growth of each cell.
  • the experimental steps refer to the method recommended in the CellTiter-Glo manual, and the cells were tested on days 1, 2, 3, and 4 after transfection.
  • the results are shown in Figure 6.
  • si-1# and si-2# can significantly inhibit the growth of HepG2 carrying H1 type
  • si-A6# significantly inhibits the growth of the cell line SNU387 carrying H2 type, but in the heterozygous cell line Huh-6, the three siRNAs cannot significantly affect cell growth.
  • the experimental results show that targeting the region described in this patent can specifically inhibit tumor growth.
  • siRNA can effectively inhibit the expression of the AURKB gene and inhibit the growth of tumor cells.
  • siRNA does not affect their normal growth.

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Abstract

An siRNA for inhibiting tumor growth and a use thereof. The siRNA targets an mRNA fragment encoded by a sequence shown in SEQ ID NO: 45 or SEQ ID NO: 46. A method for using the siRNA to inhibit tumor growth.

Description

用于抑制肿瘤生长的siRNA及其应用siRNA for inhibiting tumor growth and its application 技术领域Technical Field
本发明涉及一种等位基因特异的siRNA靶向AURKB抑制肿瘤细胞生长的方法，以及用于抑制肿瘤生长的siRNA及其应用。The invention relates to a method for inhibiting tumor cell growth by targeting AURKB with allele-specific siRNA, as well as siRNA for inhibiting tumor growth and application thereof.
背景技术Background technique
肿瘤是全球主要死亡原因之一，作为世界上的人口大国，中国的肿瘤数据不容乐观，不论是新发人数还是死亡人数，中国都远超其他国家，位居全球第一。2022年我国预计有约4820000新确诊肿瘤病例以及约3210000例死亡病例。日渐增长的新发病例与死亡病例促使我国需要更多的肿瘤预防和治疗干预来降低未来肿瘤的负担。Cancer is one of the leading causes of death in the world. As a populous country in the world, China's cancer data is not optimistic. Whether it is the number of new cases or deaths, China far exceeds other countries and ranks first in the world. In 2022, it is estimated that there will be about 4,820,000 newly diagnosed cancer cases and about 3,210,000 deaths in China. The increasing number of new cases and deaths has prompted my country to need more cancer prevention and treatment interventions to reduce the burden of cancer in the future.
单核苷酸多态性(SNP)是指在染色体序列上由于单个核苷酸位置上发生变异而引起DNA序列多态性，且在人群中发生频率大于1％。包含SNP的两种基因型为等位基因。肿瘤细胞经常发生杂合性缺失(LOH)，这就意味着发生LOH的肿瘤细胞只能依靠其中一个基因型。如果发生LOH的基因同时为必需基因，我们就可以特异地抑制该基因型表达，从而杀死肿瘤细胞，而正常细胞由于没有发生LOH，依旧可以依靠另一个基因型正常表达该必需基因而不受影响。Single nucleotide polymorphism (SNP) refers to a DNA sequence polymorphism caused by a variation in a single nucleotide position on a chromosome sequence, and the frequency of occurrence in the population is greater than 1%. The two genotypes containing SNP are alleles. Tumor cells often have loss of heterozygosity (LOH), which means that tumor cells with LOH can only rely on one of the genotypes. If the gene that has LOH is also an essential gene, we can specifically inhibit the expression of this genotype, thereby killing tumor cells, while normal cells, since LOH does not occur, can still rely on the other genotype to normally express the essential gene without being affected.
AURKB是一种有丝分裂的丝氨酸/苏氨酸蛋白激酶，属于极光激酶家族。Aurora激酶从1995年被发现以来,直到1998年才第一次应用到人体癌变组织表达的观察，在有丝分裂过程中,Aurora激酶参与纺锤体形成、中心体成熟、染色体分化和胞质分裂。AURKB是染色体乘客蛋白复合物的成员，在细胞周期进程中发挥重要作用。在一些肿瘤中观察到AURKB的失调，该基因在多种肿瘤中高表达，并与肿瘤细胞的侵袭、转移和耐药性有关。极光激酶B(AURKB)作为一个重要的肿瘤靶点，AURKB已成为吸引人的药物靶标，导致了相关小分子抑制剂的发展。尽管AURKB药物开发已经多年，目前有多达30种AURKB抑制剂在临床前或临床研究中，但是还没有药物上 市。AURKB is a mitotic serine/threonine protein kinase that belongs to the Aurora kinase family. Aurora kinase was discovered in 1995, but it was not until 1998 that it was first applied to the observation of human cancer tissue expression. During mitosis, Aurora kinase is involved in spindle formation, centrosome maturation, chromosome differentiation and cytokinesis. AURKB is a member of the chromosome passenger protein complex and plays an important role in cell cycle progression. Dysregulation of AURKB has been observed in some tumors. The gene is highly expressed in a variety of tumors and is associated with tumor cell invasion, metastasis and drug resistance. As an important tumor target, Aurora kinase B (AURKB) has become an attractive drug target, leading to the development of related small molecule inhibitors. Although AURKB drug development has been going on for many years, there are currently as many as 30 AURKB inhibitors in preclinical or clinical studies, but no drug has been marketed.
小干扰RNA(siRNA)是一类19-30个核苷酸长度的双链RNA分子，通过绑定RNA诱导沉默复合体(RISC)来特异的降解信使核糖核苷酸(mRNA)，进而降低基因表达水平。近年来，siRNA因其特异性好、效率高、易开发、可重复使用等特点，成为药物研发的热门领域，目前已有多款siRNA药物获批上市。Small interfering RNA (siRNA) is a type of double-stranded RNA molecule with a length of 19-30 nucleotides. It specifically degrades messenger ribonucleotides (mRNA) by binding to the RNA-induced silencing complex (RISC), thereby reducing gene expression levels. In recent years, siRNA has become a hot area in drug research and development due to its good specificity, high efficiency, easy development, and reusability. Currently, many siRNA drugs have been approved for marketing.
目前，针对AUKRB靶点的很多小分子抑制剂在临床前和临床研究中，但是一方面是药物不够特异，会抑制其他激酶活性，另一方面是用药副作用明显。因此，新型的抑制剂或者抑制方法需要被研发。Currently, many small molecule inhibitors targeting AUKRB are in preclinical and clinical studies, but on the one hand, the drugs are not specific enough and will inhibit the activity of other kinases, and on the other hand, the side effects of the drugs are obvious. Therefore, new inhibitors or inhibition methods need to be developed.
发明内容Summary of the invention
鉴于此，本发明提供了一种siRNA，其可以特异的靶向特定基因型的AURKB基因的表达，从而达到清除肿瘤细胞而不影响正常细胞功能的目的。本发明根据SNP设计的siRNA可以特异的抑制并杀死发生LOH的肿瘤细胞，而杂合型细胞(代表正常细胞)生长未受到明显影响。因此，该区域可成为新的siRNA药物靶向区域，用于抑制肿瘤生长，从而实现本发明的siRNA药物特异性好、效率高、易开发、可重复使用等目标。In view of this, the present invention provides a siRNA that can specifically target the expression of the AURKB gene of a specific genotype, thereby achieving the purpose of eliminating tumor cells without affecting the function of normal cells. The siRNA designed according to the SNP of the present invention can specifically inhibit and kill tumor cells that have LOH, while the growth of heterozygous cells (representing normal cells) is not significantly affected. Therefore, this area can become a new siRNA drug targeting area for inhibiting tumor growth, thereby achieving the goals of the siRNA drug of the present invention, such as good specificity, high efficiency, easy development, and reusability.
本发明的目的在于解决现阶段小分子抑制剂特异性不够以及用药副作用明显的问题。通过设计不同的siRNA分别靶向AURKB基因的两种单倍型序列，可以特异的抑制表达该单倍型的肿瘤细胞，从而减轻对杂合性正常细胞的影响。这样可以解决小分子AURKB抑制剂不能分辨基因型而产生的毒性大的问题。并且所述两种单倍型H1(CGTGCCCAT)(SEQ ID NO：45)和H2(AGTGCCCAG)(SEQ ID NO：46)中的两个SNP相隔仅7bp。因此，该区域非常适合作为siRNA靶向区域，可以提高siRNA的特异性，从而抑制发生杂合性缺失的肿瘤细胞生长。The purpose of the present invention is to solve the problem of insufficient specificity of small molecule inhibitors and obvious side effects of medication at this stage. By designing different siRNAs to target the two haplotype sequences of the AURKB gene respectively, tumor cells expressing the haplotype can be specifically inhibited, thereby reducing the impact on heterozygous normal cells. This can solve the problem of high toxicity caused by the inability of small molecule AURKB inhibitors to distinguish genotypes. And the two SNPs in the two haplotypes H1 (CGTGCCCAT) (SEQ ID NO: 45) and H2 (AGTGCCCAG) (SEQ ID NO: 46) are only 7bp apart. Therefore, this region is very suitable as a siRNA targeting region, which can improve the specificity of siRNA, thereby inhibiting the growth of tumor cells with heterozygous loss.
本文使用的术语“小干扰核酸”、“siNA或SINA”分子、“小 干扰RNA”、“siRNA”、“小干扰核酸分子”、“小干扰寡核苷酸分子”表示能够通过RNA干扰机制抑制或下调基因表达的任何核酸分子。As used herein, the terms "small interfering nucleic acid," "siNA or SINA" molecule, "small interfering RNA," "siRNA," "small interfering nucleic acid molecule," "small interfering oligonucleotide molecule" refer to any nucleic acid molecule that is capable of inhibiting or downregulating gene expression through the RNA interference mechanism.
本文使用的术语“靶序列”指H1/H2基因转录期间形成的mRNA分子(包括信使RNA(mRNA)，是RNA加工初级转录物产物的产物)的核苷酸序列的连续部分。序列的靶部分将至少足够地长，以充当siRNA指导在这个部分或其附近切割的底物。例如，靶序列总体上将是从9-36个核苷酸长度，例如，15-30个核苷酸长度，包括其之间的全部子范围。作为一个非限制性实例，靶序列可以具有从15-30个核苷酸、15-26个核苷酸、15-23个核苷酸、15-22个核苷酸、15-21个核苷酸、15-20个核苷酸、15-19个核苷酸、15-18个核苷酸、15-17个核苷酸、18-30个核苷酸、18-26个核苷酸、18-23个核苷酸、18-22个核苷酸、18-21个核苷酸、18-20个核苷酸、19-30个核苷酸、19-26个核苷酸、19-23个核苷酸、19-22个核苷酸、19-21个核苷酸、19-20个核苷酸、20-30个核苷酸、20-26个核苷酸、20-25个核苷酸、20-24个核苷酸、20-23个核苷酸、20-22个核苷酸、20-21个核苷酸、21-30个核苷酸、21-26个核苷酸、21-25个核苷酸、21-24个核苷酸、21-23个核苷酸或21-22核苷酸。The term "target sequence" as used herein refers to a continuous portion of the nucleotide sequence of an mRNA molecule (including messenger RNA (mRNA), which is a product of RNA processing primary transcript products) formed during transcription of the H1/H2 gene. The target portion of the sequence will be at least long enough to serve as a substrate for siRNA to guide cutting in this portion or its vicinity. For example, the target sequence will generally be from 9-36 nucleotides in length, for example, 15-30 nucleotides in length, including all subranges therebetween. As a non-limiting example, the target sequence can have from 15-30 nucleotides, 15-26 nucleotides, 15-23 nucleotides, 15-22 nucleotides, 15-21 nucleotides, 15-20 nucleotides, 15-19 nucleotides, 15-18 nucleotides, 15-17 nucleotides, 18-30 nucleotides, 18-26 nucleotides, 18-23 nucleotides, 18-22 nucleotides, 18-21 nucleotides, 18-20 nucleotides, 19-30 nucleotides, 19-26 nucleotides, 19-23 nucleotides, 19-22 nucleotides, 19-21 nucleotides, 19-20 nucleotides, 20-30 nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24 nucleotides, 20-23 nucleotides, 20-22 nucleotides, 20-21 nucleotides, 21-30 nucleotides, 21-26 nucleotides, 21-25 nucleotides, 21-24 nucleotides, 21-23 nucleotides or 21-22 nucleotides.
本文使用的术语“RNA”表示包含至少一个核糖核苷酸残基的分子，其包括双链RNA，单链RNA，分离的RNA，部分纯化的、纯的或合成的RNA、重组产生的RNA以及变化的RNA或天然产生的RNA的类似物。The term "RNA" as used herein refers to a molecule comprising at least one ribonucleotide residue, including double-stranded RNA, single-stranded RNA, isolated RNA, partially purified, pure or synthetic RNA, recombinantly produced RNA, and altered RNA or analogs of naturally occurring RNA.
本文使用的术语“siRNA”或“dsRNA”指一种iRNA，它包含具有杂交双链体区的RNA分子或分子复合物，所述杂交双链体区包含两条反平行和基本上互补的核酸链，所述核酸链将相对于靶RNA称作具有“有义”和“反义”方向。双链体区可以具有允许经RISC途径特异性降解所需靶RNA的任何长度，但是一般将范围从9至36碱基对长度，例如，15-30个碱基对长度。就9和36碱基对之间的双链 体时，双链体可以具有在这个范围内的任意长度，例如，9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35或36和其间的任何子范围，包括但不局限于15-30碱基对、15-26碱基对、15-23碱基对、15-22碱基对、15-21碱基对、15-20碱基对、15-19碱基对、15-18碱基对、15-17碱基对、18-30碱基对、18-26碱基对、18-23碱基对、18-22碱基对、18-21碱基对、18-20碱基对、19-30碱基对、19-26碱基对、19-23碱基对、19-22碱基对、19-21碱基对、19-20碱基对、20-30碱基对、20-26碱基对、20-25碱基对、20-24碱基对、20-23碱基对、20-22碱基对、20-21碱基对、21-30碱基对、21-26碱基对、21-25碱基对、21-24碱基对、21-23碱基对或21-22碱基对。细胞中用Dicer和相似酶加工产生的dsRNA通常具有范围19-22的碱基对长度。dsDNA的双链体区的一个链包含与靶RNA的区域基本上互补的序列。形成双链体结构的两个链可以来自具有至少一个自身互补区域的单一RNA分子，或可以由两个或更多个独立RNA分子形成。其中双链体区由单个分子的两个链组成的情况下，该分子可以具有由形成双链体结构的一个链的3'-端和相应另一个链的5'-端之间的单条核苷酸链(本文中称作“发夹环”)分隔的双链体区。发夹环可以包含至少一个非配对的核苷酸；在一些实施例中，发夹环可以包含至少3个、至少4个、至少5个、至少6个、至少7个、至少8个、至少9个、至少10个、至少20个、至少31个或更多个非配对的核苷酸。在dsRNA的两条基本上互补链由单独RNA分子构成的情况下，这些分子不需要共价连接，但是可以共价连接。在两个链借助发夹环之外的方式共价连接的情况下，连接结构称作“接头”。术语“siRNA”也在此用来指如上文所述的dsRNA。The term "siRNA" or "dsRNA" as used herein refers to an iRNA comprising an RNA molecule or a molecular complex having a hybrid duplex region comprising two antiparallel and substantially complementary nucleic acid strands, which are referred to as having "sense" and "antisense" orientations relative to the target RNA. The duplex region can have any length that allows for specific degradation of the desired target RNA via the RISC pathway, but will generally range from 9 to 36 base pairs in length, e.g., 15-30 base pairs in length. For a duplex between 9 and 36 base pairs, the length of the duplex region is preferably 15-30 base pairs. When the duplex is present, the duplex can have any length within this range, for example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 and any subranges therebetween, including but not limited to 15-30 base pairs, 15-26 base pairs, 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 1 22 base pairs, 20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs, 21-23 base pairs, or 21-22 base pairs. dsRNAs produced by processing in cells with Dicer and similar enzymes typically have a base pair length ranging from 19 to 22. One chain of the duplex region of dsDNA contains a sequence that is substantially complementary to the region of the target RNA. The two chains forming the duplex structure can be from a single RNA molecule having at least one self-complementary region, or can be formed by two or more independent RNA molecules. Wherein the duplex region is composed of two chains of a single molecule, the molecule can have a duplex region separated by a single nucleotide chain (referred to herein as a "hairpin loop") between the 3'-end of one chain forming the duplex structure and the 5'-end of the corresponding other chain. The hairpin loop can include at least one non-paired nucleotide; in some embodiments, the hairpin loop can include at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 31 or more non-paired nucleotides. In the case where the two substantially complementary chains of dsRNA are composed of separate RNA molecules, these molecules do not need to be covalently linked, but can be covalently linked. In the case where the two chains are covalently linked by means other than the hairpin loop, the connecting structure is referred to as a "joint". The term "siRNA" is also used herein to refer to dsRNA as described above.
本文使用的术语“抑制”表示基因的表达、或编码一个或多个蛋白或蛋白亚基的RNA分子或等同RNA分子的水平、或一个或多个蛋白亚基的活性被上调或下调，从而使得所述表达、水平或活性大于或 小于调节子不存在时的观察结果。例如，在某些情况下，通过给予本发明中表征的siRNA抑制AURKB基因表达至少约10％、15％、20％、25％、30％、35％、40％、45％或50％。在一些实施例中，通过给予在此所述的siRNA抑制AURKB基因至少约60％、70％或80％。在一些实施例中，通过给予如在此所述的siRNA抑制AURKB基因至少约85％、90％、95％、98％、99％、或更多。As used herein, the term "inhibit" means that the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, or the activity of one or more protein subunits is upregulated or downregulated, so that the expression, level or activity is greater or less than that observed when the regulator is not present. For example, in some cases, the expression of the AURKB gene is inhibited by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% by administering the siRNA characterized in the present invention. In some embodiments, the AURKB gene is inhibited by at least about 60%, 70% or 80% by administering the siRNA described herein. In some embodiments, the AURKB gene is inhibited by at least about 85%, 90%, 95%, 98%, 99%, or more by administering the siRNA described herein.
本文使用的术语“基因”表示编码RNA序列的核酸，其包括但不限于编码多肽的结构基因。The term "gene" as used herein refers to a nucleic acid encoding an RNA sequence, including but not limited to a structural gene encoding a polypeptide.
除非特别指出，否则“一”表示“一个(种)或多个(种)”。Unless otherwise specified, "a" or "an" means "one or more".
尽管本发明的一些实施方案重点阐述siRNA，但本文公开的内容并不解释为限于siRNA，而是还涵盖了利用小核酸分子、双链RNA(dsRNA)、小RNA(mRNA)、脱氧核糖核酸干扰(DNAi)和小发夹RNA(shRNA)分子、酶核酸分子(Enzymatic Nucleic Acid)或反义核酸分子实施的相关组合物和方法。Although some embodiments of the present invention focus on siRNA, the content disclosed herein is not to be construed as being limited to siRNA, but also covers related compositions and methods implemented using small nucleic acid molecules, double-stranded RNA (dsRNA), small RNA (mRNA), deoxyribonucleic acid interference (DNAi) and small hairpin RNA (shRNA) molecules, enzymatic nucleic acid molecules (Enzymatic Nucleic Acid) or antisense nucleic acid molecules.
本发明的第一方面，本发明涉及一种siRNA，其靶向由包含SEQ ID NO：45或SEQ ID NO：46所示的序列编码的mRNA片段。In the first aspect of the present invention, the present invention relates to a siRNA targeting an mRNA fragment encoded by a sequence shown in SEQ ID NO: 45 or SEQ ID NO: 46.
根据一个优选的实施方案，其中至少一条链由长度为19-30个核苷酸的核苷酸链构成，优选由长度为19-29个、19-28个、19-27个、19-26个、19-25个核苷酸的核苷酸链构成。According to a preferred embodiment, at least one chain consists of a nucleotide chain with a length of 19-30 nucleotides, preferably a nucleotide chain with a length of 19-29, 19-28, 19-27, 19-26, or 19-25 nucleotides.
根据一个优选的实施方案，其中所述siRNA至少包含一个修饰的核苷酸。According to a preferred embodiment, the siRNA comprises at least one modified nucleotide.
在一个实施方案中，其中所述修饰的核苷酸的至少一种选自下组：2’-O-甲基修饰的核苷酸、包含5’-硫代磷酸酯基的核苷酸、与胆甾烯衍生物或十二烷酸双癸酰胺基链接的末端核苷酸。In one embodiment, at least one of the modified nucleotides is selected from the group consisting of a 2'-O-methyl modified nucleotide, a nucleotide comprising a 5'-phosphorothioate group, a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group.
在一个实施方案中，其中所述修饰的核苷酸的至少一种选自下组：2’-脱氧修饰的核苷酸、锁定核苷酸、非碱基核苷酸、2’-氨基修饰的核苷酸、2’-烷基修饰的核苷酸、吗啉代核苷酸、磷酰胺酯核苷酸和包含非天然碱基的核苷酸。In one embodiment, at least one of the modified nucleotides is selected from the group consisting of 2'-deoxy modified nucleotides, locked nucleotides, non-basic nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidate nucleotides and nucleotides containing non-natural bases.
根据一个优选的实施方案，其中所述siRNA至少一条链包含至少一个核苷酸的3’突出端。According to a preferred embodiment, at least one strand of the siRNA comprises a 3' overhang of at least one nucleotide.
根据一个优选的实施方案，其中所述siRNA每条链的3’端修饰有悬垂碱基。According to a preferred embodiment, the 3' end of each strand of the siRNA is modified with a pendant base.
根据一个优选的实施方案，其中所述悬垂碱基由dA、dT、dG、dC中任意一个或多个脱氧核糖核苷酸组成。According to a preferred embodiment, the overhanging bases consist of any one or more deoxyribonucleotides selected from dA, dT, dG, and dC.
在一个实施方案中，其中所述悬垂碱基由dA、dT、dG、dC中任意两个脱氧核糖核苷酸组成。In one embodiment, the overhanging bases consist of any two deoxyribonucleotides selected from dA, dT, dG, and dC.
根据一个优选的实施方案，其中所述siRNA具有选自由以下组成的组的结构：SEQ ID NO：1和SEQ ID NO：2；SEQ ID NO：3和SEQ ID NO：4；SEQ ID NO：5和SEQ ID NO：6；SEQ ID NO：7和SEQ ID NO：8；SEQ ID NO：9和SEQ ID NO：10；SEQ ID NO：11和SEQ ID NO：12；SEQ ID NO：13和SEQ ID NO：14；SEQ ID NO：15和SEQ ID NO：16；SEQ ID NO：17和SEQ ID NO：18；SEQ ID NO：19和SEQ ID NO：20；SEQ ID NO：21和SEQ ID NO：22；SEQ ID NO：23和SEQ ID NO：24；SEQ ID NO：25和SEQ ID NO：26；SEQ ID NO：27和SEQ ID NO：28；SEQ ID NO：29和SEQ ID NO：30；SEQ ID NO：31和SEQ ID NO：32；SEQ ID NO：33和SEQ ID NO：34；SEQ ID NO：35和SEQ ID NO：36；SEQ ID NO：37和SEQ ID NO：38；SEQ ID NO：39和SEQ ID NO：40；SEQ ID NO：41和SEQ ID NO：42；和SEQ ID NO：43和SEQ ID NO：44。According to a preferred embodiment, the siRNA has a structure selected from the group consisting of: SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 21 and SEQ ID NO: 22 No.: SEQ ID NO: 39 and SEQ ID NO: 40; SEQ ID NO: 41 and SEQ ID NO: 42; and SEQ ID NO: 43 and SEQ ID NO: 44.
本发明的第二方面，本发明涉及一种载体，包含如本发明第一方面所述的siRNA。The second aspect of the present invention relates to a vector comprising the siRNA as described in the first aspect of the present invention.
本发明的第三方面，本发明涉及一种细胞，包含如本发明第一方面所述的siRNA。The third aspect of the present invention relates to a cell comprising the siRNA as described in the first aspect of the present invention.
本发明的第四方面，本发明涉及一种用于抑制肿瘤生长药物组合物，包含如本发明第一方面所述的siRNA和药学上可接受的赋形剂。In a fourth aspect, the present invention relates to a pharmaceutical composition for inhibiting tumor growth, comprising the siRNA as described in the first aspect of the present invention and a pharmaceutically acceptable excipient.
根据一个优选的实施方案，其中所述肿瘤选自由以下组成的组： 胶质瘤、白血病、脑癌、食道癌、胃癌、肺癌、肝癌、膀胱癌、胰腺癌、宫颈癌、头颈癌、卵巢癌、黑色素瘤、淋巴瘤、乳腺癌、肠癌、鼻咽癌、子宫内膜癌、前列腺癌。According to a preferred embodiment, the tumor is selected from the group consisting of: glioma, leukemia, brain cancer, esophageal cancer, gastric cancer, lung cancer, liver cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, breast cancer, intestinal cancer, nasopharyngeal cancer, endometrial cancer, and prostate cancer.
在一个实施方案中，其中所述肿瘤选自由脑癌、肺癌和肝癌组成的组。In one embodiment, wherein the tumor is selected from the group consisting of brain cancer, lung cancer and liver cancer.
本发明的第五方面，本发明涉及一种用于抑制肿瘤生长药物组合物，其包含抑制AURKB表达的siRNA和药学上可接受的赋形剂。In a fifth aspect, the present invention relates to a pharmaceutical composition for inhibiting tumor growth, comprising siRNA for inhibiting AURKB expression and a pharmaceutically acceptable excipient.
根据一个优选的实施方案，其中所述肿瘤选自由以下组成的组：胶质瘤、白血病、脑癌、食道癌、胃癌、肺癌、肝癌、膀胱癌、胰腺癌、宫颈癌、头颈癌、卵巢癌、黑色素瘤、淋巴瘤、乳腺癌、肠癌、鼻咽癌、子宫内膜癌、前列腺癌。According to a preferred embodiment, the tumor is selected from the group consisting of: glioma, leukemia, brain cancer, esophageal cancer, gastric cancer, lung cancer, liver cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, breast cancer, intestinal cancer, nasopharyngeal cancer, endometrial cancer, and prostate cancer.
在一个实施方案中，其中所述肿瘤选自由脑癌、肺癌和肝癌组成的组。In one embodiment, wherein the tumor is selected from the group consisting of brain cancer, lung cancer and liver cancer.
本发明的第六方面，本发明涉及一种降低靶细胞中AURKB表达的方法，所述方法通过施用本发明第一方面所述的siRNA来进行。In a sixth aspect, the present invention relates to a method for reducing the expression of AURKB in target cells, wherein the method is performed by administering the siRNA described in the first aspect of the present invention.
本发明的第七方面，本发明涉及一种抑制肿瘤生长的方法，所述方法通过向有此需要的受试者施用siRNA来进行，其中所述siRNA靶向由包含SEQ ID NO：45或SEQ ID NO：46所示的序列编码的mRNA片段。In the seventh aspect of the present invention, the present invention relates to a method for inhibiting tumor growth, which is carried out by administering siRNA to a subject in need thereof, wherein the siRNA targets an mRNA fragment encoded by a sequence shown in SEQ ID NO: 45 or SEQ ID NO: 46.
根据一个优选的实施方案，其中所述siRNA的至少一条链由19-30个核苷酸的核苷酸链构成，优选由长度为19-29个、19-28个、19-27个、19-26个、19-25个核苷酸的核苷酸链构成。According to a preferred embodiment, at least one strand of the siRNA consists of a nucleotide chain of 19-30 nucleotides, preferably a nucleotide chain of 19-29, 19-28, 19-27, 19-26, or 19-25 nucleotides in length.
根据一个优选的实施方案，其中所述siRNA至少包含一个修饰的核苷酸。According to a preferred embodiment, the siRNA comprises at least one modified nucleotide.
根据一个优选的实施方案，其中所述siRNA每条链的3’端修饰有悬垂碱基。According to a preferred embodiment, the 3' end of each strand of the siRNA is modified with a pendant base.
根据一个优选的实施方案，其中所述悬垂碱基由dA、dT、dG、dC中任意一个或多个脱氧核糖核苷酸组成。According to a preferred embodiment, the overhanging bases consist of any one or more deoxyribonucleotides selected from dA, dT, dG, and dC.
在一个实施方案中，其中所述悬垂碱基由dA、dT、dG、dC中任意两个脱氧核糖核苷酸组成。In one embodiment, the overhanging bases consist of any two deoxyribonucleotides selected from dA, dT, dG, and dC.
根据一个优选的实施方案，其中所述siRNA具有选自由以下组成的组的结构：SEQ ID NO：1和SEQ ID NO：2；SEQ ID NO：3和SEQ ID NO：4；SEQ ID NO：5和SEQ ID NO：6；SEQ ID NO：7和SEQ ID NO：8；SEQ ID NO：9和SEQ ID NO：10；SEQ ID NO：11和SEQ ID NO：12；SEQ ID NO：13和SEQ ID NO：14；SEQ ID NO：15和SEQ ID NO：16；SEQ ID NO：17和SEQ ID NO：18；SEQ ID NO：19和SEQ ID NO：20；SEQ ID NO：21和SEQ ID NO：22；SEQ ID NO：23和SEQ ID NO：24；SEQ ID NO：25和SEQ ID NO：26；SEQ ID NO：27和SEQ ID NO：28；SEQ ID NO：29和SEQ ID NO：30；SEQ ID NO：31和SEQ ID NO：32；SEQ ID NO：33和SEQ ID NO：34；SEQ ID NO：35和SEQ ID NO：36；SEQ ID NO：37和SEQ ID NO：38；SEQ ID NO：39和SEQ ID NO：40；SEQ ID NO：41和SEQ ID NO：42；和SEQ ID NO：43和SEQ ID NO：44。According to a preferred embodiment, the siRNA has a structure selected from the group consisting of: SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 21 and SEQ ID NO: 22 No.: SEQ ID NO: 39 and SEQ ID NO: 40; SEQ ID NO: 41 and SEQ ID NO: 42; and SEQ ID NO: 43 and SEQ ID NO: 44.
根据一个优选的实施方案，其中所述肿瘤选自由以下组成的组：胶质瘤、白血病、脑癌、食道癌、胃癌、肺癌、肝癌、膀胱癌、胰腺癌、宫颈癌、头颈癌、卵巢癌、黑色素瘤、淋巴瘤、乳腺癌、肠癌、鼻咽癌、子宫内膜癌、前列腺癌。According to a preferred embodiment, the tumor is selected from the group consisting of: glioma, leukemia, brain cancer, esophageal cancer, gastric cancer, lung cancer, liver cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, breast cancer, intestinal cancer, nasopharyngeal cancer, endometrial cancer, and prostate cancer.
在一个实施方案中，其中所述肿瘤选自由脑癌、肺癌和肝癌组成的组。In one embodiment, wherein the tumor is selected from the group consisting of brain cancer, lung cancer and liver cancer.
本发明的第八方面，本发明涉及一种抑制肿瘤生长的方法，所述方法包括向有此需要的受试者施用抑制AURKB表达的siRNA。In an eighth aspect, the present invention relates to a method for inhibiting tumor growth, comprising administering siRNA that inhibits AURKB expression to a subject in need thereof.
根据一个优选的实施方案，其中所述肿瘤选自由以下组成的组：胶质瘤、白血病、脑癌、食道癌、胃癌、肺癌、肝癌、膀胱癌、胰腺癌、宫颈癌、头颈癌、卵巢癌、黑色素瘤、淋巴瘤、乳腺癌、肠癌、鼻咽癌、子宫内膜癌、前列腺癌。According to a preferred embodiment, the tumor is selected from the group consisting of: glioma, leukemia, brain cancer, esophageal cancer, gastric cancer, lung cancer, liver cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, breast cancer, intestinal cancer, nasopharyngeal cancer, endometrial cancer, and prostate cancer.
在一个实施方案中，其中所述肿瘤选自由脑癌、肺癌和肝癌组成 的组。In one embodiment, wherein the tumor is selected from the group consisting of brain cancer, lung cancer and liver cancer.
本发明的第九方面，本发明涉及一种siRNA在制备抑制肿瘤生长的药物中的应用，所述siRNA靶向由包含SEQ ID NO：45或SEQ ID NO：46所示的序列编码的mRNA片段。In the ninth aspect of the present invention, the present invention relates to the use of siRNA in the preparation of a drug for inhibiting tumor growth, wherein the siRNA targets an mRNA fragment encoded by a sequence shown in SEQ ID NO: 45 or SEQ ID NO: 46.
根据一个优选的实施方案，其中所述siRNA的至少一条链由19-30个核苷酸的核苷酸链构成，优选由长度为19-29个、19-28个、19-27个、19-26个、19-25个核苷酸的核苷酸链构成。According to a preferred embodiment, at least one strand of the siRNA consists of a nucleotide chain of 19-30 nucleotides, preferably a nucleotide chain of 19-29, 19-28, 19-27, 19-26, or 19-25 nucleotides in length.
根据一个优选的实施方案，其中所述siRNA至少包含一个修饰的核苷酸。According to a preferred embodiment, the siRNA comprises at least one modified nucleotide.
根据一个优选的实施方案，其中所述siRNA每条链的3’端修饰有悬垂碱基。According to a preferred embodiment, the 3' end of each strand of the siRNA is modified with a pendant base.
根据一个优选的实施方案，其中所述悬垂碱基由dA、dT、dG、dC中任意一个或多个脱氧核糖核苷酸组成。According to a preferred embodiment, the overhanging bases consist of any one or more deoxyribonucleotides selected from dA, dT, dG, and dC.
在一个实施方案中，其中所述悬垂碱基由dA、dT、dG、dC中任意两个脱氧核糖核苷酸组成。根据一个优选的实施方案，其中所述siRNA具有选自由以下组成的组的结构：SEQ ID NO：1和SEQ ID NO：2；SEQ ID NO：3和SEQ ID NO：4；SEQ ID NO：5和SEQ ID NO：6；SEQ ID NO：7和SEQ ID NO：8；SEQ ID NO：9和SEQ ID NO：10；SEQ ID NO：11和SEQ ID NO：12；SEQ ID NO：13和SEQ ID NO：14；SEQ ID NO：15和SEQ ID NO：16；SEQ ID NO：17和SEQ ID NO：18；SEQ ID NO：19和SEQ ID NO：20；SEQ ID NO：21和SEQ ID NO：22；SEQ ID NO：23和SEQ ID NO：24；SEQ ID NO：25和SEQ ID NO：26；SEQ ID NO：27和SEQ ID NO：28；SEQ ID NO：29和SEQ ID NO：30；SEQ ID NO：31和SEQ ID NO：32；SEQ ID NO：33和SEQ ID NO：34；SEQ ID NO：35和SEQ ID NO：36；SEQ ID NO：37和SEQ ID NO：38；SEQ ID NO：39和SEQ ID NO：40；SEQ ID NO：41和SEQ ID NO：42；和SEQ ID NO：43和SEQ ID NO：44。In one embodiment, the overhanging bases are composed of any two deoxyribonucleotides selected from dA, dT, dG, and dC. According to a preferred embodiment, the siRNA has a structure selected from the group consisting of: SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 21 and SEQ ID NO: 22 No.: SEQ ID NO: 39 and SEQ ID NO: 40; SEQ ID NO: 41 and SEQ ID NO: 42; and SEQ ID NO: 43 and SEQ ID NO: 44.
根据一个优选的实施方案，其中所述肿瘤选自由以下组成的组：胶质瘤、白血病、脑癌、食道癌、胃癌、肺癌、肝癌、膀胱癌、胰腺癌、宫颈癌、头颈癌、卵巢癌、黑色素瘤、淋巴瘤、乳腺癌、肠癌、鼻咽癌、子宫内膜癌、前列腺癌。According to a preferred embodiment, the tumor is selected from the group consisting of: glioma, leukemia, brain cancer, esophageal cancer, gastric cancer, lung cancer, liver cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, breast cancer, intestinal cancer, nasopharyngeal cancer, endometrial cancer, and prostate cancer.
在一个实施方案中，其中所述肿瘤选自由脑癌、肺癌和肝癌组成的组。In one embodiment, wherein the tumor is selected from the group consisting of brain cancer, lung cancer and liver cancer.
本发明所述siRNA、组合物及其应用的优异技术效果主要在于以下几个方面：The excellent technical effects of the siRNA, composition and application thereof of the present invention are mainly in the following aspects:
(1)特异性高：通过设计不同的siRNA分别靶向AURKB基因的两种单倍型序列，可以特异的抑制表达该单倍型的肿瘤细胞，并且所述两种单倍型H1和H2中的两个SNP相隔仅7bp。因此，靶向该区域，其可以提高siRNA的特异性，从而减轻对杂合性正常细胞的影响。(1) High specificity: By designing different siRNAs to target the two haplotype sequences of the AURKB gene, tumor cells expressing the haplotype can be specifically inhibited, and the two SNPs in the two haplotypes H1 and H2 are only 7 bp apart. Therefore, targeting this region can improve the specificity of siRNA, thereby reducing the impact on heterozygous normal cells.
(2)易开发、可重复使用：通过特异的靶向特定基因型的AURKB基因的表达，从而达到清除肿瘤细胞而不影响正常细胞功能的目的。该区域可成为新的siRNA药物靶向区域，用于抑制肿瘤生长，从而实现本发明的siRNA药物特异性好、效率高、易开发、可重复使用等目标。(2) Easy to develop and reusable: By specifically targeting the expression of the AURKB gene of a specific genotype, the purpose of eliminating tumor cells is achieved without affecting the function of normal cells. This region can become a new siRNA drug targeting region for inhibiting tumor growth, thereby achieving the goals of the siRNA drug of the present invention, such as good specificity, high efficiency, easy development, and reusability.
(3)副作用和毒性弱：小分子AURKB抑制剂不能分辨基因型而产生的毒性大，而本发明的siRNA可以区分两种单倍型H1和H2从而很好的解决上述问题。(3) Weak side effects and toxicity: Small molecule AURKB inhibitors cannot distinguish genotypes and thus produce high toxicity, while the siRNA of the present invention can distinguish between the two haplotypes H1 and H2, thus effectively solving the above problem.
附图简要说明BRIEF DESCRIPTION OF THE DRAWINGS
图1：AURKB在多种肿瘤中为必需基因。CAS-9值小于0表示抑制肿瘤生长，小于-3表示该基因敲除后肿瘤细胞快速死亡，该基因为必需基因；Figure 1: AURKB is an essential gene in many tumors. A CAS-9 value less than 0 indicates inhibition of tumor growth, and a value less than -3 indicates that tumor cells die rapidly after gene knockout, indicating that the gene is an essential gene.
图2：与正常组织表达比较，肝癌，肺癌，脑胶质瘤中AURKB都有显著的表达量上调；Figure 2: Compared with the expression in normal tissues, AURKB expression was significantly upregulated in liver cancer, lung cancer, and brain glioma;
图3：多个癌种中AURKB高表达患者总体生存预后差。其中图3A显示为领星数据；图3B显示为TCGA数据；Figure 3: Patients with high AURKB expression in multiple cancer types have poor overall survival prognosis. Figure 3A shows Lingxing data; Figure 3B shows TCGA data;
图4：AURKB(NM_004217)基因中新发现的siRNA可靶向区域位于17号染色体8205000-8205040之间(GRCh38.p14，NC_000017.11)。方框位置为两个SNP位点，包含在该区域内；Figure 4: The newly discovered siRNA targetable region in the AURKB (NM_004217) gene is located between 8205000-8205040 on chromosome 17 (GRCh38.p14, NC_000017.11). The boxed positions are two SNP sites contained in this region;
图5A-5C：siRNA转染结果；Figures 5A-5C: siRNA transfection results;
图6：Cell Titer-Glo(CTG)法细胞活力实验结果。Figure 6: Cell viability test results using the Cell Titer-Glo (CTG) method.
具体实施方式Detailed ways
实施例1：AURKB为多种肿瘤的必需基因 Example 1: AURKB is an essential gene for multiple tumors
根据肿瘤细胞CRISP-Cas9数据发现，AURKB是属于必需基因(包含在Achilles_common_essentials必需基因列表中)。另外，根据DepMap数据库CRISPR敲除试验数据证实，在脑癌、肺癌和肝癌细胞系中敲除AURKB后，CRISPR分数都为负数，且基本都小于-2(图1)，因此，AURKB基因在多种肿瘤(如肝细胞癌，肺癌以及脑癌等)中为必需基因。According to the CRISP-Cas9 data of tumor cells, AURKB is an essential gene (included in the Achilles_common_essentials essential gene list). In addition, according to the CRISPR knockout test data of the DepMap database, after knocking out AURKB in brain cancer, lung cancer and liver cancer cell lines, the CRISPR scores were all negative and basically less than -2 (Figure 1). Therefore, the AURKB gene is an essential gene in many tumors (such as hepatocellular carcinoma, lung cancer and brain cancer, etc.).
实施例2：在肝癌，肺癌，脑癌中，AURKB在肿瘤组织中表达量上调 Example 2: In liver cancer, lung cancer, and brain cancer, AURKB expression is upregulated in tumor tissues
TCGA数据显示在在肝癌和肺癌中，AURKB表达量均高于正常组织。在公开网站GEPIA2(http://gepia2.cancer-pku.cn/#analysis)中选择AURKB基因，选择相应的癌种，再选择Box Plot后，网页就可以显示肿瘤与正常组织的AURKB基因的表达对比图。与正常组织表达比较，肝癌，肺癌，脑胶质瘤中AURKB都有显著的表达量上调。表示AURKB与肿瘤发生有密切关系(图2)。TCGA data show that the expression of AURKB in liver cancer and lung cancer is higher than that in normal tissues. Select the AURKB gene in the public website GEPIA2 (http://gepia2.cancer-pku.cn/#analysis), select the corresponding cancer type, and then select Box Plot. The webpage can display the expression comparison chart of the AURKB gene in tumor and normal tissues. Compared with the expression in normal tissues, the expression of AURKB in liver cancer, lung cancer, and brain glioma is significantly upregulated. This indicates that AURKB is closely related to tumor occurrence (Figure 2).
实施例3：GC和TCGA数据显示，AURKB表达低的肝细胞癌，肺癌和脑癌患者整体生存较好 Example 3: GC and TCGA data show that patients with low AURKB expression in hepatocellular carcinoma, lung cancer and brain cancer have better overall survival
领星数据显示，中国脑癌和肝癌患者中，相比于AURKB基因表达高的患者，AURKB基因表达低的患者总体生存较好(图3A)。同时，TCGA数据(http://gepia2.cancer-pku.cn/#survival)显示肺癌、肝癌和脑癌的患者中也有相同趋势，AURKB基因表达低(以中位数分组)的患者总体生存均优于AURKB表达高的患者(图3B)。结果提示，敲低AURKB可能可以延长患者总生存期。According to the data from Lingxing, among Chinese patients with brain cancer and liver cancer, patients with low AURKB gene expression had better overall survival than those with high AURKB gene expression (Figure 3A). At the same time, TCGA data (http://gepia2.cancer-pku.cn/#survival) showed the same trend in patients with lung cancer, liver cancer, and brain cancer. The overall survival of patients with low AURKB gene expression (grouped by median) was better than that of patients with high AURKB expression (Figure 3B). The results suggest that knocking down AURKB may prolong the overall survival of patients.
实施例4：AURKB基因新的siRNA可靶向区域 Example 4: New siRNA targetable regions of the AURKB gene
在AURKB外显子上发现了SNP单倍型，在AURKB(NM_004217)基因中新发现的siRNA可靶向区域位于17号染色体8205000-8205040之间(GRCh38.p14，NC_000017.11)，该区域为全新的siRNA靶向区域(图4)，结果显示相距7bp的两个SNP可明显提高siRNA的抑制效率。SNP haplotypes were found in the AURKB exons. The newly discovered siRNA targetable region in the AURKB (NM_004217) gene is located between 8205000-8205040 on chromosome 17 (GRCh38.p14, NC_000017.11). This region is a completely new siRNA targeting region (Figure 4). The results showed that two SNPs 7 bp apart can significantly improve the inhibition efficiency of siRNA.
此外，领星中国人群基因数据显示，AURKB发生杂合性缺失的中国癌症人群比例大于20％，如下表1所示。In addition, the genetic data of the Chinese population from Lingxing showed that the proportion of Chinese cancer population with heterozygous loss of AURKB was greater than 20%, as shown in Table 1 below.
表1.Haplotype LOH人群比率Table 1. Haplotype LOH population ratio
肝细胞癌Hepatocellular carcinoma 肺癌Lung cancer 脑癌Brain cancer
27.8％27.8% 23.4％23.4% 21.2％21.2%
实施例5：siRNA的设计与抑制效率 Example 5: siRNA design and inhibition efficiency
根据靶向区域与SNP信息，设计一系列可以覆盖两个基因型的siRNA并进行了转染试验，观察其干扰效率。Based on the targeted region and SNP information, a series of siRNAs that can cover the two genotypes were designed and transfection experiments were carried out to observe their interference efficiency.
针对该区域设计的一系列上述siRNA，对其进行筛选，得到下述敲低效率好的siRNA，如表2所示，其为针对单倍型H1(CGTGCCCAT)(SEQ ID NO.45)设计的系列siRNA序列；如表3所示，其为针对单倍型H2(AGTGCCCAG)(SEQ ID NO.46)设计的系列siRNA序列。A series of the above siRNAs designed for this region were screened to obtain the following siRNAs with good knockdown efficiency, as shown in Table 2, which are a series of siRNA sequences designed for haplotype H1 (CGTGCCCAT) (SEQ ID NO.45); as shown in Table 3, which are a series of siRNA sequences designed for haplotype H2 (AGTGCCCAG) (SEQ ID NO.46).
表2针对单倍型H1(CGTGCCCAT)的siRNA序列Table 2 siRNA sequences targeting haplotype H1 (CGTGCCCAT)
Figure PCTCN2022126237-appb-000001
Figure PCTCN2022126237-appb-000001
表3针对单倍型H2(CAGTGCCCAGG)的siRNA序列Table 3 siRNA sequences targeting haplotype H2 (CAGTGCCCAGG)
Figure PCTCN2022126237-appb-000002
Figure PCTCN2022126237-appb-000002
Figure PCTCN2022126237-appb-000003
Figure PCTCN2022126237-appb-000003
为了验证各个siRNA的干扰效率，选择3株肝细胞癌细胞系代表三种常见的基因型：HepG2(H1型)，Huh-6(杂合型，包含H1和H2两种基因型)和SNU387(H2型)。将3株细胞分别转染表3和表4中的siRNA以及阴性对照siRNA(锐博生物)和阳性对照siRNA(锐博生物)。转染使用Lipofectamine TM RNAiMAXP(Thermo Fisher Scientific)试剂，转染操作按照Lipofectamine RNAiMAX推荐步骤进行反向转染试验。转染24小时后收集细胞，采用qRT-PCR的方法检测siRNA的干扰效率。如图5所示，不同siRNA有着不同的干扰效率。靶向H1型的siRNA si-1#和si-2#在携带H1的HepG2细胞中干扰效率最好，可以抑制60％的基因表达，其余的如si-5#、si-6#、si-7#、si-8#和si-11#能达到40％的抑制效率。si-1#和si-2#在杂合的携带H1和H2的Huh-6中的抑制效率为50％，在携带H2的SNU387中只能 抑制大约20％。靶向H2型的siRNA中si-A6#可以抑制SNU387约40％的基因表达，而在HepG2和Huh6中抑制率变差。实验结果证实了siRNA对不同基因型表达的特异性抑制。 In order to verify the interference efficiency of each siRNA, three hepatocellular carcinoma cell lines were selected to represent three common genotypes: HepG2 (H1 type), Huh-6 (heterozygous, including two genotypes, H1 and H2) and SNU387 (H2 type). The three cell lines were transfected with siRNAs in Tables 3 and 4, as well as negative control siRNA (Reibo Bio) and positive control siRNA (Reibo Bio). Lipofectamine TM RNAiMAXP (Thermo Fisher Scientific) reagent was used for transfection, and the transfection operation was performed according to the recommended steps of Lipofectamine RNAiMAX for reverse transfection experiments. Cells were collected 24 hours after transfection, and the interference efficiency of siRNA was detected by qRT-PCR. As shown in Figure 5, different siRNAs have different interference efficiencies. siRNAs si-1# and si-2# targeting H1 type have the best interference efficiency in HepG2 cells carrying H1, which can inhibit 60% of gene expression, while the rest, such as si-5#, si-6#, si-7#, si-8# and si-11#, can achieve 40% inhibition efficiency. The inhibition efficiency of si-1# and si-2# in the heterozygous Huh-6 carrying H1 and H2 was 50%, and it could only inhibit about 20% in SNU387 carrying H2. Among the siRNAs targeting H2, si-A6# could inhibit about 40% of the gene expression in SNU387, while the inhibition rate became worse in HepG2 and Huh6. The experimental results confirmed the specific inhibition of siRNA on the expression of different genotypes.
实施例6：siRNA靶向该区域可以有效抑制AURKB基因在肿瘤细胞中的表达，并抑制肿瘤的生长，而对杂合细胞生长没有影响 Example 6: siRNA targeting this region can effectively inhibit the expression of AURKB gene in tumor cells and inhibit tumor growth, but has no effect on the growth of hybrid cells
选择3个siRNA(si-1#，si-2#，si-A6#)进行CellTiter-Glo(CTG)法生长实验，观察AURKB干扰对各个细胞生长的影响。试验步骤参照CellTiter-Glo手册推荐方法，分别与转染后第1,2,3,4天对细胞进行检测。结果如图6所示，si-1#和si-2#可以显著抑制携带H1型的HepG2的生长，si-A6#显著抑制携带H2型的细胞株SNU387的生长，但是在杂合型细胞株Huh-6中，三个siRNA均不能显著影响细胞生长。实验结果表明，靶向本专利描述区域可以特异的抑制肿瘤的生长。对于发生LOH的肿瘤细胞，siRNA可以有效的抑制AURKB基因的表达，并抑制肿瘤细胞的生长，对未发生LOH的杂合型正常细胞，siRNA不影响其正常生长。Three siRNAs (si-1#, si-2#, si-A6#) were selected for the CellTiter-Glo (CTG) growth experiment to observe the effect of AURKB interference on the growth of each cell. The experimental steps refer to the method recommended in the CellTiter-Glo manual, and the cells were tested on days 1, 2, 3, and 4 after transfection. The results are shown in Figure 6. si-1# and si-2# can significantly inhibit the growth of HepG2 carrying H1 type, and si-A6# significantly inhibits the growth of the cell line SNU387 carrying H2 type, but in the heterozygous cell line Huh-6, the three siRNAs cannot significantly affect cell growth. The experimental results show that targeting the region described in this patent can specifically inhibit tumor growth. For tumor cells with LOH, siRNA can effectively inhibit the expression of the AURKB gene and inhibit the growth of tumor cells. For heterozygous normal cells without LOH, siRNA does not affect their normal growth.
应当理解，尽管本发明已根据其优选实施例进行了示例性描述，但不应限于上述实施例，对于本领域的技术人员来说，本发明可以有各种更改和变化。靶向AURKB基因H1/H2单倍型的siRNA的序列变换及核苷酸修饰等可以根据具体的需要进行相应的调整和改变。因此对于本领域技术人员来说，在不脱离本发明的构思和原则之内，还可做出若干简单替换，这些均应包含在本发明的保护范围之内。It should be understood that although the present invention has been described exemplarily according to its preferred embodiments, it should not be limited to the above embodiments. For those skilled in the art, the present invention may have various modifications and variations. The sequence transformation and nucleotide modification of the siRNA targeting the AURKB gene H1/H2 haplotype can be adjusted and changed accordingly according to specific needs. Therefore, for those skilled in the art, several simple substitutions can be made without departing from the concept and principle of the present invention, which should all be included in the scope of protection of the present invention.

Claims (34)

  1. siRNA，其靶向包含由SEQ ID NO：45或SEQ ID NO：46所示的序列编码的mRNA片段。siRNA that targets an mRNA fragment encoded by the sequence shown by SEQ ID NO: 45 or SEQ ID NO: 46.
  2. 如权利要求1所述的siRNA，其中至少一条链由长度为19-30个核苷酸的核苷酸链构成，优选由长度为19-29个、19-28个、19-27个、19-26个、19-25个核苷酸的核苷酸链构成。The siRNA as claimed in claim 1, wherein at least one chain consists of a nucleotide chain with a length of 19-30 nucleotides, preferably a nucleotide chain with a length of 19-29, 19-28, 19-27, 19-26, or 19-25 nucleotides.
  3. 如权利要求1所述的siRNA，其中所述siRNA至少包含一个修饰的核苷酸。The siRNA of claim 1, wherein the siRNA comprises at least one modified nucleotide.
  4. 如权利要求1所述的siRNA，其中所述siRNA每条链的3’端修饰有悬垂碱基。The siRNA according to claim 1, wherein the 3' end of each strand of the siRNA is modified with an overhanging base.
  5. 如权利要求4所述的siRNA，其中所述悬垂碱基由dA、dT、dG、dC中任意一个或多个脱氧核糖核苷酸组成。The siRNA according to claim 4, wherein the overhanging bases consist of any one or more deoxyribonucleotides selected from dA, dT, dG, and dC.
  6. 如权利要求1所述的siRNA，其中所述siRNA具有选自由以下组成的组的结构：The siRNA of claim 1, wherein the siRNA has a structure selected from the group consisting of:
    SEQ ID NO：1和SEQ ID NO：2；SEQ ID NO: 1 and SEQ ID NO: 2;
    SEQ ID NO：3和SEQ ID NO：4；SEQ ID NO: 3 and SEQ ID NO: 4;
    SEQ ID NO：5和SEQ ID NO：6；SEQ ID NO: 5 and SEQ ID NO: 6;
    SEQ ID NO：7和SEQ ID NO：8；SEQ ID NO: 7 and SEQ ID NO: 8;
    SEQ ID NO：9和SEQ ID NO：10；SEQ ID NO: 9 and SEQ ID NO: 10;
    SEQ ID NO：11和SEQ ID NO：12；SEQ ID NO: 11 and SEQ ID NO: 12;
    SEQ ID NO：13和SEQ ID NO：14；SEQ ID NO: 13 and SEQ ID NO: 14;
    SEQ ID NO：15和SEQ ID NO：16；SEQ ID NO: 15 and SEQ ID NO: 16;
    SEQ ID NO：17和SEQ ID NO：18；SEQ ID NO: 17 and SEQ ID NO: 18;
    SEQ ID NO：19和SEQ ID NO：20；SEQ ID NO: 19 and SEQ ID NO: 20;
    SEQ ID NO：21和SEQ ID NO：22；SEQ ID NO: 21 and SEQ ID NO: 22;
    SEQ ID NO：23和SEQ ID NO：24；SEQ ID NO: 23 and SEQ ID NO: 24;
    SEQ ID NO：25和SEQ ID NO：26；SEQ ID NO: 25 and SEQ ID NO: 26;
    SEQ ID NO：27和SEQ ID NO：28；SEQ ID NO: 27 and SEQ ID NO: 28;
    SEQ ID NO：29和SEQ ID NO：30；SEQ ID NO: 29 and SEQ ID NO: 30;
    SEQ ID NO：31和SEQ ID NO：32；SEQ ID NO: 31 and SEQ ID NO: 32;
    SEQ ID NO：33和SEQ ID NO：34；SEQ ID NO: 33 and SEQ ID NO: 34;
    SEQ ID NO：35和SEQ ID NO：36；SEQ ID NO: 35 and SEQ ID NO: 36;
    SEQ ID NO：37和SEQ ID NO：38；SEQ ID NO: 37 and SEQ ID NO: 38;
    SEQ ID NO：39和SEQ ID NO：40；SEQ ID NO: 39 and SEQ ID NO: 40;
    SEQ ID NO：41和SEQ ID NO：42；和SEQ ID NO: 41 and SEQ ID NO: 42; and
    SEQ ID NO：43和SEQ ID NO：44。SEQ ID NO: 43 and SEQ ID NO: 44.
  7. 一种载体，包含如权利要求1-6中任一项所述的siRNA。A vector comprising the siRNA according to any one of claims 1 to 6.
  8. 一种细胞，包含如权利要求1-6中任一项所述的siRNA。A cell comprising the siRNA according to any one of claims 1 to 6.
  9. 一种用于抑制肿瘤生长药物组合物，包含权利要求1-6中任一项所述的siRNA和药学上可接受的赋形剂。A pharmaceutical composition for inhibiting tumor growth, comprising the siRNA according to any one of claims 1 to 6 and a pharmaceutically acceptable excipient.
  10. 如权利要求9所述的药物组合物，其中所述肿瘤选自由以下组成的组：胶质瘤、白血病、脑癌、食道癌、胃癌、肺癌、肝癌、膀胱癌、胰腺癌、宫颈癌、头颈癌、卵巢癌、黑色素瘤、淋巴瘤、乳腺癌、肠癌、鼻咽癌、子宫内膜癌、前列腺癌。The pharmaceutical composition of claim 9, wherein the tumor is selected from the group consisting of glioma, leukemia, brain cancer, esophageal cancer, gastric cancer, lung cancer, liver cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, breast cancer, intestinal cancer, nasopharyngeal cancer, endometrial cancer, and prostate cancer.
  11. 如权利要求9所述的组合物，其中所述肿瘤选自由脑癌、肺 癌和肝癌组成的组。The composition of claim 9, wherein the tumor is selected from the group consisting of brain cancer, lung cancer and liver cancer.
  12. 一种用于抑制肿瘤生长的药物组合物，其包含抑制AURKB表达的siRNA和药学上可接受的赋形剂。A pharmaceutical composition for inhibiting tumor growth comprises siRNA for inhibiting AURKB expression and a pharmaceutically acceptable excipient.
  13. 如权利要求12所述的药物组合物，其中所述肿瘤选自由以下组成的组：胶质瘤、白血病、脑癌、食道癌、胃癌、肺癌、肝癌、膀胱癌、胰腺癌、宫颈癌、头颈癌、卵巢癌、黑色素瘤、淋巴瘤、乳腺癌、肠癌、鼻咽癌、子宫内膜癌、前列腺癌。The pharmaceutical composition of claim 12, wherein the tumor is selected from the group consisting of glioma, leukemia, brain cancer, esophageal cancer, gastric cancer, lung cancer, liver cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, breast cancer, intestinal cancer, nasopharyngeal cancer, endometrial cancer, and prostate cancer.
  14. 如权利要求13所述的组合物，其中所述肿瘤选自由脑癌、肺癌和肝癌组成的组。The composition of claim 13, wherein the tumor is selected from the group consisting of brain cancer, lung cancer and liver cancer.
  15. 降低靶细胞中AURKB表达的方法，所述方法通过施用权利要求1-6中任一项所述的siRNA来进行。A method for reducing AURKB expression in a target cell, the method being performed by administering the siRNA according to any one of claims 1 to 6.
  16. 一种抑制肿瘤生长的方法，所述方法通过向有此需要的受试者施用siRNA来进行，所述siRNA靶向由包含SEQ ID NO：45或SEQ ID NO：46所示的序列编码的mRNA片段。A method for inhibiting tumor growth, the method being performed by administering siRNA to a subject in need thereof, wherein the siRNA targets an mRNA fragment encoded by a sequence shown in SEQ ID NO: 45 or SEQ ID NO: 46.
  17. 如权利要求16所述的方法，其中所述siRNA的至少一条链由长度为19-30个核苷酸的核苷酸链构成,优选由长度为19-29个、19-28个、19-27个、19-26个、19-25个核苷酸的核苷酸链构成。The method of claim 16, wherein at least one strand of the siRNA consists of a nucleotide chain with a length of 19-30 nucleotides, preferably a nucleotide chain with a length of 19-29, 19-28, 19-27, 19-26, or 19-25 nucleotides.
  18. 如权利要求16所述的方法，其中所述siRNA至少包含一个修饰的核苷酸。The method of claim 16, wherein the siRNA comprises at least one modified nucleotide.
  19. 如权利要求16所述的方法，其中所述siRNA每条链的3’端 修饰有悬垂碱基。The method of claim 16, wherein the 3' end of each strand of the siRNA is modified with an overhanging base.
  20. 如权利要求16所述的方法，其中所述悬垂碱基由dA、dT、dG、dC中任意一个或多个脱氧核糖核苷酸组成。The method of claim 16, wherein the overhanging bases consist of any one or more deoxyribonucleotides of dA, dT, dG, and dC.
  21. 如权利要求16所述的方法，其中所述siRNA具有选自由以下组成的组的结构：The method of claim 16, wherein the siRNA has a structure selected from the group consisting of:
    SEQ ID NO：1和SEQ ID NO：2；SEQ ID NO: 1 and SEQ ID NO: 2;
    SEQ ID NO：3和SEQ ID NO：4；SEQ ID NO: 3 and SEQ ID NO: 4;
    SEQ ID NO：5和SEQ ID NO：6；SEQ ID NO: 5 and SEQ ID NO: 6;
    SEQ ID NO：7和SEQ ID NO：8；SEQ ID NO: 7 and SEQ ID NO: 8;
    SEQ ID NO：9和SEQ ID NO：10；SEQ ID NO: 9 and SEQ ID NO: 10;
    SEQ ID NO：11和SEQ ID NO：12；SEQ ID NO: 11 and SEQ ID NO: 12;
    SEQ ID NO：13和SEQ ID NO：14；SEQ ID NO: 13 and SEQ ID NO: 14;
    SEQ ID NO：15和SEQ ID NO：16；SEQ ID NO: 15 and SEQ ID NO: 16;
    SEQ ID NO：17和SEQ ID NO：18；SEQ ID NO: 17 and SEQ ID NO: 18;
    SEQ ID NO：19和SEQ ID NO：20；SEQ ID NO: 19 and SEQ ID NO: 20;
    SEQ ID NO：21和SEQ ID NO：22；SEQ ID NO: 21 and SEQ ID NO: 22;
    SEQ ID NO：23和SEQ ID NO：24；SEQ ID NO: 23 and SEQ ID NO: 24;
    SEQ ID NO：25和SEQ ID NO：26；SEQ ID NO: 25 and SEQ ID NO: 26;
    SEQ ID NO：27和SEQ ID NO：28；SEQ ID NO: 27 and SEQ ID NO: 28;
    SEQ ID NO：29和SEQ ID NO：30；SEQ ID NO: 29 and SEQ ID NO: 30;
    SEQ ID NO：31和SEQ ID NO：32；SEQ ID NO: 31 and SEQ ID NO: 32;
    SEQ ID NO：33和SEQ ID NO：34；SEQ ID NO: 33 and SEQ ID NO: 34;
    SEQ ID NO：35和SEQ ID NO：36；SEQ ID NO: 35 and SEQ ID NO: 36;
    SEQ ID NO：37和SEQ ID NO：38；SEQ ID NO: 37 and SEQ ID NO: 38;
    SEQ ID NO：39和SEQ ID NO：40；SEQ ID NO: 39 and SEQ ID NO: 40;
    SEQ ID NO：41和SEQ ID NO：42；和SEQ ID NO: 41 and SEQ ID NO: 42; and
    SEQ ID NO：43和SEQ ID NO：44。SEQ ID NO: 43 and SEQ ID NO: 44.
  22. 如权利要求16所述的方法，其中所述肿瘤选自由以下组成的组：胶质瘤、白血病、脑癌、食道癌、胃癌、肺癌、肝癌、膀胱癌、胰腺癌、宫颈癌、头颈癌、卵巢癌、黑色素瘤、淋巴瘤、乳腺癌、肠癌、鼻咽癌、子宫内膜癌、前列腺癌。The method of claim 16, wherein the tumor is selected from the group consisting of glioma, leukemia, brain cancer, esophageal cancer, gastric cancer, lung cancer, liver cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, breast cancer, intestinal cancer, nasopharyngeal cancer, endometrial cancer, and prostate cancer.
  23. 如权利要求22所述的方法，其中所述肿瘤选自由脑癌、肺癌和肝癌组成的组。The method of claim 22, wherein the tumor is selected from the group consisting of brain cancer, lung cancer and liver cancer.
  24. 抑制肿瘤生长的方法，所述方法包括向有此需要的受试者施用抑制AURKB表达的siRNA。A method of inhibiting tumor growth, comprising administering to a subject in need thereof an siRNA that inhibits AURKB expression.
  25. 如权利要求24所述的方法，其中所述肿瘤选自由以下组成的组：胶质瘤、白血病、脑癌、食道癌、胃癌、肺癌、肝癌、膀胱癌、胰腺癌、宫颈癌、头颈癌、卵巢癌、黑色素瘤、淋巴瘤、乳腺癌、肠癌、鼻咽癌、子宫内膜癌、前列腺癌。The method of claim 24, wherein the tumor is selected from the group consisting of glioma, leukemia, brain cancer, esophageal cancer, gastric cancer, lung cancer, liver cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, breast cancer, intestinal cancer, nasopharyngeal cancer, endometrial cancer, and prostate cancer.
  26. 如权利要求25所述的方法，其中所述肿瘤选自由脑癌、肺癌和肝癌组成的组。The method of claim 25, wherein the tumor is selected from the group consisting of brain cancer, lung cancer and liver cancer.
  27. 一种siRNA在制备抑制肿瘤生长的药物中的应用，所述siRNA靶向由包含SEQ ID NO：45或SEQ ID NO：46所示的序列编码的mRNA片段。A use of siRNA in the preparation of a drug for inhibiting tumor growth, wherein the siRNA targets an mRNA fragment encoded by a sequence shown in SEQ ID NO: 45 or SEQ ID NO: 46.
  28. 如权利要求27所述的应用，其中所述siRNA的至少一条链由长度为19-30个核苷酸的核苷酸链构成,优选由长度为19-29个、19-28个、19-27个、19-26个、19-25个核苷酸的核苷酸链构成。The use as claimed in claim 27, wherein at least one chain of the siRNA is composed of a nucleotide chain with a length of 19-30 nucleotides, preferably a nucleotide chain with a length of 19-29, 19-28, 19-27, 19-26, or 19-25 nucleotides.
  29. 如权利要求27所述的应用，其中所述siRNA至少包含一个修饰的核苷酸。The use according to claim 27, wherein the siRNA comprises at least one modified nucleotide.
  30. 如权利要求27所述的应用，其中所述siRNA每条链的3’端修饰有悬垂碱基。The use as claimed in claim 27, wherein the 3' end of each strand of the siRNA is modified with a pendant base.
  31. 如权利要求27所述的应用，其中所述悬垂碱基由dA、dT、dG、dC中任意一个或多个脱氧核糖核苷酸组成。The use according to claim 27, wherein the overhanging bases are composed of any one or more deoxyribonucleotides of dA, dT, dG, and dC.
  32. 如权利要求27所述的应用，其中所述siRNA具有选自由以下组成的组的结构：The use according to claim 27, wherein the siRNA has a structure selected from the group consisting of:
    SEQ ID NO：1和SEQ ID NO：2；SEQ ID NO: 1 and SEQ ID NO: 2;
    SEQ ID NO：3和SEQ ID NO：4；SEQ ID NO: 3 and SEQ ID NO: 4;
    SEQ ID NO：5和SEQ ID NO：6；SEQ ID NO: 5 and SEQ ID NO: 6;
    SEQ ID NO：7和SEQ ID NO：8；SEQ ID NO: 7 and SEQ ID NO: 8;
    SEQ ID NO：9和SEQ ID NO：10；SEQ ID NO: 9 and SEQ ID NO: 10;
    SEQ ID NO：11和SEQ ID NO：12；SEQ ID NO: 11 and SEQ ID NO: 12;
    SEQ ID NO：13和SEQ ID NO：14；SEQ ID NO: 13 and SEQ ID NO: 14;
    SEQ ID NO：15和SEQ ID NO：16；SEQ ID NO: 15 and SEQ ID NO: 16;
    SEQ ID NO：17和SEQ ID NO：18；SEQ ID NO: 17 and SEQ ID NO: 18;
    SEQ ID NO：19和SEQ ID NO：20；SEQ ID NO: 19 and SEQ ID NO: 20;
    SEQ ID NO：21和SEQ ID NO：22；SEQ ID NO: 21 and SEQ ID NO: 22;
    SEQ ID NO：23和SEQ ID NO：24；SEQ ID NO: 23 and SEQ ID NO: 24;
    SEQ ID NO：25和SEQ ID NO：26；SEQ ID NO: 25 and SEQ ID NO: 26;
    SEQ ID NO：27和SEQ ID NO：28；SEQ ID NO: 27 and SEQ ID NO: 28;
    SEQ ID NO：29和SEQ ID NO：30；SEQ ID NO: 29 and SEQ ID NO: 30;
    SEQ ID NO：31和SEQ ID NO：32；SEQ ID NO: 31 and SEQ ID NO: 32;
    SEQ ID NO：33和SEQ ID NO：34；SEQ ID NO: 33 and SEQ ID NO: 34;
    SEQ ID NO：35和SEQ ID NO：36；SEQ ID NO: 35 and SEQ ID NO: 36;
    SEQ ID NO：37和SEQ ID NO：38；SEQ ID NO: 37 and SEQ ID NO: 38;
    SEQ ID NO：39和SEQ ID NO：40；SEQ ID NO: 39 and SEQ ID NO: 40;
    SEQ ID NO：41和SEQ ID NO：42；和SEQ ID NO: 41 and SEQ ID NO: 42; and
    SEQ ID NO：43和SEQ ID NO：44。SEQ ID NO: 43 and SEQ ID NO: 44.
  33. 如权利要求27所述的应用，其中所述肿瘤选自由以下组成的组：胶质瘤、白血病、脑癌、食道癌、胃癌、肺癌、肝癌、膀胱癌、胰腺癌、宫颈癌、头颈癌、卵巢癌、黑色素瘤、淋巴瘤、乳腺癌、肠癌、鼻咽癌、子宫内膜癌、前列腺癌。The use as claimed in claim 27, wherein the tumor is selected from the group consisting of: glioma, leukemia, brain cancer, esophageal cancer, gastric cancer, lung cancer, liver cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, breast cancer, intestinal cancer, nasopharyngeal cancer, endometrial cancer, and prostate cancer.
  34. 如权利要求33所述的应用，其中所述肿瘤选自由脑癌、肺癌和肝癌组成的组。The use according to claim 33, wherein the tumor is selected from the group consisting of brain cancer, lung cancer and liver cancer.
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