WO2021037265A1 - 一种抑制MCM7基因表达的siRNA、组合物及其应用 - Google Patents

一种抑制MCM7基因表达的siRNA、组合物及其应用 Download PDF

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WO2021037265A1
WO2021037265A1 PCT/CN2020/112563 CN2020112563W WO2021037265A1 WO 2021037265 A1 WO2021037265 A1 WO 2021037265A1 CN 2020112563 W CN2020112563 W CN 2020112563W WO 2021037265 A1 WO2021037265 A1 WO 2021037265A1
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cancer
sirna
cells
mcm7
composition
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French (fr)
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梁纯
王俊
张文熙
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恩智（广州）医药科技有限公司
恩康药业科技（广州）有限公司
佛山英特医药科技有限公司
广州英特基因科技有限公司
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Priority to EP20856954.1A priority Critical patent/EP4023229A4/en
Priority to US17/639,331 priority patent/US20220325283A1/en
Publication of WO2021037265A1 publication Critical patent/WO2021037265A1/zh

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  • the invention belongs to the field of biomedicine, and particularly relates to a siRNA, a composition and an application thereof for inhibiting the expression of MCM7 gene.
  • the MCM complex is composed of MCM2-MCM7 subunits, has helicase activity in the cell, opens the DNA double strand before DNA replication, and participates in the initiation of DNA replication (Bik Tye, Annual Review of Biochemistry, 1999). Moreover, the MCM complex also plays an important regulatory role in cell proliferation, DNA damage repair and cell cycle.
  • siRNA Small interfering RNA
  • RNA interference RNA interference
  • siRNA Since different sites on the target gene have different secondary structures (binary results) and different thermodynamic properties due to different sequences, the possibility and degree of interference by siRNA at different sites will vary greatly. In addition, the same siRNA may have different activities in different types of cells. Therefore, for any target gene in any cell, the design, testing and acquisition of highly active siRNA is a process of discovery and invention.
  • siRNA used to inhibit the MCM7 gene of cancer cells such as liver cancer, gastric cancer, prostate cancer, etc.
  • the primary objective of the present invention is to provide an siRNA that inhibits the expression of MCM7 gene.
  • Another object of the present invention is to provide applications of the above-mentioned siRNA.
  • Another object of the present invention is to provide a method for siRNA to specifically target MCM7 protein to treat cancer.
  • RNA interference fragments for the MCM7 gene, but most of the siRNAs have low interference efficiency and cannot effectively carry out late-stage tumor treatment research.
  • the inventor designed siRNA with the MCM7 gene as a target.
  • the siRNA inhibits the expression of human MCM7 gene to inhibit the synthesis of MCM7 protein, thereby hindering the formation of the entire MCM complex (MCM2-MCM7), thereby inhibiting DNA replication and cell proliferation to achieve the purpose of cancer prevention or treatment.
  • Inhibition of the expression of any subunit of the MCM complex by siRNA can inhibit the formation of the complex, thereby inhibiting cell proliferation and producing anti-tumor effects.
  • a siRNA which can inhibit the expression of MCM7 gene and is composed of a sense strand and an antisense strand,
  • siRNA is selected from:
  • siRNA-1 sense strand: 5'-GUGGAGAAUUGACCUUAGA-3' (SEQ ID NO: 9);
  • Antisense strand 5'-UCUAAGGUCAGUUCUCCAC-3' (SEQ ID NO: 10);
  • nucleotides can use conventional methods in the art Modification or modification, such as 2'-OH in one or more nucleotides is changed to 2'-methoxy, sulfur is substituted for the central oxygen atom of phosphate, or the sense strand or antisense strand is added with cholesterol, etc. , And have the same or similar functions and activities.
  • deoxyribonucleotides dT or dN in a single-stranded suspension structure need to be added to the 3'ends of the sense strand and antisense strand of the siRNA-1.
  • the siRNA prevents or treats diseases by inhibiting the expression of MCM7 gene, and the diseases are preferably tumors/cancers.
  • the tumor/cancer is selected from liver cancer, stomach cancer, prostate cancer, breast cancer, lung cancer, pancreatic cancer, cervical cancer, endometrial cancer, colorectal cancer, lung cancer, nasopharyngeal cancer, ovarian cancer, skin cancer, and esophageal cancer Or brain tumor.
  • the tumor may slow down or stop growing, shrink or disappear due to the inhibition of MCM7 gene expression.
  • the MCM7 gene is selected from the human MCM7 gene.
  • sequence of the siRNA sequence can be modified or modified at part or all of the nucleotide positions, such as 2'-OH being changed to 2'-methoxy, sulfur replacing the central oxygen atom of the phosphate, or The sense strand or antisense strand plus cholesterol, etc., as long as it does not affect its binding and inhibition to the target.
  • siRNA in yet another aspect of the present invention, the application of siRNA in the preparation of drugs or compositions for the prevention or treatment of tumors/cancers is proposed.
  • the tumor/cancer is selected from liver cancer, stomach cancer, prostate cancer, breast cancer, lung cancer, pancreatic cancer, cervical cancer, endometrial cancer, colorectal cancer, lung cancer, nasopharyngeal cancer, ovarian cancer, skin cancer, and esophageal cancer Or brain tumor.
  • the tumor/cancer may slow down or stop growing, shrink or disappear due to the inhibition of MCM7 gene expression.
  • the concentration of siRNA is 5 to 150 nM, preferably 10 to 100 nM, more preferably 15 to 60 nM, and most preferably 20 to 40 nM.
  • the siRNA can inhibit the expression of MCM7 protein and is composed of a sense strand and an antisense strand
  • siRNA is selected from:
  • siRNA-1 sense strand: 5'-GUGGAGAAUUGACCUUAGA-3' (SEQ ID NO: 9);
  • Antisense strand 5'-UCUAAGGUCAGUUCUCCAC-3' (SEQ ID NO: 10);
  • siRNA-1 has more than 80%, or better, more than 90% homology with the sense strand or antisense strand sequence of the siRNA-1; or one or more of its nucleotides can be modified by conventional methods in the art Or modification, for example, 2'-OH is changed to 2'-methoxy group, sulfur is substituted for the central oxygen atom of phosphate, or the sense strand or antisense strand is added with cholesterol, etc., and have the same or similar functions and activities. Further, two deoxyribonucleotides dT or dN in a single-stranded suspension structure need to be added to the 3'end of the siRNA.
  • the MCM7 gene is selected from the human MCM7 gene.
  • siRNA can be modified or modified at part or all of the nucleotide positions, such as 2'-OH being changed to 2'-methoxy, sulfur substituted for the central oxygen atom of phosphate, or the sense strand or Antisense chain plus cholesterol, etc., as long as it does not affect its binding and inhibition to the target.
  • a drug or composition for preventing or treating tumor/cancer includes:
  • an expression system that can express any of the siRNAs described above, such as a shRNA expression system;
  • the tumor/cancer is selected from liver cancer, stomach cancer, prostate cancer, breast cancer, lung cancer, pancreatic cancer, cervical cancer, endometrial cancer, colorectal cancer, lung cancer, nasopharyngeal cancer, ovarian cancer, skin cancer, and esophageal cancer Or brain tumor.
  • the tumor may slow down or stop growing, shrink or disappear due to the inhibition of MCM7 gene expression.
  • the concentration of siRNA is 5 to 150 nM, preferably 10 to 100 nM, more preferably 15 to 60 nM, and most preferably 20 to 40 nM.
  • the above-mentioned medicine or composition also includes
  • the pharmaceutically acceptable carriers and/or adjuvants include, but are not limited to, buffers, emulsifiers, suspending agents, stabilizers, preservatives, physiological salt, excipients, fillers, coagulants and blending agents, Surfactant, diffuser, defoamer.
  • the other active ingredients for the prevention or treatment of tumors include: chemotherapeutic agents, radiotherapy agents or antibody drugs.
  • 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, bacterial carrier plasmid Expression RNA method or virus expression RNA method.
  • the form of the siRNA drug or its composition is not particularly limited, and can be selected from any form of solid, liquid, gel, semi-liquid, and aerosol.
  • the MCM7 gene is selected from the human MCM7 gene.
  • siRNA can effectively inhibit the expression of MCM7 gene and the synthesis of MCM7 protein for the purpose of treating diseases, including tumors.
  • a method for inhibiting the expression of MCM7 gene comprises administering the siRNA of the present invention or the drug or composition of the present invention to an individual in need.
  • a method for preventing or treating tumors/cancers comprises administering the siRNA of the present invention or the medicament or composition of the present invention to an individual in need.
  • the tumor may slow down or stop growing, shrink or disappear due to the inhibition of MCM7 gene expression.
  • 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 the sense strand into the RNA interference complex protein, thereby increasing the efficiency of siRNA-1 to inhibit the target MCM7 gene.
  • the present invention provides a siRNA targeting MCM7 gene. Compared with conventional gene knockout technology, the present invention is simple to operate and has a short test period; the inhibitory effect of siRNA at the mRNA and protein level is as high as 90%, and the inhibitory efficiency is extremely high. Good specificity; at the same time, it can effectively inhibit the DNA replication, proliferation and cloning ability 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.
  • Figure 1 shows the silencing effect of MCM7-specific siRNA-1 at the mRNA level of HepG2 and Hep3B liver cancer cells MCM7.
  • Panels A and B of Figure 2 show the silencing effect of MCM7-specific siRNA-1 at the level of MCM7 protein in HepG2 and Hep3B liver cancer cells, where ⁇ -actin ( ⁇ -actin) is an internal reference protein.
  • Figure 3 is a fluorescence microscope image of EdU positive cells that inhibited DNA replication of HepG2 liver cancer cells by siRNA-1, where Figure 3A, C and E are respectively EdU positive cells of negative control cells after NC transfection of HepG2 liver cancer cells Figure, Hochst-transfected nuclear DNA image, EdU-positive cell fluorescence microscope image, and Hochst-transfected nuclear DNA image overlapped; Figure 3B, D, and F are the EdU-positive cells after siRNA-1 transfection of HepG2 liver cancer cells Fluorescence microscope image, Hochst stained cell nuclear DNA image, EdU positive cell fluorescence microscope image, and Hochst stained cell nuclear DNA image overlapped.
  • Figure 4 is a statistical diagram of the proportion of HepG2 cells that are positive for EdU incorporation.
  • Figures 5A to E are 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.
  • Figure 6 shows the results of siRNA-1 inhibiting the cloning of various cancer cells.
  • Figures A to E are respectively: HepG2 liver cancer cells, Hep3B liver cancer cells, SGC-7907 gastric cancer cells, and PC3 were transfected with siRNA-1 or negative control NC Comparison of the number of cancer cell clones after prostate cancer cells and MCF7 breast cancer cells.
  • Figure 7 shows the ability of siRNA-1 to inhibit the cloning of various cancer cells.
  • Figures A to E are respectively: HepG2 liver cancer cells, Hep3B liver cancer cells, SGC-7907 gastric cancer cells, and PC3 prostate cancer cells were transfected with siRNA-1 or negative control NC After cells and MCF7 breast cancer cells, the ratio of the total clonal area of cancer cells to the total area of the hole.
  • the reagents and instruments used in the following examples are all reagents and instruments known in the art and can be obtained commercially; the experimental methods used are all conventional methods. The recorded experimental content can undoubtedly implement the program and obtain the corresponding experimental results.
  • siRNA sequence (1 21 nucleotides) expressed by the human MCM7 gene transcript (NM_001278595.1), namely the sense strand and antisense strand of the siRNA.
  • the base sequence is:
  • siRNA-1 sense strand 5'-GUGGAGAAUUGACCUUAGA dTdT-3' (SEQ ID NO.1),
  • siRNA-1 antisense strand 5'-UCUAAGGUCAGUUCUCCAC dTdT-3' (SEQ ID NO. 2).
  • NC negative control RNA
  • Sense chain 5'-CUCUUAGCCAAUAUUCGCU dTdT-3' (SEQ ID NO.3);
  • Antisense strand 5'-AGCGAAUAUUGGCUAAGAGdTdT-3' (SEQ ID NO.4).
  • deoxyribonucleotides in a single-stranded suspension structure are added to the 3'ends of the siRNA sequence of the present invention and the control RNA sense strand and antisense strand to enhance the stability of the siRNA in vivo and in vitro, Prevent degradation by nucleases.
  • siRNA sequence of the present invention can be modified or modified at part or all of the nucleotide positions, as long as it does not affect its binding and inhibition to the target.
  • Lipofectamine RNAiMax is used as the transfection reagent, and the steps are in accordance with the operating procedures of Thermo Fisher Scientific.
  • the cell lines used are HepG2 (purchased from ATCC, ATCC HB-8065) and Hep3B liver cancer cell line (purchased from ATCC, ATCC HB-8064), SGC-7907 gastric cancer cell line (purchased from Shanghai Institute of Biological Sciences), PC3 prostate cancer cell line (purchased from ATCC, ATCC CRL-1435) and MCF7 breast cancer cell line (purchased from ATCC, ATCC HTB-22).
  • the transfection steps are as follows: the above-mentioned different cancer cell lines are respectively seeded in 12-well plates, and cultured overnight at 37°C and 5% CO 2 to make the cell growth density reach 40-50%.
  • the siRNA and negative control RNA (NC) prepared by the present invention were respectively transfected into different cells. After transfection, the cells were collected, and qRT-PCR and western blotting were used to further detect the interference effect of siRNA-1.
  • Methods Collect cells after transfection, extract RNA, perform real-time fluorescent quantitative PCR after reverse transcription, and detect the expression of MCM7 mRNA after siRNA-1 treatment of cancer cells.
  • the siRNA-1 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. After 24 hours of transfection, the cells were collected, and an appropriate amount of cells was re-transfected. Inoculated in a 6-well plate, and collected cells 72 hours later to extract total RNA. Reverse transcription, and perform fluorescence quantitative real-time PCR to detect MCM7 mRNA.
  • RNA is stored in the upper water phase
  • THUNDERBRID SYBR qPCR Mix After diluting the reverse-transcribed cDNA sample at an appropriate ratio, use THUNDERBRID SYBR qPCR Mix to configure the following PCR reaction system:
  • the sequence of the upstream primer is 5’-GTGAAGGATCCTGCGACACA-3’ (SEQ ID NO.5);
  • the downstream primer sequence is 5'-ACACGCGTTCTTTTGTTCCG-3' (SEQ ID NO.6);
  • sequence of the upstream primer of the internal reference is 5'-AGAAGAGCTACGAGCTGCCTGACG-3' (SEQ ID NO. 7);
  • the downstream primer sequence of the internal reference is 5'-GGACTCCATGCCCAGGAAGGAA-3' (SEQ ID NO. 8).
  • Figures 1A and B show that compared with the control group NC, siRNA-1 can effectively inhibit the expression of MCM7 mRNA in HepG2 and Hep3B liver cancer cells after transfecting cancer cells with a silencing effect of more than 90%.
  • the siRNA-1 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. After 24 hours of transfection, the cells were collected and an appropriate amount was taken. The cells were re-seeded in a 12-well plate, and 72 hours later, the cells were collected for western blotting.
  • Figures 2A and B show that, compared with the control group NC, siRNA-1 can effectively inhibit the expression of MCM7 protein in HepG2 and Hep3B cells after transfecting cancer cells, and the inhibitory effect can reach more than 90%.
  • the siRNA-1 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. After 24 hours of transfection, the cells were collected and an appropriate amount was taken. The cells were re-seeded in 96-well plates. After 12 hours, the mimosine reagent was added to the cells and incubated for 24 hours to synchronize the cells at the junction of G1 and S phases.
  • Figures 3 and 4 show that after siRNA-1 transfection of HepG2 liver cancer cells, the proportion of EdU incorporated into positive cells was significantly reduced compared with the negative control, indicating that siRNA-1 significantly inhibited the DNA replication of cancer cells.
  • the data of Hep3B cells is similar to that of HepG2 cells.
  • the fresh medium used Gibco RPMI 1640.
  • Example 6 MCM7 siRNA inhibits cancer cell proliferation
  • siRNA-1 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-seeded them in a 12-well plate, and count for five consecutive days, and select one well for cell count every day. Draw the cell growth curve after transfection.
  • 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.
  • Example 7 MCM7 siRNA inhibits cancer cell clone formation
  • the siRNA-1 and negative control RNA (NC) prepared by the present invention were respectively transfected into the above-mentioned different cancer cell lines, and the cells were collected 24 hours after transfection.
  • the cells were seeded in a 6-well plate with a cell density of 0.4 ⁇ 10 3 cells/well. After 14 days of culture, it was fixed with methanol and stained with crystal violet.
  • MCM7 siRNA of the present invention in the preparation of compounds for the prevention or treatment of tumors, said tumors/cancers selected from liver cancer, stomach 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.
  • said tumors/cancers selected from liver cancer, stomach 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.
  • 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 90% or more, 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 cloning formation is of great significance for the development of new anti-cancer gene compounds and improving the therapeutic effect of cancer, and has significant clinical application prospects and economic value.

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Abstract

一种抑制MCM7基因表达的siRNA、组合物及其应用，设计及验证的siRNA能高效抑制MCM7基因表达，从而抑制癌症细胞的DNA合成、细胞增殖和细胞克隆的生成，起到预防和治疗肿瘤的目的。为癌症预防或治疗提供了一种新的靶点和候选化合物。

Description

一种抑制MCM7基因表达的siRNA、组合物及其应用 技术领域
本发明属于生物医药领域，特别涉及一种抑制MCM7基因表达的siRNA、组合物及其应用。
背景技术
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。
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，不需要依赖点蛋白的晶体结构。因此科学家们研究了一系列利用RNA干扰(RNA interference，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基因治疗的方法。
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(二元结果)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、测试和获得是一个探索发明的过程。
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。
发明内容
本发明的首要目的在于提供一种抑制MCM7基因表达的siRNA。
本发明的另一目的在于提供上述siRNA的应用。
本发明的再一目的在于提供一种siRNA特异性靶向MCM7蛋白治疗癌症的方法。
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。发明人经过创造性探索研究，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，针对靶基因不同位点的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、两端自 由能大小、碱基分布等因素有关。
发明人进一步探索发现，通过改变所述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siRNA抑制靶标MCM7基因的效率。
为达到上述目的，本发明所采取的技术方案是：
发明人以MCM7基因为靶点设计了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癌症预防或治疗的目的。
通过siRNA对MCM复合体任何一个亚基的表达抑制，可以抑制复合体的形成，进而抑制细胞增殖产生抗肿瘤作用。
本发明一个方面，提出了一种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：
siRNA-1：正义链：5'-GUGGAGAAUUGACCUUAGA-3'(SEQ ID NO:9)；
反义链：5'-UCUAAGGUCAGUUCUCCAC-3'(SEQ ID NO:10)；
或与所述siRNA-1限定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；或其中的一个或多个核苷酸可以利用本领域的常规方法进行修饰或修改，如一个或多个核苷酸中的2’-OH改为2’-甲氧基，硫取代磷酸根的中心氧原子，或者所述正义链或反义链加上胆固醇等，且具有相同或相似的功能和活性。
进一步的，所述siRNA-1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。
进一步的，所述siRNA通过抑制MCM7基因表达而预防或治疗疾病，所述疾病优选为肿瘤/癌症。
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。
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。
进一步的，所述MCM7基因选自人MCM7基因。
进一步的，所述的siRNA序列，其序列可以在部分或全部核苷酸位点进行修饰或修改，如2’-OH改为2’-甲氧基，硫取代磷酸根的中心氧原子，或者所述正义链或反义链加上胆固醇等，只要不影响其对靶标的结合及抑制。
本发明的又一个方面，提出了siRNA在制备预防或治疗肿瘤/癌症的药物或组合物中的应 用。
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。
进一步的，所述肿瘤/癌症可因MCM7基因表达抑制而减慢或停止生长、缩小或消失。
进一步的，所述药物或组合物中，siRNA的浓度为5～150nM，优选10～100nM，更优选15～60nM，最优选20～40nM。
还可以具有下列附加技术特征：
所述siRNA可抑制MCM7蛋白表达，由正义链和反义链组成，
其中，所述siRNA选自：
siRNA-1：正义链：5'-GUGGAGAAUUGACCUUAGA-3'(SEQ ID NO:9)；
反义链：5'-UCUAAGGUCAGUUCUCCAC-3'(SEQ ID NO:10)；
或与所述siRNA-1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；或其中的一个或多个核苷酸可以利用本领域的常规方法修饰或修改，如2’-OH改为2’-甲氧基，硫取代磷酸根的中心氧原子，或者所述正义链或反义链加上胆固醇等，且具有相同或相似的功能和活性。进一步的，所述siRNA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。
进一步的，所述MCM7基因选自人MCM7基因。
进一步的，所述siRNA可以在部分或全部核苷酸位点进行修饰或修改，如2’-OH改为2’-甲氧基，硫取代磷酸根的中心氧原子，或者所述正义链或反义链加上胆固醇等，只要不影响其对靶标的结合及抑制。
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：
上述任一所述的siRNA；
或可表达上述任一所述的siRNA的表达体系，如shRNA表达系统；
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。
进一步的，所述肿瘤可因MCM7基因表达抑制而减慢或停止生长、缩小或消失。
进一步的，所述药物或组合物中，siRNA的浓度为5～150nM，优选10～100nM，更优选15～60nM，最优选20～40nM。
进一步的，上述药物或组合物，还包括
药学上可接受的载体和/或辅料；和/或
预防或治疗肿瘤的其它活性成分。
进一步的，所述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。
进一步的，所述预防或治疗肿瘤的其它活性成分包括：化疗剂、放疗剂或抗体药物。
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。
进一步的，所述siRNA药物或其组合物的形式无特殊限制，可以选自固体、液体、凝胶、半流质、气雾任一形式。
进一步的，所述MCM7基因选自人MCM7基因。
利用siRNA可以有效抑制MCM7基因表达和MCM7蛋白的合成，起到治疗疾病，包括肿瘤的目的。
本发明的又一个方面，提出了一种抑制MCM7基因表达的方法，包括向需要的个体施用本发明的siRNA或本发明的药物或组合物。
本发明的又一个方面，提出了一种预防或治疗肿瘤/癌症的方法，包括向需要的个体施用本发明的siRNA或本发明的药物或组合物。其中，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。
本发明的有益效果是：
本发明通过改变所述siRNA-1正义链的5’端顺数的第9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。
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。
附图说明
图1为MCM7特异性的siRNA-1在HepG2和Hep3B肝癌细胞MCM7 mRNA水平的沉默效果。
图2的A、B图为MCM7特异性的siRNA-1在HepG2和Hep3B肝癌细胞MCM7蛋白水平的沉默效果，其中β-actin(β-肌动蛋白)为内部参照蛋白。
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。
图4为EdU掺入显阳性的HepG2细胞比例统计图。
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乳腺癌细胞的增殖的生长曲线。
图6为siRNA-1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。
图7为siRNA-1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。
具体实施方式
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。
如无明确说明，以下实施例中使用的试剂和仪器，都是本领域已知试剂和仪器，可以通过商购方式获得；所使用的实验方法都是常规方法，本领域技术人员根据实施例中记载的实验内容可以毫无疑问地实施所述方案并获得相应的实验结果。
实施例1 siRNA设计
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：
siRNA-1正义链：5'-GUGGAGAAUUGACCUUAGA dTdT-3'(SEQ ID NO.1),
siRNA-1反义链：5'-UCUAAGGUCAGUUCUCCAC dTdT-3'(SEQ ID NO.2)。
阴性对照RNA(NC)的碱基序列为：
正义链：5'-CUCUUAGCCAAUAUUCGCU dTdT-3'(SEQ ID NO.3)；
反义链：5′-AGCGAAUAUUGGCUAAGAGdTdT-3'(SEQ ID NO.4)。
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。
本发明siRNA序列可以在部分或全部核苷酸位点进行修饰或修改，只要不影响其对靶标的结合及抑制。
实施例2 siRNA在细胞中的转染
采用脂质体Lipofectamine RNAiMax作为转染试剂，步骤按照Thermo Fisher Scientific公司的操作规程。
采用的细胞系为HepG2(购自ATCC，ATCC HB-8065)和Hep3B肝癌细胞系(购自ATCC，ATCC HB-8064)、SGC-7907胃癌细胞系(购自上海生科院细胞研究所)、PC3前列腺癌细胞系(购自ATCC，ATCC CRL-1435)和MCF7乳腺癌细胞系(购自ATCC，ATCC HTB-22)。
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。
转染步骤如下：将上述不同的癌细胞系分别接种于12孔板中，37℃、5％CO 2培养过夜，使细胞生长密度达到40～50％。参照Lipofectamine RNAiMax(Thermo Fisher Scientific)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。转染后收集细胞，采用qRT-PCR和蛋白印迹(western blotting)进一步检测siRNA-1的干扰效果。
实施例3 siRNA抑制MCM7 mRNA表达检测
方法：转染后收集细胞，提取RNA，逆转录后进行实时荧光定量PCR，检测siRNA-1处理癌细胞后MCM7 mRNA的表达量。
将本发明制备的siRNA-1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，并进行荧光定量实时PCR检测MCM7 mRNA。
1.总RNA提取
(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；
(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；
(3)加入0.2ml氯仿/ml TRIzol，剧烈振荡混匀15sec，室温静置10min；
(4)12000rpm，4℃，离心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；
(6)吸取上层水相至新的EP管中，注意避免将中间蛋白吸出；
(7)加入预冷的异丙醇0.5ml/ml TRIzol，颠倒混匀，室温静置10min；
(8)12000rpm，4℃，离心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；
(10)弃上清，超净台中鼓风干燥3分钟左右，RNA呈半透明状；
(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。
2.反转录成cDNA
高温预变形5min后冰上急冻，反转录反应体系：
Figure PCTCN2020112563-appb-000001
反应条件：37℃15min，50℃5min，98℃5min，4℃保持。合成的cDNA可立即用于下游实验或-20℃冰箱保存。
3.荧光定量实时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：
Figure PCTCN2020112563-appb-000002
其中：
上游引物序列为5’-GTGAAGGATCCTGCGACACA-3’(SEQ ID NO.5)；
下游引物序列为5’-ACACGCGTTCTTTTGTTCCG-3’(SEQ ID NO.6)；
内参上游引物序列为5’-AGAAGAGCTACGAGCTGCCTGACG-3’(SEQ ID NO.7)；
内参下游引物序列为5’-GGACTCCATGCCCAGGAAGGAA-3’(SEQ ID NO.8)。
结果：图1A和B表明，相对于对照组NC，siRNA-1转染癌细胞后，分别有效抑制HepG2、Hep3B肝癌细胞中的MCM7 mRNA表达量，沉默效果达90％以上。
实施例4 siRNA抑制MCM7蛋白表达检测
方法：将本发明制备的siRNA-1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。
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；
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；
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；
4、次日，TBST洗3遍，每遍10min；
5、选用与一抗宿主种属相对应的HRP标记的抗小鼠IgG二抗(Pierce)，以适当的稀释比例，与PVDF膜室温孵育1小时；
6、TBST洗3遍，每遍10min；
7、ECL液显影，检测MCM7蛋白在肿瘤细胞中的表达情况。
结果：图2A和B显示，相对于对照组NC，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，抑制效果可达90％以上。
实施例5 siRNA抑制癌细胞DNA复制
方法：将本发明制备的siRNA-1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。
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。
结果：图3和图4表明，siRNA-1转染HepG2肝癌细胞后，与阴性对照相比，EdU掺入阳性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。Hep3B细胞数据与HepG2细胞相似。
所述新鲜培养基采用的是：Gibco RPMI 1640。
实施例6 MCM7 siRNA抑制癌细胞增殖
方法：将本发明制备的siRNA-1和阴性对照RNA(NC)分别转染到不同的癌细胞系，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。
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。
实施例7 MCM7 siRNA抑制癌细胞克隆生成
方法：将本发明制备的siRNA-1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0.4×10 3个/孔。培养14天后，用甲醇固定，并使用结晶紫染色。
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。
实施例8 MCM7 siRNA应用
本发明MCM7 siRNA在制备预防或治疗肿瘤化合物中的应用，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。
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化合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。
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。

Claims (10)

  1. 一种siRNA，其特征在于，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：
    siRNA-1：正义链：5'-GUGGAGAAUUGACCUUAGA-3'(SEQ ID NO:9)；
    反义链：5'-UCUAAGGUCAGUUCUCCAC-3'(SEQ ID NO:10)；
    或与所述siRNA-1的正义链或反义链序列具有80％以上同源性，优选地90％以上同源性；或其中的一个或多个核苷酸可以进行修饰或修改，且具有相同或相似的功能和活性，优选地，一个或多个核苷酸中的2’-OH改为2’-甲氧基，硫取代磷酸根的中心氧原子，或者所述正义链或反义链加上胆固醇。
  2. 根据权利要求1所述的siRNA，其特征在于，所述siRNA-1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。
  3. 根据权利要求1或2所述siRNA在制备预防或治疗肿瘤/癌症的药物或组合物中的应用。
  4. 根据权利要求3所述的应用，其特征在于，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。
  5. 一种预防或治疗肿瘤的药物或组合物，其特征在于，所述药物或组合物包括：
    权利要求1或2所述siRNA；
    或可表达权利要求1或2所述siRNA的表达体系，优选为shRNA表达系统。
  6. 根据权利要求5所述的药物或组合物，其特征在于，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。
  7. 根据权利要求5所述的药物或组合物，其特征在于，还包括：
    药学上可接受的载体和/或辅料；和/或
    预防或治疗肿瘤的其它活性成分。
  8. 根据权利要求7所述的药物或组合物，所述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。
  9. 根据权利要求7所述的药物或组合物，其特征在于，所述预防或治疗肿瘤的其它活性成分包括：化疗剂、放疗剂或抗体药物。
  10. 根据权利要求5～9任一项所述的药物或组合物，其特征在于，所述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。
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