WO2023245771A1 - Helz2基因在肿瘤防治药物中的应用 - Google Patents

Helz2基因在肿瘤防治药物中的应用 Download PDF

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WO2023245771A1
WO2023245771A1 PCT/CN2022/105592 CN2022105592W WO2023245771A1 WO 2023245771 A1 WO2023245771 A1 WO 2023245771A1 CN 2022105592 W CN2022105592 W CN 2022105592W WO 2023245771 A1 WO2023245771 A1 WO 2023245771A1
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helz2
gene
seq
mice
drugs
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陈华标
李涛
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江苏康可得生物技术股份有限公司
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Definitions

  • the present invention relates to gene therapy drug preparation technology, specifically to the application of Helz2 gene in tumor prevention and treatment drugs.
  • Helz2 (Helicase with zinc finger 2) [OMIM 611265], also known as PRIC285 or PDIP1, is a 2649 amino acid nuclease protein and a peroxisome proliferator-activated receptor alpha interaction complex (Peroxisome proliferator activated receptor ⁇ interacting complex, part of PRIC). Helz2 consists of two ATP-binding sequences, an RNaseB domain and dual DNA/RNA helicase sequences, and is involved in a variety of mechanisms related to gene regulation, including gene transcription, mRNA processing and DNA repair. It acts as a nuclear transcriptional coactivator of PPAR ⁇ and PPAR ⁇ as well as other nuclear receptors (RXRA, THRA, THRB).
  • Helz2 is also involved in cellular lipid, carbohydrate metabolism and liver metabolic processes, and is closely related to adipocyte differentiation and primary biliary cirrhosis.
  • the expression of Helz2 is up-regulated in human and mouse non-alcoholic fatty liver disease (NAFLD), and its deletion activates the expression of liver long functional leptin receptor (Functional leptin receptor long form, Leprb). And inhibited NAFLD development and weight gain in obese mice. It has been reported that Helz2 binding partner PPAR ⁇ can regulate the expression of mouse inflammatory genes such as Ccl3, Ccl7, Cxcl10 and Tgtp.
  • PPAR ⁇ also plays an important role in the innate immune response and is associated with immune-related diseases, such as HCV/HIV infection, osteoarthritis, and acne.
  • Helz2 has the ability to bind proteins important in immune responses, such as BCL6 and ISG15.
  • Helz2 was found to have antiviral function in mammals, and the gene was identified as an interferon stimulated gene (ISG) with conserved components of antiviral immunity.
  • ISG interferon stimulated gene
  • a mediating role of Helz2 in the IFN antiviral response was discovered, specifically involving the upregulation of Helz2 transcription and nuclear protein, as well as the activation of the transcriptional program.
  • the CRISPR/Cas9 genome editing system is part of the bacterial adaptive immune system and is used to defend against invasive nucleic acids from phages and plasmids.
  • the CRISPR/Cas9 gene editing tool is not only powerful, but also has strong specificity and high efficiency, and can perform gene editing accurately and quickly. Although there are studies reporting the use of this technology to interfere with Helz2 gene expression at the cellular level. However, the CRISPR/Cas9 system has not been used to obtain homozygous Helz2 gene knockout mouse models and corresponding research applications. There is also no research and application of targeting the Helz2 gene or its expression products in tumor treatment.
  • RNA interference is one of the most powerful targeted therapies that inhibits specific gene expression at the post-transcriptional level.
  • siRNA has the following limitations: insufficient cellular uptake, poor stability, easy degradation in vivo and in vitro, and low delivery efficiency in clinical experiments.
  • viral vector-mediated shRNA has long-lasting and stable gene silencing capabilities.
  • the shRNA lentiviral vector is a type of recombinant retroviral vector that can stably express shRNA in various mammalian cells. Such vectors have been validated in clinical databases of relevant tumor animal models. Therefore, lentivirus-mediated shRNA strategy is an attractive candidate for tumor treatment.
  • the technical problem to be solved by the present invention is to provide a method for constructing a targeted Helz2 gene knockout animal model, as well as the application of targeted interference with the Helz2 gene in the preparation or screening of tumor drugs and in tumor prevention and treatment drugs.
  • the first aspect of the present invention is a method for constructing a Helz2 gene knockout mouse model, which uses CRISPR/Cas9 technology to knock out the Helz2 gene in mouse fertilized eggs, and then uses microinjection and breeding to obtain Homozygous mouse model with systemic gene knockout of Helz2 (Helz2-Cas9-KO). It provides an ideal animal model for studying the mechanism between the Helz2 gene and tumors, and also provides support for the application of the Helz2-Cas9-KO mouse model in the preparation or screening of drugs for the treatment and/or prevention of tumors.
  • the method for constructing the Helz2 gene knockout mouse model includes the following steps:
  • Step 1 Design and construct sgRNA targeting the sgRNA recognition sequence of the mouse Helz2 gene; after analyzing the Helz2 gene to determine the specific knockout region, design a pair of corresponding gRNA sequences based on the target gene and synthesize the sgRNA sequence; sgRNA The sequence includes,
  • SEQ ID No.1 5’-GCCCCAGAGTTACCAGATGGAGG-3’;
  • SEQ ID No.2 5’-CCTACACCCGACAGAGGTGTAGG-3’;
  • SEQ ID No.3 5’-AGCAGTGACAGTCTTATGGGTGG-3’;
  • step two the mRNA obtained in step one and the Cas9 plasmid are injected into mouse fertilized eggs by microinjection, and the F0 generation mice are obtained after culturing.
  • Step 3 Screen out positive F0 generation mice through PCR and sequencing identification
  • Step 4 F0 generation mice are sexually mature and bred to obtain positive F1 generation mice.
  • the second aspect of the present invention is the application of the Helz2 systemic gene knockout homozygous mouse model obtained by the above method in the preparation or screening of anti-tumor and/or tumor prevention drugs.
  • the applications include screening of drug targets, screening of drugs, pharmacodynamic evaluation of drugs, and safety evaluation of drugs.
  • the third aspect of the present invention is an sgRNA sequence used to construct a Helz2 gene knockout animal model, which includes one or more of the following SEQ ID No. 1-SEQ ID No. 4 gene sequences,
  • SEQ ID No.1 5’-GCCCCAGAGTTACCAGATGGAGG-3’;
  • SEQ ID No.2 5’-CCTACACCCGACAGAGGTGTAGG-3’;
  • SEQ ID No.3 5’-AGCAGTGACAGTCTTATGGGTGG-3’;
  • the fourth aspect of the present invention is a vector containing the Helz2 gene targeting, and the target sequence for interfering with the Helz2 gene is one or more of the following gene sequences:
  • SEQ ID No.6 5’-GCACGATGCTGTATGGCTTTG-3’;
  • the vector is one of lentiviral vector complex, adenovirus, adeno-associated virus, N-acetylgalactosamine (GalNAc), liposomes (LNPs), polymers (Polymers), oncolytic viruses, or other biological acceptable gene vectors.
  • the fifth aspect of the present invention is the use of the above-mentioned vector containing the Helz2 gene targeting in the preparation of biological agents that inhibit the expression of the Helz2 gene.
  • the sixth aspect of the present invention is a drug for treating and/or preventing tumors, which contains a product targeting the Helz2 gene or its expression.
  • the present invention provides Helz2, a key target for potential treatment and/or prevention of tumors, and constructs a Helz2-Cas9-KO mouse model with the help of CRISPR/Cas9 gene technology and shRNA interference technology respectively and invents a method for targeting Helz2 gene to treat tumors. new treatments.
  • a Helz2-Cas9-KO mouse model with the help of CRISPR/Cas9 gene technology and shRNA interference technology respectively and invents a method for targeting Helz2 gene to treat tumors. new treatments.
  • the present invention can provide a reliable animal model for elucidating the mechanism of action of the Helz2 gene in the occurrence and development of tumors, and can also provide a target for clinical drug screening and preparation of the Helz2 gene for the treatment and/or prevention of tumors, and also provides a target A new therapy for treating tumors using the Helz2 gene has broad application prospects.
  • Figure 1 shows the strategic design diagram of Helz2-Cas9-KO mice.
  • FIG. 2 shows the electrophoresis results.
  • B6 is a negative control, which is B6 genomic DNA
  • N is a blank control, a control without template
  • TRANS 2K PLUS II bands 8000bp, 5000bp, 3000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp.
  • Figure 3 is the survival curve of mesothelioma AE17 tumor-bearing Helz2-Cas9-KO mice in Example 1.
  • Figure 4 is the survival curve of mesothelioma 40L tumor-bearing Helz2-Cas9-KO mice in Example 2.
  • Figure 5 is the survival curve of ovarian cancer ID8 tumor-bearing Helz2-Cas9-KO mice in Example 3.
  • Figure 6 shows the survival curve and tumor growth of lung cancer Lewis tumor-bearing Helz2-Cas9-KO mice in Example 4.
  • Figure 7 is the survival curve of breast cancer E0771 tumor-bearing Helz2-Cas9-KO mice in Example 5.
  • Figure 8 is the survival curve of wild-type mice bearing tumors in sh-NC-AE17 and sh-Helz2-AE17 cells in Example 7.
  • Figure 9 is a model diagram of sh-Helz2 treatment of AE17 tumor-bearing wild-type mice in Example 8.
  • Figure 10 is the survival curve of AE17 tumor-bearing wild-type mice treated with sh-Helz2 in Example 8.
  • Figure 11 shows the expression of Helz2 in pan-cancer in Example 9.
  • FIGS 1 and 2 show the Helz2-Cas9-KO mouse strategy design and electrophoresis identification results in the embodiments of the present invention.
  • Example 1 Establishment of mouse model of mesothelioma cell line AE17
  • mice aged 8 to 10 weeks were divided into two groups, namely wild-type mice and Helz2 knockout mice.
  • the mesothelioma cell line AE17 cells in the logarithmic growth phase were digested with trypsin, washed once with PBS, and the viable cell concentration was adjusted to contain 1 ⁇ 10 5 cells per 100 ⁇ l volume.
  • 100 ⁇ l of AE17 cell suspension was inoculated into the abdominal cavity of wild-type mice and Helz2 knockout mice respectively. Observe the mice every day.
  • mice aged 8 to 10 weeks were divided into two groups, namely wild-type mice and Helz2 knockout mice.
  • the mesothelioma cell line 40L cells in the logarithmic growth phase were digested with trypsin and washed once with PBS, and the viable cell concentration was adjusted to contain 2 ⁇ 10 5 cells per 100 ⁇ l volume.
  • 100 ⁇ l of 40L cell suspension was inoculated into the abdominal cavity of wild-type mice and Helz2 knockout mice respectively. Observe the mice every day.
  • Example 3 Establishment of mouse model of ovarian cancer cell line ID8
  • mice aged 8 to 10 weeks were divided into two groups, namely wild-type mice and Helz2 knockout mice.
  • the ovarian cancer cell line ID8 cells in the logarithmic growth phase were digested with trypsin, washed once with PBS, and the viable cell concentration was adjusted to contain 1 ⁇ 10 6 cells per 100 ⁇ l volume.
  • 100 ⁇ l of ID8 cell suspension was inoculated into the abdominal cavity of wild-type mice and Helz2 knockout mice respectively. Observe the mice every day.
  • mice aged 8 to 10 weeks were divided into two groups, namely wild-type mice and Helz2 knockout mice.
  • the Lewis cells of the lung cancer cell line in the logarithmic growth phase were digested with trypsin and washed once with PBS, and the viable cell concentration was adjusted to contain 2 ⁇ 10 6 cells per 100 ⁇ l volume.
  • 100 ⁇ l of Lewis cell suspension was inoculated subcutaneously in the right shoulder blades of wild-type mice and Helz2 knockout mice. Observe the mice every day.
  • mice aged 8 to 10 weeks were divided into two groups, namely wild-type mice and Helz2 knockout mice.
  • the breast cancer cell line E0771 cells in the logarithmic growth phase were digested with trypsin, washed once with PBS, and the viable cell concentration was adjusted to 8 ⁇ 10 4 cells per 100 ⁇ l volume.
  • 100 ⁇ l of E0771 cell suspension was inoculated into the abdominal cavity of wild-type mice and Helz2 knockout mice respectively. Observe the mice every day.
  • Example 6 Lentivirus infection to construct mesothelioma cell line AE17 cells with stable Helz2 gene knockdown (sh-Helz2-AE17)
  • AE17 cells in the logarithmic growth phase were seeded in 96 wells at 2000 cells per well.
  • sh-Helz2 was used to infect AE17 cells.
  • Each group had 3 duplicate wells, with sh-NC as the Control group (sh-NC-AE17); dilute the lentivirus stock solution with a titer of 10 8 into three gradients: stock solution, 10-fold dilution, and 100-fold dilution; take 10 ⁇ l of each of the three virus liquids with different concentrations to each In the three duplicate wells of the group, add 1:1000 polybrene (final concentration: 5 ⁇ g/ml) to each well, mix crosswise, and return the cells to the cell culture incubator for incubation; replace with fresh culture medium after 24 hours; infection 72- After 96 hours, observe the expression of fluorescence, and determine the optimal dilution factor of infected cells through preliminary experiments.
  • puromycin on the amount of dead cells, culture AE17 cells in a 24-well plate in advance to the logarithmic growth phase, and add different concentrations of puromycin (1 ⁇ g/ml, 2 ⁇ g/ml, 3 ⁇ g/ml, 4 ⁇ g/ml, 5 ⁇ g/ml).
  • AE17 sh-Helz2-AE17 cells
  • Example 7 Construction of lentivirus-infected AE17 cells (sh-NC-AE17 cells and sh-Helz2-AE17 cells) tumor-bearing wild-type mouse model
  • wild-type mice aged 8 to 10 weeks were divided into two groups, namely the sh-NC-AE17 group and the sh-Helz2-AE17 group. Then, the sh-NC-AE17 cells and sh-Helz2-AE17 cells in the logarithmic growth phase were trypsinized, washed once with PBS, and the viable cell concentration was adjusted to 3 ⁇ 10 5 cells per 100 ⁇ l volume. Finally, 100 ⁇ l of sh-NC-AE17 cells and sh-Helz2-AE17 cell solution were inoculated into the abdominal cavity of wild-type mice. Observe the mice every day.
  • mice in the sh-NC-AE17 group began to die on the 35th day after bearing tumors, and all died on the 48th day; one mouse in the sh-Helz2-AE17 group died on the 63rd day after bearing tumors, and the rest There were no obvious ascites or abnormalities in the mice, and the observation ended on the 182nd day.
  • Example 8 shRNA interference-mediated targeted Helz2 gene therapy for AE17 tumor-bearing wild-type mouse model
  • mice aged 8 to 10 weeks were divided into four groups, namely the sh-NC control group and the sh-Helz2-1, sh-Helz2-2, and sh-Helz2-3 treatment groups. Then, the mesothelioma cell line AE17 cells in the logarithmic growth phase were digested with trypsin, washed once with PBS, and the viable cell concentration was adjusted to contain 1 ⁇ 10 5 cells per 100 ⁇ l volume. Finally, 100 ⁇ l of AE17 cell suspension was inoculated into the abdominal cavity of wild-type mice.
  • Example 9 In order to explore the expression of Helz2 gene in other types of tumors, a total of 15,776 samples were analyzed using the databases TCGA and GTEx. The results showed that bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), cervical squamous cell carcinoma, Carcinoma and adenocarcinoma (CESC), cholangiocarcinoma (CHOL), colon cancer (COAD), esophageal cancer (ESCA), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), clear cell renal cell carcinoma (KIRC), renal papillary cell carcinoma (KIRP), acute myeloid leukemia (LAML), brain low-grade glioma (LGG), hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), ovarian serous cyst Adenocarcinoma (OV), pancreatic cancer (PAAD), pheochromocytoma and paragangliom

Abstract

提供了Helz2基因在肿瘤防治药物中的应用。还提供了Helz2基因敲除小鼠模型的构建方法，其是通过CRISPR/Cas9技术在小鼠受精卵上进行Helz2基因敲除，再通过显微注射和配繁，获得Helz2全身性基因敲除的纯合小鼠模型。还提供了Helz2全身性基因敲除的纯合小鼠模型和靶向Helz2基因治疗肿瘤的新疗法，为阐明Helz2在肿瘤发生发展中的作用机制提供动物模型，也为临床上针对Helz2基因在治疗和/或预防肿瘤的药物筛选和制备中提供靶点。

Description

Helz2基因在肿瘤防治药物中的应用 技术领域
本发明涉及基因治疗药物制备技术，具体是关于Helz2基因在肿瘤防治药物中的应用。
背景技术
Helz2(Helicase with zinc finger 2)[OMIM 611265]，又称PRIC285或PDIP1，是一种2649个氨基酸的核解酶蛋白，也是过氧化物酶体增殖物激活受体α相互作用复合体(Peroxisome proliferator activated receptorαinteracting complex，PRIC)的一部分。Helz2由两个ATP结合序列、一个RNaseB结构域和双DNA/RNA解旋酶序列组成，其参与了多种与基因调控相关的机制，包括基因转录、mRNA加工和DNA修复。它可作为PPARα和PPARγ以及其他核受体(RXRA、THRA、THRB)的核转录共激活因子。
Helz2还参与细胞脂质、糖类代谢和肝脏代谢过程，与脂肪细胞分化和原发性胆汁性肝硬化密切相关。Helz2在人和小鼠非酒精性脂肪性肝病(Nonalcoholic fatty liver disease，NAFLD)中的表达上调，其缺失激活了肝脏长型功能性瘦素受体(Functional leptin receptor long form，Leprb)的表达，并抑制了肥胖小鼠的NAFLD发展和体重增加。据报道，Helz2的结合体PPARγ可以调节小鼠炎症基因如Ccl3、Ccl7、Cxcl10和Tgtp的表达。PPARγ在先天免疫反应中也发挥了重要作用并与免疫相关疾病有关，例如HCV/HIV感染、骨关节炎、痤疮。就其本身而言，Helz2能结合对免疫反应具有重要作用蛋白的能力，例如BCL6和ISG15。另外，Helz2被发现在哺乳动物中具有抗病毒功能，该基因被鉴定为是具有抗病毒免疫保守成分的干扰素刺激基因(Interferon stimulated gene，ISG)。此外，还发现了Helz2在IFN抗病毒反应中的介导作用，具体涉及Helz2转录和细胞核蛋白的上调，以及转录程序的激活。
然而，利用Helz2基因敲除小鼠模型在抗肿瘤方面的研究及应用未见报道。为了进一步阐明Helz2表达水平下降对肿瘤发生发展的影响，需要构建Helz2基因敲除小鼠，并对其进行肿瘤建模，为探究Helz2影响肿瘤细胞生长机制，抑制肿瘤发生发展的作用以及治疗和/或预防肿瘤的新方法提供动物模型。
CRISPR/Cas9基因组编辑系统是细菌适应性免疫系统的一部分，用于抵御来自噬 菌体和质粒的侵入性核酸。CRISPR/Cas9基因编辑工具不仅功能强大，而且具有特异性强、效率高等特点，可以准确、快速地进行基因编辑。虽然有研究报道在细胞水平利用这一技术干扰Helz2基因表达的研究。但是未有利用CRISPR/Cas9系统获得纯合子Helz2基因敲除小鼠模型与相应的研究应用。也未有靶向Helz2基因或其表达的产物在肿瘤治疗当中的研究及应用。
基因靶向治疗正在成为一种重要的肿瘤治疗方法。RNA干扰(RNA interference，RNAi)是在转录后水平抑制特定基因表达的最强大的靶向疗法之一。人工合成RNAi主要有两种类型：siRNA和shRNA。之前研究已经证明了siRNA具有以下局限性：细胞对其摄取能力不足、稳定性差、在体内外易于被降解和在临床实验中递送效率低。然而，病毒载体介导的shRNA具有长效而稳定的基因沉默能力。shRNA的慢病毒载体是一类重组逆转录病毒载体，该载体能在哺乳动物各类细胞中稳定表达shRNA。这类载体已在相关肿瘤动物模型的临床数据库中得到验证。因此，慢病毒介导的shRNA策略是一种具有吸引力的肿瘤治疗候选疗法。
发明内容
本发明所要解决的技术问题是，提供一种构建靶向Helz2基因敲除动物模型的方法，以及靶向干扰Helz2基因在肿瘤药物的制备或筛选和在肿瘤防治药物中的应用。
本发明的第一个方面，为一种Helz2基因敲除小鼠模型的构建方法，其通过CRISPR/Cas9技术在小鼠受精卵上进行Helz2基因敲除，再通过显微注射和配繁，获得Helz2全身性基因敲除的纯合小鼠模型(Helz2-Cas9-KO)。为探究Helz2基因与肿瘤之间的机理研究提供了理想的动物模型，也为Helz2-Cas9-KO小鼠模型在制备或筛选治疗和/或预防肿瘤药物中的应用提供了支撑。
进一步的，所述Helz2基因敲除小鼠模型的构建方法包括如下步骤：
步骤一，靶向小鼠Helz2基因的sgRNA识别序列设计和构建sgRNA；通过对Helz2基因进行分析，确定具体的敲除区域后，根据靶基因设计一对相应的gRNA序列，并合成sgRNA序列；sgRNA序列包括，
SEQ ID No.1：5’-GCCCCAGAGTTACCAGATGGAGG-3’；
SEQ ID No.2：5’-CCTACACCCGACAGAGGTGTAGG-3’；
SEQ ID No.3：5’-AGCAGTGACAGTCTTATGGGTGG-3’；
SEQ ID No.4：5’-GGCATACAGAGGATTGCCACAGG-3’。
将合成的sgRNA序列在体外转录成mRNA；
步骤二，将步骤一中获得的mRNA与Cas9质粒一并通过显微注射的方式注射入小鼠受精卵中，培育后获得F0代小鼠。
步骤三，通过PCR和测序鉴定筛选出阳性F0代小鼠；
步骤四，F0代小鼠性成熟配繁，获得阳性F1代小鼠。
本发明的第二个方面，为上述方法获得的Helz2全身性基因敲除的纯合小鼠模型在制备或筛选抗肿瘤和/或预防肿瘤药物中的应用。所述应用包括药物靶标的筛选、药物的筛选、药物的药效学评价和药物的安全性评价。
本发明的第三个方面，为一种用于构建Helz2基因敲除动物模型的sgRNA序列，其包括下述SEQ ID No.1-SEQ ID No.4基因序列中的一种或多种，
SEQ ID No.1：5’-GCCCCAGAGTTACCAGATGGAGG-3’；
SEQ ID No.2：5’-CCTACACCCGACAGAGGTGTAGG-3’；
SEQ ID No.3：5’-AGCAGTGACAGTCTTATGGGTGG-3’；
SEQ ID No.4：5’-GGCATACAGAGGATTGCCACAGG-3’。
本发明的第四个方面，为一种含有靶向Helz2基因的载体，其靶向干扰Helz2基因的靶点序列为下述基因序列中的一种或多种，
SEQ ID No.5：5’-GCTATCAAGTCTGTCACTACT-3’；
SEQ ID No.6：5’-GCACGATGCTGTATGGCTTTG-3’；
SEQ ID No.7：5’-GGGCCTCATTGACACTCAAAG-3’。
所述载体为慢病毒载体复合物、腺病毒、腺相关病毒、N-乙酰半乳糖胺(GalNAc)、脂质体(LNPs)、聚合物(Polymers)、溶瘤病毒之一，或其它生物学上可接受的基因载体。
本发明的第五个方面，为上述含有靶向Helz2基因的载体在制备抑制Helz2基因表达的生物制剂中的应用。
本发明的第六个方面，为一种治疗和/或预防肿瘤药物，其含有靶向Helz2基因或其表达的产物。
本发明的有益效果：
本发明提供了一种潜在治疗和/或预防肿瘤的关键靶点Helz2，借助于CRISPR/Cas9基因技术和shRNA干扰技术分别构建了Helz2-Cas9-KO小鼠模型和发明了靶向Helz2基因治疗肿瘤的新疗法。通过Helz2-Cas9-KO小鼠体内荷瘤实验和野生型小鼠荷瘤后经shRNA干扰介导的靶向Helz2基因治疗证实了敲除Helz2基因能够显 著抑制肿瘤细胞的生长，并延长了小鼠的生存期。
本发明可为阐明Helz2基因在肿瘤发生发展中的作用机制提供可靠的动物模型，也为临床上针对Helz2基因在治疗和/或预防肿瘤的药物筛选和制备中提供靶点，并且还提供了靶向Helz2基因治疗肿瘤的新疗法，应用前景广泛。
附图说明
图1为Helz2-Cas9-KO小鼠策略设计图。
图2为电泳结果图。其中：B6为阴性对照，是B6基因组DNA；N为空白对照，无模板的对照；TRANS 2K PLUS II条带：8000bp、5000bp、3000bp、2000bp、1000bp、750bp、500bp、250bp、100bp。
图3为实施例1中间皮瘤AE17荷瘤Helz2-Cas9-KO小鼠生存曲线。
图4为实施例2中间皮瘤40L荷瘤Helz2-Cas9-KO小鼠生存曲线。
图5为实施例3中卵巢癌ID8荷瘤Helz2-Cas9-KO小鼠生存曲线。
图6为实施例4中肺癌Lewis荷瘤Helz2-Cas9-KO小鼠生存曲线和肿瘤生长情况。
图7为实施例5中乳腺癌E0771荷瘤Helz2-Cas9-KO小鼠生存曲线。
图8为实施例7中sh-NC-AE17和sh-Helz2-AE17细胞荷瘤野生型小鼠生存曲线。
图9为实施例8中sh-Helz2治疗AE17荷瘤野生型小鼠模式图。
图10为实施例8中sh-Helz2治疗AE17荷瘤野生型小鼠生存曲线。
图11为实施例9中Helz2在泛癌中的表达情况。
具体实施方式
下面结合附图和具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此。
图1和图2显示了本发明实施例中Helz2-Cas9-KO小鼠策略设计图和电泳鉴定结果图。
实施例1：间皮瘤细胞株AE17细胞小鼠模型建立
首先，将8～10周龄小鼠分为两组，分别为野生型小鼠(wild-type)组和Helz2敲基因小鼠(knockout)组。然后，将处于对数生长期的间皮瘤细胞株AE17细胞经胰酶消化后，PBS洗涤1次，调整活细胞浓度为每100μl体积中含1×10 5个细胞。最后，取100μl AE17细胞悬液分别接种于野生型小鼠(wild-type)和Helz2敲基因小鼠(knockout)腹腔中。每天观察小鼠情况。
结果显示：野生型小鼠荷瘤后于第50天开始死亡，至第127天全部死亡；Helz2 敲基因小鼠荷瘤后一直均无明显腹水和异常以及死亡，观察截止于第174天。以上结果表明Helz2敲基因小鼠较野生型小鼠荷瘤后能够显著延长小鼠生存期(P＜0.001)，提示Helz2敲基因小鼠具有显著的抗间皮瘤AE17能力，见图3。
实施例2：间皮瘤细胞株40L细胞小鼠模型建立
首先，将8～10周龄小鼠分为两组，分别为野生型小鼠(wild-type)组和Helz2敲基因小鼠(knockout)组。然后，将处于对数生长期的间皮瘤细胞株40L细胞经胰酶消化后，PBS洗涤1次，调整活细胞浓度为每100μl体积中含2×10 5个细胞。最后，取100μl 40L细胞悬液分别接种于野生型小鼠(wild-type)和Helz2敲基因小鼠(knockout)腹腔中。每天观察小鼠情况。
结果显示：野生型小鼠荷瘤后于第43天开始死亡，至第66天全部死亡；Helz2敲基因小鼠荷瘤后一直均无明显腹水和异常以及死亡，观察截止于第166天。以上结果表明Helz2敲基因小鼠较野生型小鼠荷瘤后能够明显延长小鼠生存期(P＜0.001)，提示Helz2敲基因小鼠具有显著的抗间皮瘤40L能力，见图4。
实施例3：卵巢癌细胞株ID8细胞小鼠模型建立
首先，将8～10周龄小鼠分为两组，分别为野生型小鼠(wild-type)组和Helz2敲基因小鼠(knockout)组。然后，将处于对数生长期的卵巢癌细胞株ID8细胞经胰酶消化后，PBS洗涤1次，调整活细胞浓度为每100μl体积中含1×10 6个细胞。最后，取100μl ID8细胞悬液分别接种于野生型小鼠(wild-type)和Helz2敲基因小鼠(knockout)腹腔中。每天观察小鼠情况。
结果显示：野生型小鼠荷瘤后于第50天开始死亡，至第66天全部死亡；Helz2敲基因小鼠荷瘤后第65天开始死亡，最后死亡的Helz2敲基因小鼠于第86天死亡，还剩一只Helz2敲基因小鼠一直均无明显腹水和异常以及死亡，观察截止于第156天。以上结果表明Helz2敲基因小鼠较野生型小鼠荷瘤后能够明显延长小鼠生存期(P＝0.006)，提示Helz2敲基因小鼠具有显著的抗卵巢癌ID8能力，见图5。
实施例4：肺癌细胞株Lewis细胞小鼠模型建立
首先，将8～10周龄小鼠分为两组，分别为野生型小鼠(wild-type)组和Helz2敲基因小鼠(knockout)组。然后，将处于对数生长期的肺癌细胞株Lewis细胞经胰酶消化后，PBS洗涤1次，调整活细胞浓度为每100μl体积中含2×10 6个细胞。最后，取100μl Lewis细胞悬液分别接种于野生型小鼠(wild-type)和Helz2敲基因 小鼠(knockout)右肩胛皮下处。每天观察小鼠情况。
结果显示：野生型小鼠荷瘤后于第26天开始死亡，至第34天全部死亡；Helz2敲基因小鼠荷瘤后第31天开始死亡，至第68天全部死亡。以上结果表明Helz2敲基因小鼠较野生型小鼠荷瘤后能够明显延长小鼠生存期(P＝0.021)，有效抑制肿瘤生长，提示Helz2敲基因小鼠具有显著的抗卵巢癌抗肺癌Lewis能力，见图6。
实施例5：乳腺癌细胞株E0771细胞小鼠模型建立
首先，将8～10周龄小鼠分为两组，分别为野生型小鼠(wild-type)组和Helz2敲基因小鼠(knockout)组。然后，将处于对数生长期的乳腺癌细胞株E0771细胞经胰酶消化后，PBS洗涤1次，调整活细胞浓度为每100μl体积中含8×10 4个细胞。最后，取100μl E0771细胞悬液分别接种于野生型小鼠(wild-type)和Helz2敲基因小鼠(knockout)腹腔中。每天观察小鼠情况。
结果显示：野生型小鼠荷瘤后于第26天开始死亡，至第33天全部死亡；Helz2敲基因小鼠荷瘤后一直均无明显腹水和异常以及死亡，观察截止于第90天。以上结果表明Helz2敲基因小鼠较野生型小鼠荷瘤后能够明显延长小鼠生存期(P＝0.001)，提示Helz2敲基因小鼠具有显著的抗乳腺癌E0771能力，见图7。
实施例6：慢病毒感染构建稳定敲减Helz2基因的间皮瘤细胞株AE17细胞(sh-Helz2-AE17)
将对数生长期的AE17细胞按每孔2000个细胞接种于96孔中，待细胞密度为40％-60％时进行sh-Helz2感染AE17细胞，每组3个复孔，以sh-NC作为对照组(sh-NC-AE17)；将滴度为10 8的慢病毒原液稀释为三个梯度：原液、10倍稀释、100倍稀释；将三个不同浓度的病毒液体，各取10μl至每组的3个复孔中，每孔加入1:1000的polybrene(终浓度为5μg/ml)，十字混匀，将细胞放回细胞培养箱孵育；24小时后更换为新鲜培养基；感染72-96小时后，观察荧光的表达情况，通过预实验确定感染细胞的最佳稀释倍数，实验发现AE17细胞感染的最佳浓度为原液，因此采用原液浓度进行正式感染；在感染前进行72小时最低杀死细胞量的嘌呤霉素预实验，提前在24孔板培养AE17细胞至对数生长期，加入不同浓度的嘌呤霉素(1μg/ml、2μg/ml、3μg/ml、4μg/ml、5μg/ml、6μg/ml、7μg/ml、8μg/ml、9μg/ml、10μg/ml)，72小时后挑选最低杀死细胞浓度；实验挑选的最低杀死细胞浓度是3μg/ml；AE17细胞感染后连续培养一周，期间将AE17细胞从96孔板消化转移至6孔板，进行嘌呤霉素筛选，筛选培养1个月，即可获得稳定敲减Helz2基因的AE17 (sh-Helz2-AE17细胞)。
实施例7：构建慢病毒感染的AE17细胞(sh-NC-AE17细胞和sh-Helz2-AE17细胞)荷瘤野生型小鼠模型
首先，将8～10周龄野生型小鼠分为两组，分别为sh-NC-AE17组和sh-Helz2-AE17组。然后，将处于对数生长期的sh-NC-AE17细胞和sh-Helz2-AE17细胞经胰酶消化后，PBS洗涤1次，调整活细胞浓度为每100μl体积中含3×10 5个细胞。最后，取100μl的sh-NC-AE17细胞和sh-Helz2-AE17细胞液分别接种于野生型小鼠腹腔中。每天观察小鼠情况。
结果显示：sh-NC-AE17组小鼠荷瘤后于第35天开始死亡，至第48天全部死亡；sh-Helz2-AE17组小鼠荷瘤后于第63天死亡一只小鼠，剩余小鼠无明显腹水和异常，观察截止于第182天。以上结果发现与sh-NC-AE17组相比，sh-Helz2-AE17组小鼠能够显著延长荷瘤小鼠的生存期(P＝0.025)，提示经过慢病毒sh-Helz2感染后的AE17细胞体内成瘤能力显著减弱，见图8。
实施例8：shRNA干扰介导的靶向Helz2基因治疗AE17荷瘤野生型小鼠模型
首先，将8～10周龄小鼠分为四组，分别为sh-NC对照组和sh-Helz2-1、sh-Helz2-2、sh-Helz2-3治疗组。然后，将处于对数生长期的间皮瘤细胞株AE17细胞经胰酶消化后，PBS洗涤1次，调整活细胞浓度为每100μl体积中含1×10 5个细胞。最后，取100μl AE17细胞悬液分别接种于野生型小鼠腹腔中。
将10μl的滴度为10 8慢病毒原液sh-NC和sh-Helz2分别加入到90μl的无菌PBS中稀释，即获得100μl的慢病毒使用液。于接种肿瘤细胞第12小时，使用慢病毒使用液sh-NC和sh-Helz2分别注射到荷瘤野生型小鼠腹腔中，注射体积为上述稀释后100μl的慢病毒使用液。此为第1次使用慢病毒sh-Helz2治疗。于第1次治疗间隔3天，按第1次方式重复再次治疗。此为第2次使用慢病毒sh-Helz2治疗。于第2次治疗间隔3天，按第1次方式重复再次治疗。此为第3次使用慢病毒sh-Helz2治疗。于第3次治疗间隔3天，按第1次方式重复再次治疗。此为第4次使用慢病毒sh-Helz2治疗。于第4次治疗间隔7天，按第1次方式重复再次治疗。此为第5次使用慢病毒sh-Helz2治疗。于第5次治疗间隔7天，按第1次方式重复再次治疗。此为第6次使用慢病毒sh-Helz2治疗。治疗间隔时间如图9所示，一共治疗6次。
结果显示：sh-NC对照组荷瘤小鼠于第51天开始死亡，至第63天全部死亡；sh-Helz2-1治疗组荷瘤小鼠于第52天开始死亡，至第92天全部死亡(P＝0.197)； sh-Helz2-2治疗组荷瘤小鼠于第74天开始死亡，剩余一只小鼠无明显腹水和异常，观察截止于第113天(P＝0.025)；sh-Helz2-3治疗组荷瘤小鼠于第63天开始死亡，剩余两只小鼠无明显腹水和异常，观察截止于第113天(P＝0.063)。以上结果表明与sh-NC对照组相比，sh-Helz2治疗组能够显著延长荷瘤小鼠的生存期，提示针对Helz2基因为靶点开发的慢病毒运载的shRNA(sh-Helz2)能够有效的治疗荷瘤小鼠，提升生存时间和生存质量，见图10。
实施例9：为了探究Helz2基因在其它类型肿瘤中的表达情况，利用数据库TCGA和GTEx共分析了15776例样本，结果发现，膀胱尿路上皮癌(BLCA)、乳腺浸润癌(BRCA)、宫颈鳞癌和腺癌(CESC)、胆管癌(CHOL)、结肠癌(COAD)、食管癌(ESCA)、多形成性胶质细胞瘤(GBM)、头颈鳞状细胞癌(HNSC)、肾透明细胞癌(KIRC)、肾乳头状细胞癌(KIRP)、急性髓细胞样白血病(LAML)、脑低级别胶质瘤(LGG)、肝细胞肝癌(LIHC)、肺腺癌(LUAD)、卵巢浆液性囊腺癌(OV)、胰腺癌(PAAD)、嗜铬细胞瘤和副神经节瘤(PCPG)、前列腺癌(PRAD)、直肠腺癌(READ)、肉瘤(SARC)、皮肤黑色素瘤(SKCM)、胃癌(STAD)、睾丸癌(TGCT)、甲状腺癌(THCA)和子宫内膜癌(UCEC)共25类肿瘤中均高表达Helz2(*P＜0.05；**P＜0.01；***P＜0.001)，见图11，提示在这些肿瘤中，针对Helz2基因靶点进行干扰也具有同实施例8一样具有治疗肿瘤的作用。

Claims (9)

  1. 一种含有靶向Helz2基因的载体，其特征是：其靶向干扰Helz2基因的靶点序列为下述基因序列中的一种或多种，
    SEQ ID No.5：5’-GCTATCAAGTCTGTCACTACT-3’；
    SEQ ID No.6：5’-GCACGATGCTGTATGGCTTTG-3’；
    SEQ ID No.7：5’-GGGCCTCATTGACACTCAAAG-3’。
  2. 根据权利要求1所述的含有靶向Helz2基因的载体，其特征是：所述载体为慢病毒载体复合物、腺病毒、腺相关病毒、N-乙酰半乳糖胺、脂质体、聚合物、溶瘤病毒之一，或其他生物学上可接受的基因载体。
  3. 一种权利要求1所述的含有靶向Helz2基因的载体在制备抑制Helz2基因表达的生物制剂中的应用。
  4. 一种治疗和/或预防肿瘤药物，其特征在于：其含有靶向干扰Helz2基因或其表达的产物。
  5. 一种Helz2基因敲除小鼠模型的构建方法，其特征是：通过CRISPR/Cas9技术在小鼠受精卵上进行Helz2基因敲除，再通过显微注射和配繁获得Helz2全身性基因敲除的小鼠模型。
  6. 根据权利要求5所述的Helz2基因敲除小鼠模型的构建方法，其特征是：用于敲除小鼠Helz2基因的sgRNA序列包括下述SEQ ID No.1-SEQ ID No.4基因序列中的一种或多种，
    SEQ ID No.1：5-GCCCCAGAGTTACCAGATGGAGG-3’；
    SEQ ID No.2：5’-CCTACACCCGACAGAGGTGTAGG-3’；
    SEQ ID No.3：5’-AGCAGTGACAGTCTTATGGGTGG-3’；
    SEQ ID No.4：5’-GGCATACAGAGGATTGCCACAGG-3’。
  7. 一种权利要求5所述Helz2全身性基因敲除的纯合小鼠模型在在制备或筛选抗肿瘤和/或预防肿瘤药物中的应用。
  8. 根据权利要求7所述的应用，其特征是：所述应用包括药物靶标的筛选、药物的筛选、药物的药效学评价和药物的安全性评价。
  9. 一种用于构建Helz2基因敲除动物模型的sgRNA序列，其特征是：包括下述SEQ ID No.1-SEQ ID No.4基因序列中的一种或多种，
    SEQ ID No.1：5’-GCCCCAGAGTTACCAGATGGAGG-3’；
    SEQ ID No.2：5’-CCTACACCCGACAGAGGTGTAGG-3’；
    SEQ ID No.3：5’-AGCAGTGACAGTCTTATGGGTGG-3’；
    SEQ ID No.4：5’-GGCATACAGAGGATTGCCACAGG-3’。
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