WO2019052000A1 - 一种靶向STAT3信号通路miRNA及其制备方法和应用 - Google Patents

一种靶向STAT3信号通路miRNA及其制备方法和应用 Download PDF

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WO2019052000A1
WO2019052000A1 PCT/CN2017/112216 CN2017112216W WO2019052000A1 WO 2019052000 A1 WO2019052000 A1 WO 2019052000A1 CN 2017112216 W CN2017112216 W CN 2017112216W WO 2019052000 A1 WO2019052000 A1 WO 2019052000A1
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mirna
stat3
nucleotide sequence
mir
diseases
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钱政江
李翔
李霞
陈瑜
李燕娇
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中国科学院深圳先进技术研究院
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Definitions

  • the present invention relates to the field of biomedicine, in particular to a miRNA targeting STAT3 signaling pathway and a preparation method and application thereof, in particular to a miRNA-miR-1181 targeting STAT3 signaling pathway and application thereof, in particular to hsa-miR-1182 Use in the preparation of a drug caused by abnormal activation of a STAT3 mediated signaling pathway.
  • STAT3 Signal transducer and activator of transcription 3
  • STAT3 are important intracellular proteins with signal transduction and transcriptional activation. They are composed of six regions: N-terminal amino acid conserved sequence, helix region, and DNA binding region. , SH3 domain, SH2 domain and C-terminal transcriptional activation region. After being stimulated by exogenous signals such as growth factors and cytokines, the cells transmit signals via tyrosine kinase-associated receptors and tyrosine kinase JAK, thereby activating the STAT3 signaling pathway and ultimately acting on specific DNA fragments in the nucleus. , regulate target gene transcription.
  • the STAT3 signal transduction pathway is closely related to cell growth, differentiation and programmed death and angiogenesis.
  • STAT3 kinase signaling pathway is abnormally activated, and cardiovascular diseases such as heart failure, myocardial infarction, cardiac load hypertrophy caused by stress load, myocardial protection induced by ischemic preconditioning, ischemia reperfusion, hypertension and angina pectoris Occurrence and development are closely related, and have great potential as a new target for the treatment of cardiovascular diseases.
  • cardiovascular diseases such as heart failure, myocardial infarction, cardiac load hypertrophy caused by stress load, myocardial protection induced by ischemic preconditioning, ischemia reperfusion, hypertension and angina pectoris Occurrence and development are closely related, and have great potential as a new target for the treatment of cardiovascular diseases.
  • cardiovascular diseases such as heart failure, myocardial infarction, cardiac load hypertrophy caused by stress load, myocardial protection induced by ischemic preconditioning, ischemia reperfusion, hypertension and angina pectoris Occurrence and development are closely related, and have
  • CN 1778918A discloses an siRNA which inhibits expression of a Stat3 gene, and a preparation method thereof, which comprises a pBS/U6 vector; a DNA fragment encoding an siRNA which specifically inhibits expression of the Stat3 gene.
  • the inhibitor utilization rate is low and the biological stability is poor.
  • the currently studied STAT3 kinase signaling pathway inhibitors also include peptide and peptidomimetic inhibitors, natural product inhibitors, and small molecule inhibitors discovered by computer drug virtual screening techniques.
  • Peptide and peptidomimetic inhibitors are phosphopeptides designed by using the amino acid residue sequence of the related molecule phosphorylation product on the STAT3 signaling pathway as a template, which can block the transmission of STAT3 signaling pathway and achieve inhibitory effects, but such inhibition
  • the agent is easily metabolized in the body and has low bioavailability.
  • Natural product inhibitors mainly include natural chemical products such as terpenoids and flavonoids, which are natural active molecules that inhibit the STAT3 signaling pathway. However, the process of obtaining such biologically active compounds is complicated, the process is cumbersome, and it is not specific. The STAT3 signaling pathway is inhibited.
  • the present application provides a STAT3 signaling pathway miRNA having endogenous and specific targeting inhibition of STAT3 (signal transducer and transcriptional activator 3) A signaling pathway capable of producing a drug that inhibits diseases caused by abnormal activation of a STAT3-mediated signaling pathway.
  • the application provides a miRNA that targets a STAT3 signaling pathway, the nucleotide sequence of which is SEQ ID NO. 1 or a nucleotide sequence having at least 90% identity thereto.
  • the nucleotide sequence shown in SEQ ID NO. 1 is as follows: 5'-CCGUCGCCGCCACCCGAGCCG-3'.
  • the nucleotide sequence of the miRNA is the nucleotide sequence shown in SEQ ID NO.
  • the activation or expression of STAT3 signaling pathway by platelet-derived growth factor BB can be inhibited;
  • PDGFBB platelet-derived growth factor BB
  • PDGFBB platelet-derived growth factor BB
  • the preparation method of the miRNA includes a method for whole-gene synthesis and clonal expression of a mature sequence, and the method for expressing the clone is a conventional technique in the art, and a person skilled in the art can construct according to a suitable method as needed. This is not particularly limited.
  • the method of constructing the miRNA comprises the following steps:
  • the nucleotide sequence of the primer is shown in SEQ ID NO. 2-3;
  • the nucleotide sequence shown in SEQ ID NO. 2 is as follows: 5'-CCGCTCGAGCGCCCGATGACGCGGAGT-3';
  • the nucleotide sequence shown in SEQ ID NO. 3 is as follows: 5'-CCGGAATTCGGTCCCTGGGCAGTATCG-3'.
  • the application provides the use of a miRNA according to the first aspect, in the manufacture of a medicament or kit for the diagnosis, prevention, treatment or prognosis of a disease.
  • the disease is a disease caused by abnormal activation of a STAT3 mediated signaling pathway.
  • the disease is any one or a combination of at least two of cardiovascular and cerebrovascular diseases, gastrointestinal diseases, inflammatory diseases, leukemias, prostate diseases, nervous system diseases or tumors.
  • the cardiovascular and cerebrovascular diseases include cardio-cerebral vascular diseases such as cardiac hypertrophy, cerebral ischemia-reperfusion injury, heart failure, myocardial infarction, ischemia reperfusion, hypertension, and atherosclerosis; Malignant tumors such as esophageal cancer or ovarian cancer.
  • Cerebral ischemia-reperfusion refers to ligating the left anterior descending branch of the coronary artery to cause myocardial ischemia for a period of time, and then releasing the ligature to resuspend the myocardium.
  • the application provides a lentiviral vector comprising the nucleotide sequence of the miRNA of the first aspect.
  • the present application provides a recombinant lentivirus, which comprises co-transfecting a mammalian cell comprising the lentiviral vector of the third aspect with a packaging helper plasmid to obtain a recombinant lentivirus.
  • the mammalian cell is a HEK293T cell.
  • the present application provides a pharmaceutical composition comprising the miRNA of the first aspect.
  • the effective dose of the miRNA is 10-200 mg/kg, for example, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/ Kg, 90 mg/kg, 100 mg/kg, 120 mg/kg, 130 mg/kg, 150 mg/kg, 160 mg/kg, 180 mg/kg or 200 mg/kg.
  • the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable virus, carrier or adjuvant.
  • a pharmaceutically acceptable virus, carrier or adjuvant is selected from the group consisting of chitosan, cholesterol, liposomes or nanoparticles.
  • the present application provides a kit for the diagnosis and/or prognosis of a disease, the kit comprising the miRNA of the first aspect and/or a probe or primer for specifically detecting the miRNA.
  • nucleotide sequence of the probe or primer is as shown in SEQ ID NO. 2-3;
  • the nucleotide sequence shown in SEQ ID NO. 2 is as follows: 5'-CCGCTCGAGCGCCCGATGACGCGGAGT-3';
  • the nucleotide sequence shown in SEQ ID NO. 3 is as follows: 5'-CCGGAATTCGGTCCCTGGGCAGTATCG-3'.
  • This application finds that small miRNAs target STAT3 and inhibit the activity of this signaling pathway by inhibiting the expression of STAT3 molecules. It can be used as a novel STAT3 kinase inhibitor for the treatment of abnormal activation of STAT3 kinase signaling pathway.
  • the miRNA of the present application is a human endogenous miRNA, which is less toxic to the human body and can be better utilized by the human body, and can be introduced into the human body by intravenous injection and transported to a specific site through blood circulation, thereby achieving therapeutic purposes;
  • the miRNA of the present application can be used as a novel STAT3 kinase signaling pathway inhibitor for the treatment of cardiovascular diseases, gastrointestinal diseases, inflammatory diseases, prostate, nervous system diseases and malignant tumors;
  • the preparation method of the miRNA of the present application is simple, and can adopt the whole gene synthesis or the clone expression, and the prepared miRNA can be introduced into the human body by intravenous injection, and transported to a specific part by blood circulation, thereby correspondingly inhibiting. effect.
  • Figure 1A shows the 3'-UTR binding site of miR-1181 and STAT3;
  • Figure 1B shows the effect of overexpression of miR-1181 on STAT3-UTR or STAT3-UTR-mut luciferase activity, wherein STAT3-UTR-mut is a mutant vector constructed based on the binding site for 3'-UTR;
  • Figure 1C shows the effect of overexpression of miR-1181 on total protein expression of STAT3 in human pulmonary artery smooth muscle cells, wherein Mimic Ctrl is a chemically synthesized miR-1181 analog;
  • FIG. 1D shows the effect of overexpression of miR-1181 on phosphorylation-activated STAT3 protein expression in PDGFBB-stimulated human pulmonary artery smooth muscle cells.
  • Mimic Ctrl is a chemically synthesized structurally similar but nonsense sequence.
  • Mimic Ctrl-1 is not treated with PDGF.
  • Mimic Ctrl-2 is processed with PDGF;
  • FIG. 2A is a typical fluorescent picture of the effect of overexpression of miR-1181 on cell proliferation after PDGFB stimulates human pulmonary artery smooth muscle cells by EdU.
  • Mimic Ctrl is a chemically synthesized structurally similar but meaningless sequence.
  • Mimic Ctrl-1 does not use PDGF.
  • Processing, Mimic Ctrl-2 is processed with PDGF;
  • FIG. 2B is a statistical result of the effect of overexpression of miR-1181 on cell proliferation after PDGFB stimulates human pulmonary artery smooth muscle cells by EdU.
  • Mimic Ctrl is a chemically synthesized structurally similar but meaningless sequence.
  • Mimic Ctrl-1 is not treated with PDGF.
  • Mimic Ctrl-2 is processed with PDGF;
  • FIG. 2C shows the effect of overexpression of miR-1181 on PCNA protein expression after PDGFBB stimulates human pulmonary artery smooth muscle cells.
  • Mimic Ctrl is a chemically synthesized structurally similar but meaningless sequence.
  • Mimic Ctrl-1 is not treated with PDGF,
  • Mimic Ctrl -2 was treated with PDGF.
  • the mature sequence of miR-1181 is 5'-CCGUCGCCGCCACCCGAGCCG-3' (22 bp, SEQ ID NO. 1) can be obtained by:
  • miR-1181 The analog of miR-1181 (miR-1181mimic) was synthesized by Guangzhou Ruibo Biotechnology Co., Ltd. as double-stranded RNA, which can mimic the high level expression of endogenous mature miR-1181; standard purification, stored at -20 °C.
  • the 20 M storage solution was dissolved in RNase-free water, placed at -80 ° C until use, and diluted to the desired concentration during use.
  • Upstream primer 5'-CCGCTCGAGCGCCCGATGACGCGGAGT-3' (SEQ ID NO. 2);
  • the sequence of the downstream primer is 5'-CCGGAATTCGGTCCCTGGGCAGTATCG-3' (SEQ ID NO. 3);
  • PCR amplification was carried out by designing primers.
  • the PCR reaction conditions were: denaturation at 95 ° C for 2 minutes, 95 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 40 seconds for 35 cycles, 72 °C extension for 3 minutes;
  • the prepared DNA vector can be transfected into cells to transiently express miRNA, i.e., miR-1181.
  • the resulting lentiviral vector can be used for lentiviral packaging, as described below:
  • the cell line required for lentiviral packaging is the HEK293T cell line, and the desired plasmid includes the lentiviral expression vector pLVX-miR-1181 and a lentiviral packaging plasmid.
  • Cell transfection was carried out by conventional calcium phosphate transfection method, mixed with 2M CaCl 2 and plasmid, then added dropwise to 2 ⁇ HBS solution and gently shaken, and finally the mixed solution was slowly and uniformly added to the cell culture solution. Change the culture solution after 12 hours of transfection;
  • the packaged lentivirus can be used to infect selected cells, and cells stably stabilizing miR-1181 can be obtained by screening with the antibiotic puromycin.
  • HEK293A cells purchased from ATCC, Manassas, VA
  • DMEN medium containing 10% fetal bovine serum
  • cells grown in good condition were collected, and seeded in a 24-well plate at 6 ⁇ 10 4 /well.
  • Human pulmonary artery smooth muscle cells were purchased from Lonza (Walkersville, MD), cultured in SMCM complete medium containing 5% serum, and cells grown in good condition were collected, centrifuged, and inoculated into a 60 mm dish at 6 ⁇ 10 5 . Incubate at 37 ° C, 5% CO 2 for 24 hours;
  • miR-1181 analog (mimic) was used to overexpress or inhibit the expression of miR-1181, and cel-miR-67 was used as a negative control, all of which were purchased from Guangzhou Ruibo Bio.
  • the cells were seeded in a Petri dish, and when the cell density reached 70%, transfection was carried out by Lipofectamine 2000 (Invitrogen), the medium was changed after 6 hours of transfection, and the culture was continued for 24 hours for the experiment.
  • the luciferase activity assay was performed using the dual luciferase assay kit (E1810, Promega) reference specification, and the corrected luciferase activity value was obtained by dividing the firefly luciferase value by the internal reference renilla luciferase reading.
  • the luciferase activity values of the 3'-UTR vector of CSTAT3 were compared with the control group.
  • the 3'-UTR binding site of miR-1181 and STAT3 is shown in Figure 1A, and the fluorescein test results are shown in Figure 1B.
  • the STAT3 luciferase activity overexpressing the miR-1181 group is significant.
  • the decrease indicates that miR-1181 may bind to the 3'-UTR of STAT3; the 3'-UTR mutation vector STAT3-UTR-mut is constructed based on the binding site, and the dual luciferase activity is detected again using the mutant vector.
  • Mutation of the STAT3 binding site results in restoration of luciferase activity compared to wild-type UTR.
  • the total protein of the cells was collected, and the protein was quantified and subjected to polyacrylamide gel electrophoresis.
  • the expression of STAT3 was detected by STAT3 protein antibody, as shown in Fig. 1C.
  • Overexpression of miR-1181 was inhibited compared with the control. Expression of endogenous STAT3 in human pulmonary artery smooth muscle cells; phosphorylation of STAT3 by STAT3 phosphorylation antibody compared with control (Ctrl-1 for PDGF untreated indicates normal cell function, and Ctrl-2 for PDGF treatment indicates abnormal cell function) ), as shown in Figure 1D, overexpression of miR-1181 significantly inhibited phosphorylation of STAT3.
  • Example 3 miR-1181 inhibits platelet-derived growth factor BB (PDGFBB)-induced proliferation of human pulmonary artery smooth muscle cells.
  • PDGFBB platelet-derived growth factor BB
  • miR-1181 in the activation of the STAT3 signaling pathway by platelet-derived growth factor BB (PDGFBB), which leads to the regulation of dysfunction of human pulmonary artery smooth muscle cells: lentivirus infection with miR-1181 is used to increase miR in human pulmonary artery smooth muscle cells. At the level of 1181, cell proliferation levels were then determined by cell proliferation marker proteins PCNA and EdU cell proliferation assays.
  • PDGFBB platelet-derived growth factor BB
  • the EdU cell proliferation assay kit (Ribo Biotechnology) was used for the EdU labeling, and the experimental procedure was carried out according to the kit instructions. The main steps were as follows: 1) Cell transfection: 1 ⁇ 10 4 cells were seeded in a 48-well plate and cultured for 24 hours.
  • Fig. 2A, Fig. 2B and Fig. 2C The results are shown in Fig. 2A, Fig. 2B and Fig. 2C. It can be seen from Fig. 2A and Fig. 2B that, under the stimulation of PDGFBB, compared with the control (Ctrl-1 is PDGF untreated, indicating that the cell function is normal, Ctrl-2 is PDGF treatment). It indicates that the cell function is abnormal.)
  • Overexpression of miR-1181 reduced the proliferation of human pulmonary artery smooth muscle cells by 30%.
  • overexpression of miR-1181 also promoted the proliferation of human pulmonary artery smooth muscle markers.
  • the expression of white PCNA was down-regulated by 33%; indicating that miR-1181 has a significant inhibitory effect on growth factor-induced proliferation of pulmonary artery smooth muscle cells.
  • the present application found that small miRNAs targeting STAT3 inhibit the activity of this signaling pathway by inhibiting the expression of STAT3 molecules, and can be used as a novel STAT3 kinase inhibitor for the treatment of abnormal activation by the STAT3 kinase signaling pathway.
  • the disease caused.

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Abstract

本申请涉及一种靶向STAT3信号通路miRNA及其制备方法和应用，具体涉及一种靶向抑制STAT3基因的小分子RNA-miR-1181及其应用，所述miRNA的核苷酸序列为SEQ ID NO.1或与其具有至少90％同一性的核苷酸序列。本申请miRNA具有内源性、特异性靶向抑制STAT3(信号转导子和转录激活因子3)信号通路，能够制备抑制由于STAT3介导的信号通路异常激活所引起疾病的药物。

Description

一种靶向STAT3信号通路miRNA及其制备方法和应用 技术领域
本申请涉及生物医学领域，尤其涉及一种靶向STAT3信号通路miRNA及其制备方法和应用，具体涉及一种靶向STAT3信号通路的miRNA-miR-1181及其应用，特别涉及hsa-miR-1182在制备由STAT3介导的信号通路异常激活所引起疾病的药物中的应用。
背景技术
信号转导子和转录激活因子3(STAT3)是细胞内重要的具有信号转导功能和转录活化功能的蛋白，在结构上由6个区域组成：N端氨基酸保守序列、螺旋区、DNA结合区、SH3结构域、SH2结构域及C端的转录活化区。细胞在接受生长因子、细胞因子等外源信号刺激后，经由酪氨酸激酶相关受体以及酪氨酸激酶JAK等分子的信号传递，从而激活STAT3信号通路，最终作用于细胞核内特异的DNA片段，调控靶基因转录。STAT3信号转导通路与细胞生长、分化和程序性死亡及血管形成密切相关。
大量研究结果表明，STAT3激酶信号通路异常激活，与心力衰竭、心肌梗死、压力负荷导致的心肌肥大、缺血预处理诱导的心肌保护、局部缺血再灌注、高血压及心绞痛等心血管疾病的发生和发展密切相关，极具潜力作为心血管疾病治疗的药物新靶点。此外，该信号通路持续活化还与胃肠道疾病、炎症性疾病、白血病、前列腺、神经系统疾病和各种肿瘤(如肝癌、胃癌、乳腺癌、卵巢癌)等疾病的形成相关。当前，开发STAT3激酶信号通路的抑制剂已经成为新药研究的热点，然而由于目前很多抑制剂具有生物稳定性差、利用度较低、制备工艺繁琐等缺点，至今还没有针对STAT3信号通路的有效抑制物。
CN 1778918A公开了抑制Stat3基因表达的siRNA及其制备方法，该siRNA表达质粒包括pBS/U6载体；编码特异性抑制Stat3基因表达的siRNA的DNA片段。但该抑制剂利用度交底，生物稳定性较差。
目前研究较多的STAT3激酶信号通路抑制剂还包括肽和拟肽类抑制剂，天然产物类抑制剂以及通过计算机药物虚拟筛选技术发现的小分子抑制剂。
(1)肽和拟肽类抑制剂是以STAT3信号通路上相关分子磷酸化产物的氨基酸残基序列为模板设计的磷酸肽，可阻断STAT3信号通路的传递从而达到抑制效果，但此类抑制剂易在体内代谢失活，且生物利用度较低。(2)天然产物类抑制剂主要包括萜类和黄酮类等天然化学产物，对STAT3信号通路具有抑制作用的天然活性分子，但获取该类生物活性化合物工艺复杂，流程繁琐，且不能特异的对STAT3信号通路进行抑制。(3)通过计算机药物虚拟筛选技术，能够获得一系列对STAT3信号通路具有抑制活性的小分子化合物，但很多筛选的小分子化合物合成较为困难，且很难保持高的生物抑制活性。
因此，现有技术还有待于改进和发展，需研发效果更好的STAT3激酶信号通路抑制剂。
发明内容
以下是对本文详细描述的主题的概述。本概述并非是为了限制权利要求的保护范围。
针对现有技术的不足，本申请提供了一种靶向STAT3信号通路miRNA及其制备方法和应用，所述miRNA具有内源性、特异性靶向抑制STAT3(信号转导子和转录激活因子3)信号通路，能够制备抑制由于STAT3介导的信号通路异常激活所引起疾病的药物。
为达此目的，本申请采用以下技术方案：
第一方面，本申请提供一种靶向抑制STAT3信号通路的miRNA，所述miRNA的核苷酸序列为SEQ ID NO.1或与其具有至少90％同一性的核苷酸序列。
所述SEQ ID NO.1所示的核苷酸序列如下：5’-CCGUCGCCGCCACCCGAGCCG-3’.
根据本申请，所述miRNA的核苷酸序列为SEQ ID NO.1所示的核苷酸序列。
本申请中，通过在人肺动脉平滑肌细胞中过表达本申请miRNA，即miR-1181，可抑制血小板衍生生长因子BB(PDGFBB)对STAT3信号通路的激活或表达；通过细胞功能实验，发现过表达本申请miRNA，即miR-1181可抑制血小板衍生生长因子BB(PDGFBB)激活STAT3信号通路诱导的，人肺动脉平滑肌细胞的异常增殖。
本申请中，所述miRNA的制备方法包括成熟序列全基因合成和克隆表达的方法，所述克隆表达的方法为本领域的常规技术，本领域技术人员可以根据需要采用合适的方法进行构建，在此不作特殊限定。
在一个具体的实施例中，所述miRNA的构建方法包括如下步骤：
(1)设计引物，以人基因组DNA为模板，进行PCR扩增；
(2)将PCR扩增的片段插入plv4/EGFP慢病毒载体的XhoI和EcoRI之间；
(3)将构建的慢病毒载体转染细胞，表达miRNA。
所述引物的核苷酸序列如SEQ ID NO.2-3所示；
所述SEQ ID NO.2所示的核苷酸序列如下：5’-CCGCTCGAGCGCCCGATGACGCGGAGT-3’；
所述SEQ ID NO.3所示的核苷酸序列如下：5’-CCGGAATTCGGTCCCTGGGCAGTATCG-3’.
第二方面，本申请提供如第一方面所述的miRNA在制备用于诊断、预防、治疗或预后评估疾病的药物或试剂盒的用途。
根据本申请，所述疾病为STAT3介导的信号通路异常激活引起的疾病。
根据本申请，所述疾病为心脑血管疾病，胃肠道疾病、炎症性疾病、白血病、前列腺疾病、神经系统疾病或肿瘤中的任意一种或至少两种的组合。
本申请中，所述心脑血管疾病包括心肌肥厚、脑缺血再灌注损伤、心力衰竭、心肌梗死、局部缺血再灌注、高血压及动脉粥样硬化等心脑血管疾病；所述肿瘤包括食道癌或卵巢癌等恶性肿瘤。
本申请中的“脑缺血再灌注”是指结扎小鼠冠状动脉左前降支造成心肌缺血一段时间之后，松开结扎线使心肌达到再灌注。
第三方面，本申请提供一种慢病毒载体，所述慢病毒载体包含如第一方面所述的miRNA的核苷酸序列。
第四方面，本申请提供一种重组慢病毒，将包含如第三方面所述的慢病毒载体与包装辅助质粒共转染哺乳动物细胞得到重组慢病毒。
根据本申请，所述哺乳动物细胞为HEK293T细胞。
第五方面，本申请提供一种药物组合物，所述组合物包括如第一方面所述的miRNA。
根据本申请，所述miRNA的有效剂量为10-200mg/kg，例如可以是10mg/kg、20mg/kg、30mg/kg、40mg/kg、50mg/kg、60mg/kg、70mg/kg、80mg/kg、90mg/kg、100mg/kg、120mg/kg、130mg/kg、150mg/kg、160mg/kg、180mg/kg或200mg/kg。
根据本申请，所述药物组合物还包括药学上可接受的病毒、载体或辅料中的任意一种或至少两种的组合。
本申请中，包括药学上可接受的病毒、载体或辅料选自壳聚糖、胆固醇、脂质体或纳米颗粒等。
第六方面，本申请提供一种用于诊断和/或预后评估疾病的试剂盒，所述试剂盒包括如第一方面所述的miRNA和/或用于特异性检测miRNA的探针或引物。
根据本申请，所述探针或引物的核苷酸序列如SEQ ID NO.2-3所示；
所述SEQ ID NO.2所示的核苷酸序列如下：5’-CCGCTCGAGCGCCCGATGACGCGGAGT-3’；
所述SEQ ID NO.3所示的核苷酸序列如下：5’-CCGGAATTCGGTCCCTGGGCAGTATCG-3’.
与现有技术相比，本申请具有如下有益效果：
(1)本申请发现小miRNA以STAT3为靶点，通过抑制STAT3分子的表达从而抑制该信号通路的活性，可作为一种新的STAT3激酶抑制剂用于治疗由STAT3激酶信号通路异常激活所引起的疾病；
(2)本申请的miRNA是人体内源性miRNA，对人体的毒性较小，能够较好的被人体利用，可通过静脉注射导入人体内，通过血液循环运输到特定部位，从而达到治疗目的；
(3)本申请的miRNA可作为一种新的STAT3激酶信号通路抑制剂用于治疗心血管疾病，胃肠道疾病、炎症性疾病、前列腺、神经系统疾病和恶性肿瘤等；
(4)本申请的miRNA制备方法简单，既可以采用全基因合成也可以采用克隆表达，制备得到的miRNA可通过静脉注射导入人体内，通过血液循环运输到特定的部位，从而起到相应的抑制效果。
附图说明
图1A为miR-1181与STAT3的3’-UTR结合位点；
图1B为过表达miR-1181对STAT3-UTR或STAT3-UTR-mut荧光素酶活性的影响，其中，STAT3-UTR-mut为基于结合位点构建3’-UTR的突变载体；
图1C为过表达miR-1181对人肺动脉平滑肌细胞STAT3总蛋白表达影响，其中，Mimic Ctrl为化学合成的miR-1181类似物；
图1D为过表达miR-1181对PDGFBB刺激人肺动脉平滑肌细胞后，磷酸化激活状态STAT3蛋白表达影响，其中，Mimic Ctrl为化学合成的结构相似但无意义的序列，Mimic Ctrl-1未用PDGF处理，Mimic Ctrl-2用PDGF处理；
图2A为EdU检测PDGFBB刺激人肺动脉平滑肌细胞后，过表达miR-1181对细胞增殖影响的典型荧光图片，其中，Mimic Ctrl为化学合成的结构相似但无意义的序列，Mimic Ctrl-1未用PDGF处理，Mimic Ctrl-2用PDGF处理；
图2B为EdU检测PDGFBB刺激人肺动脉平滑肌细胞后，过表达miR-1181对细胞增殖影响的统计结果，其中，Mimic Ctrl为化学合成的结构相似但无意义的序列，Mimic Ctrl-1未用PDGF处理，Mimic Ctrl-2用PDGF处理；
图2C为PDGFBB刺激人肺动脉平滑肌细胞后，过表达miR-1181对PCNA蛋白表达的影响，其中，Mimic Ctrl为化学合成的结构相似但无意义的序列，Mimic Ctrl-1未用PDGF处理，Mimic Ctrl-2用PDGF处理。
具体实施方式
为更进一步阐述本申请所采取的技术手段及其效果，以下结合附图并通过具体实施方式来进一步说明本申请的技术方案，但本申请并非局限在实施例范围内。
除非特别说明，以下实施例中所采用的各种原料均来源于市售，所采用的 方法均为常规技术手段。
实施例1 miRNA miR-1181的制备
miR-1181的成熟序列为5’-CCGUCGCCGCCACCCGAGCCG-3’(22bp，SEQ ID NO.1)可通过以下方式制得：
1)化学合成
miR-1181的类似物(miR-1181mimic)由广州瑞博生物科技有限公司合成，为双链RNA，能模拟内源性成熟miR-1181高水平表达；标准纯化，于-20℃保存。用无RNA酶的水溶解配置成20M的储存液，置于-80℃备用，使用时稀释成所需浓度。
2)病毒载体介导的miR-1181的表达
(1)在miRBase(http：//www.mirbase.org/)数据库中得到miR-1181的成熟序列和前体序列(pre-miRNA)信息。在人基因组数据库中找到成熟序列两个约300bp的侧翼序列，依据侧翼序列设计引物，并分别加上XhoI和EcoRI的酶切位点和保护碱基，设计引物：
上游引物5’-CCGCTCGAGCGCCCGATGACGCGGAGT-3’(SEQ ID NO.2)；
下游引物的序列为5’-CCGGAATTCGGTCCCTGGGCAGTATCG-3’(SEQ ID NO.3)；
(2)以人基因组DNA为模板，通过设计的引物进行PCR扩增，PCR反应条件为：95℃变性2分钟，95℃30秒，55℃30秒，72℃40秒进行35个循环，72℃延伸3分钟；
(3)将扩增的PCR产物琼脂糖凝胶调用后，用胶回收试剂盒(E.Z.N.A Gel Extraction Kit，Omgega)进行片段回收，用限制性内切酶XhoI和EcoRI(NEB)进行双酶切，将酶切后的PCR片段按DNA纯化试剂盒(E.Z.N.A Cycle-Pure Kit， Omgega)说明进行回收后作为插入片段，插入PLV4/EGFP慢病毒载体中；载体以限制性内切酶XhoI和EcoRI(NEB)进行双酶切后进行回收、纯化作为载体片段；
(4)将插入片段与载体片段利用T4连接酶(Promega)于16℃连接2h，然后将连接产物转化至大肠杆菌感受态STBL3，37℃过夜培养后挑取生长良好的单菌落在含有载体相应抗生素的LB培养基中扩增，提取质粒后进行测序验证；
(5)制得的DNA载体可转染入细胞，瞬时表达miRNA，即miR-1181。
制得的慢病毒载体可用于慢病毒包装，步骤如下所述：
(1’)慢病毒包装所需细胞系为HEK293T细胞系，所需质粒包括慢病毒表达载体pLVX-miR-1181和慢病毒包装质粒。细胞转染采用常规磷酸钙转染方法，用2M CaCl2与质粒混匀，然后逐滴加入2×HBS溶液并轻摇混匀，最后将混合溶液缓慢均匀加入细胞培养液中。转染12小时后更换培养液；
(2’)转染48-72小时后，收集含有病毒颗粒的细胞培养液，4000rpm室温离心10分钟，收集上清液并分装置于-80℃备用；
(3’)包装好的慢病毒可用于感染所选的细胞，通过抗生素嘌呤霉素筛选后获得可稳定过表达miR-1181的细胞。
实施例2 miR-1181靶向抑制STAT3分子及其信号通路
(1)细胞培养：HEK293A细胞(购自ATCC，Manassas，VA)，用含10％胎牛血清的DMEN培养基培养，收集生长状态良好的细胞，以6×104/孔接种于24孔板内，37℃，5％CO2培养24小时；
(2)人肺动脉平滑肌细胞购自Lonza(Walkersville，MD)，于含有5％血清的SMCM完全培养基中培养，收集生长状态良好的细胞，离心技术，以6×105 接种于60mm皿内，37℃，5％CO2培养24小时；
(3)转染：待细胞密度达70％时用磷酸钙法转染细胞，共转染CSTAT3的3’-UTR双荧光素酶报告载体或突变的3’-UTR双荧光素酶报告载体，miR-1181过表达载体pLVX-CMV-miR-1181或pLVX-CMV-control，转染两天后，移去培养液，PBS清洗两次后用50μL 1×裂解液裂解细胞；
(4)将化学合成的miR-1181类似物(mimic)用于过表达或抑制miR-1181的表达，以cel-miR-67作为阴性对照，均购自广州瑞博生物。将细胞种于培养皿中，当细胞密度达到70％时，通过Lipofectamine 2000(Invitrogen)转染，转染6小时后更换培养基，继续培养24小时用于实验。
荧光素酶检测
利用双荧光素酶检测试剂盒(E1810，Promega)参考说明书进行荧光素酶活性检测，用萤火虫荧光素酶数值除以内参海肾荧光素酶读值，即可得到校正的荧光素酶活性数值。将CSTAT3的3’-UTR载体的荧光素酶活性数值与对照组进行比较。
所述miR-1181与STAT3的3’-UTR结合位点如图1A所示，荧光霉素检测结果如图1B所示，与对照相比，过表达miR-1181组的STAT3荧光素酶活性显著降低，说明miR-1181可能与STAT3的3’-UTR能够靶向结合；基于结合位点构建3’-UTR的突变载体STAT3-UTR-mut，利用突变体载体再次进行双荧光素酶活性检测，与野生型UTR相比，STAT3结合位点的突变引起荧光素酶活性的恢复。
蛋白杂交检测
收集细胞总蛋白，经蛋白定量后进行聚丙烯酰胺凝胶电泳，利用STAT3蛋白抗体检测STAT3表达，如图1C所示，与对照相比，过表达miR-1181可抑制 人肺动脉平滑肌细胞内源性STAT3的表达；利用STAT3磷酸化抗体检测磷酸化STAT3，与对照相比(Ctrl-1为PDGF未处理表示细胞功能正常状态，Ctrl-2为PDGF处理表示细胞功能异常状态)，如图1D所示，过表达miR-1181可显著抑制STAT3的磷酸化。
本实施例结果表明：miR-1181通过靶向STAT3的3’-UTR抑制STAT3的表达以及STAT3激酶信号通路。
实施例3 miR-1181抑制血小板衍生生长因子BB(PDGFBB)诱导人肺动脉平滑肌细胞增殖。
为探讨miR-1181对血小板衍生生长因子BB(PDGFBB)激活STAT3信号通路，从而引起人肺动脉平滑肌细胞功能异常的调控作用：用表达miR-1181的慢病毒感染来提高人肺动脉平滑肌细胞中的miR-1181的水平，然后通过细胞增殖标志蛋白PCNA和EdU细胞增殖检测确定细胞的增殖水平。
使用EdU细胞增殖检测试剂盒(锐博生物)进行EdU标记，根据试剂盒说明书进行实验操作，主要步骤如下：1)细胞转染：接种1×104个细胞于48孔板，培养24h后进行转染实验，第二天培养24h后用无血清培养基进行饥饿处理，然后加入血小板衍生生长因子BB(PDGFBB)以及20μM EdU继续4小时；2)细胞染色拍照：将细胞用4％多聚甲醛室温固定30分钟，0.5％Triton X-100进行膜通透10分钟，PBS清洗，然后每孔加入150μL 1×Apollo染色液孵育30分钟，DNA用1×Hochest(每孔150μL)染色5分钟，在荧光显微镜下拍照。
结果如图2A、图2B和图2C所示，从图2A和图2B可见，在PDGFBB刺激下，与对照相比(Ctrl-1为PDGF未处理表示细胞功能正常状态，Ctrl-2为PDGF处理表示细胞功能异常状态)，过表达miR-1181分别使人肺动脉平滑肌细胞增殖下降30％；从图2C可见，过表达miR-1181还使人肺动脉平滑肌增殖标记蛋 白PCNA表达下调33％；说明miR-1181对生长因子引起的肺动脉平滑肌细胞增殖具有明显的抑制作用。
综上所述，本申请发现小miRNA以STAT3为靶点，通过抑制STAT3分子的表达从而抑制该信号通路的活性，可作为一种新的STAT3激酶抑制剂用于治疗由STAT3激酶信号通路异常激活所引起的疾病。
申请人声明，本申请通过上述实施例来说明本申请的详细方法，但本申请并不局限于上述详细方法，即不意味着本申请必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。

Claims (13)

  1. 一种靶向STAT3信号通路的miRNA，其核苷酸序列为SEQ ID NO.1或与其具有至少90％同一性的核苷酸序列。
  2. 根据权利要求1所述的miRNA，其核苷酸序列为SEQ ID NO.1所示的核苷酸序列。
  3. 根据权利要求1或2所述的miRNA在制备用于诊断、预防、治疗或预后评估疾病的药物或试剂盒的用途。
  4. 根据权利要求3所述的用途，其中，所述疾病为STAT3介导的信号通路异常激活引起的疾病。
  5. 根据权利要求4所述的用途，其中，所述疾病为心脑血管疾病，胃肠道疾病、炎症性疾病、白血病、前列腺疾病、神经系统疾病或肿瘤中的任意一种或至少两种的组合。
  6. 一种慢病毒载体，其包含如权利要求1或2所述的miRNA的核苷酸序列。
  7. 一种重组慢病毒，其通过将如权利要求6所述的慢病毒载体与包装辅助质粒共转染哺乳动物细胞而得到。
  8. 根据权利要求7所述的重组慢病毒，其中，所述哺乳动物细胞为HEK293T细胞。
  9. 一种药物组合物，其包括如权利要求1或2所述的miRNA。
  10. 根据权利要求9所述的药物组合物，其中，所述miRNA的有效剂量为10-200mg/kg。
  11. 根据权利要求9所述的药物组合物，其还包括药学上可接受的病毒、载体或辅料中的任意一种或至少两种的组合。
  12. 一种用于诊断和/或预后评估疾病的试剂盒，其包括如权利要求1或2 所述的miRNA和/或用于特异性检测miRNA的探针或引物。
  13. 根据权利要求12所述的试剂盒，其中，所述探针或引物的核苷酸序列如SEQ ID NO.2-3所示。
PCT/CN2017/112216 2017-09-13 2017-11-22 一种靶向STAT3信号通路miRNA及其制备方法和应用 WO2019052000A1 (zh)

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