WO2019047368A1 - 一种靶向cJun信号通路的miRNA及其制备方法和应用 - Google Patents

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

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WO2019047368A1
WO2019047368A1 PCT/CN2017/111054 CN2017111054W WO2019047368A1 WO 2019047368 A1 WO2019047368 A1 WO 2019047368A1 CN 2017111054 W CN2017111054 W CN 2017111054W WO 2019047368 A1 WO2019047368 A1 WO 2019047368A1
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mirna
cjun
nucleotide sequence
signaling pathway
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钱政江
李翔
杨海洋
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Shenzhen Institute of Advanced Technology of CAS
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Definitions

  • the present invention relates to the field of biomedicine, in particular to a miRNA targeting a cJun signaling pathway and a preparation method and application thereof, in particular to a small molecule RNA-miR-4632 targeted for inhibiting cJun gene and application thereof, in particular to hsa-
  • miR-4632 in the preparation of a drug caused by a cJun-mediated abnormal activation of a signaling pathway.
  • the cell proto-oncogene cJun is an important transcription factor in the nucleus and a member of the activation protein (AP-1) family; cJun participates in many genes in the form of a single transcription factor or AP-1. Transcriptional regulation, and thereby play various biological functions. After being stimulated by various external signals such as cytokines, growth signals, ultraviolet rays or environmental pollutants, the cells are induced to activate cJun via tyrosine kinase-related receptors and cJun amino terminal kinase JNK. A specific DNA fragment in the nucleus regulates transcription of the target gene.
  • the cJun signaling pathway is involved in the regulation of cell proliferation, differentiation, cycle and apoptosis.
  • cJun mediates the abnormal activation of signaling pathways, and the occurrence of cardiovascular and cerebrovascular diseases such as cardiac hypertrophy, cerebral ischemia-reperfusion injury, heart failure, myocardial infarction, ischemia reperfusion, hypertension and atherosclerosis. Development related.
  • cardiovascular and cerebrovascular diseases such as cardiac hypertrophy, cerebral ischemia-reperfusion injury, heart failure, myocardial infarction, ischemia reperfusion, hypertension and atherosclerosis.
  • cardiovascular and cerebrovascular diseases such as cardiac hypertrophy, cerebral ischemia-reperfusion injury, heart failure, myocardial infarction, ischemia reperfusion, hypertension and atherosclerosis. Development related.
  • the continuous activation of this signaling pathway is also closely related to the formation of gastrointestinal diseases, inflammatory diseases, prostate, nervous system diseases and malignant tumors (such as esophageal cancer, ovarian cancer). Therefore, the cJun signaling pathway has great potential as a new drug target for the treatment of related
  • CN 104080469A discloses the use of novel JNK inhibitor molecules and their use in a method for treating human or animal body by treatment, which is mainly directed to the JJ upstream signal molecule JNK, which has poor biostability and availability. Low, complicated preparation process and other shortcomings.
  • Peptide and peptidomimetic inhibitors are phosphopeptides designed by using the amino acid residue sequence of the related molecule phosphorylation product on the JNK signaling pathway as a template to block the transmission of JNK signaling to cJun to 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, natural active molecules which inhibit the JNK signaling pathway, thereby inhibiting cJun Signal transmission, but the process of obtaining such biologically active compounds is complicated, the process is cumbersome, and the cJun signaling pathway cannot be specifically inhibited;
  • a series of inhibitory activities against JNK signaling pathway can be obtained by computer drug virtual screening technology. Small molecule compounds, but many screened small molecule compounds are
  • the present application provides a miRNA targeting a cJun signaling pathway and a preparation method and application thereof, which have endogenous and specific targeting inhibition cJun (proto-oncogene) signaling pathway, A drug capable of inhibiting a disease caused by abnormal activation of a cJun-mediated signaling pathway can be prepared.
  • the application provides a miRNA that targets a cJun 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'-UGCCGCCCUCUCGCUGCUCUAG-3'.
  • the nucleotide sequence of the miRNA is the nucleotide sequence shown in SEQ ID NO.
  • the activation or expression of cJun 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'-CCGCTCGAGGACGAGCAGGACTGCGGA-3';
  • the nucleotide sequence shown in SEQ ID NO. 3 is as follows: 5'-CCGGAATTCCAAGGACCTGAGCCCCAC-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 cJun-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, tumors or pulmonary hypertension.
  • 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 comprising a recombinant lentivirus obtained by co-transfecting a lentiviral vector according to the third aspect with a packaging helper plasmid into a mammalian cell;
  • 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'-CCGCTCGAGGACGAGCAGGACTGCGGA-3';
  • the nucleotide sequence shown in SEQ ID NO. 3 is as follows: 5'-CCGGAATTCCAAGGACCTGAGCCCCAC-3'.
  • This application finds that small miRNAs target cJun and inhibit the activity of this signaling pathway by inhibiting the expression of cJun molecules. It can be used as a novel cJun kinase inhibitor for the treatment of abnormal activation of cJun 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 cJun kinase signaling pathway inhibitor for the treatment of cardiovascular diseases, gastrointestinal diseases, inflammatory diseases, prostate, nervous system diseases and malignant tumors.
  • Figure 1A shows the 3'-UTR binding site of miR-4632 and cJ Jun;
  • Figure 1B shows the effect of overexpression of miR-4632 on cJun-UTR or cJun-UTR-mut luciferase activity, wherein cJun-UTR-mut is a mutant vector constructed based on the binding site for 3'-UTR;
  • Figure 1C shows the effect of overexpression of miR-4632 on the expression of cJun total protein in human pulmonary artery smooth muscle cells, wherein Mimic Ctrl is a chemically synthesized miR-4632 analog;
  • FIG. 1D shows the effect of overexpression of miR-4632 on phosphorylation-activated cJun protein expression after PDGFBB stimulation of 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 is processed with PDGF;
  • FIG. 2A is a typical fluorescent picture of the effect of overexpression of miR-4632 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-4632 on cell proliferation after PDGFB stimulates human pulmonary artery smooth muscle cells by EdU.
  • Mimic Ctrl is a structurally similar but meaningless sequence of chemical synthesis, and Mimic Ctrl-1 is not treated with PDGF. , Mimic Ctrl-2 is processed with PDGF;
  • FIG. 2C shows the effect of overexpression of miR-4632 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-4632 is 5'-UGCCGCCCUCUCGCUGCUCUAG-3' (22 bp, SEQ ID NO. 1) can be obtained by:
  • miR-4632mimic The analog of miR-4632 (miR-4632mimic) was synthesized by Guangzhou Ruibo Biotechnology Co., Ltd. It is a double-stranded RNA that mimics high-level expression of endogenous mature miR-4632; 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.
  • the mature sequence and precursor sequence (pre-miRNA) information of miR-4632 was obtained in the miRBase (http://www.mirbase.org/) database. Two flanking sequences of about 300 bp in the mature sequence were found in the human genome database. Primers were designed based on the flanking sequences, and the restriction sites and protected bases of XhoI and EcoRI were added respectively to design primers:
  • Upstream primer 5'-CCGCTCGAGGACGAGC AGGACTGCGGA-3' (SEQ ID NO. 2);
  • the sequence of the downstream primer is 5'-CCGGAATTCCAAGGACCTGAGCCC CAC-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, ie miR-4632.
  • 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-4632 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-4632 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;
  • the chemically synthesized miR-4632 analog (mimic) was used to overexpress or inhibit the expression of miR-4632, and cel-miR-67 was used as a negative control, all purchased from Guangzhou Ruibo Bio.
  • the cells were seeded in a Petri dish. When the cell density reached 70%, transfection was performed 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 CCJUN were compared with the control group.
  • the 3'-UTR binding site of the miR-4632 and cJun is shown in FIG. 1A, and the fluorescein detection result is shown in FIG. 1B.
  • the CJUN luciferase activity of the overexpressing miR-4632 group is remarkable.
  • the decrease indicates that miR-4632 may bind to the 3'-UTR of cJun; the mutation vector cJun-UTR-mut of 3'-UTR is constructed based on the binding site, and the dual luciferase activity is detected again by using the mutant vector.
  • Mutation of the cJun binding site results in restoration of luciferase activity compared to wild-type UTR.
  • cJun protein antibody was used to detect cJun expression.
  • Fig. 1C overexpression of miR-4632 inhibited endogenous human pulmonary artery smooth muscle cells compared with the control.
  • Overexpression of miR-4632 significantly inhibited phosphorylation of cJun.
  • Example 3 miR-4632 inhibits platelet-derived growth factor BB (PDGFBB)-induced proliferation of human pulmonary artery smooth muscle cells.
  • PDGFBB platelet-derived growth factor BB
  • 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).
  • Ctrl-1 is PDGF untreated, indicating that the cell function is normal, Ctrl-2 is PDGF treatment.
  • Overexpression of miR-4632 reduced the proliferation of human pulmonary artery smooth muscle cells by 32%
  • Fig. 2C overexpression of miR-4632 also down-regulated the expression of PCNA of human pulmonary artery smooth muscle proliferation marker protein by 31%; 4632 has a significant inhibitory effect on the proliferation of pulmonary artery smooth muscle cells induced by growth factors.
  • the present application found that small miRNAs targeting cJun inhibit the activity of this signaling pathway by inhibiting the expression of cJun molecules, and can be used as a novel cJun kinase inhibitor for the treatment of abnormal activation by the cJun kinase signaling pathway.
  • the disease caused.

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Abstract

一种靶向cJun信号通路的miRNA及其制备方法和应用，具体涉及一种靶向抑制cJun基因的小分子RNA-miR-4632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。所述miRNA具有内源性、特异性靶向抑制cJun信号通路，能够制备抑制由于cJun介导的信号通路异常激活所引起疾病的药物。

Description

一种靶向cJun信号通路的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cJun信号通路的miRNA及其制备方法和应用，具体涉及一种靶向抑制cJun基因的小分子RNA-miR-4632及其应用，特别涉及hsa-miR-4632在制备由cJun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
细胞原癌基因cJun是核内重要的转录因子，同时也是核转录激活蛋白1(activation protein，AP-1)家族的组成成员；cJun以单独的转录因子或以AP-1的形式参与很多基因的转录调控，并以此发挥各种生物学功能。细胞在接受细胞因子、生长信号、紫外线或环境污染物等各种外界信号刺激后，经由酪氨酸激酶相关受体以及cJun氨基末端激酶JNK等信号分子的传递，从而诱导cJun的活化，最终作用于细胞核内特异的DNA片段，调节靶基因转录。cJun信号通路参与调控细胞增殖、分化、周期和凋亡等功能。
研究表明，cJun介导信号通路的异常活化，与心肌肥厚、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的发生和发展相关。此外，该信号通路持续活化还与胃肠道疾病、炎症性疾病、前列腺、神经系统疾病和恶性肿瘤(如食道癌、卵巢癌)等形成密切相关。因此，cJun信号通路极具潜力作为治疗相关疾病的药物新靶点。
然而，当前开发针对cJun信号通路的特异性抑制剂相对较少。CN 104080469A公开了新的JNK抑制剂分子的用途及其在通过治疗处理人或动物体的方法中的用途，其主要针对于cJun上游信号分子JNK，生物稳定性差、利用度较 低、制备工艺繁琐等缺点。
其他针对cJun信号通路的特异性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JNK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JNK信号传递至cJun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JNK信号通路具有抑制作用的天然活性分子，从而抑制cJun信号的传递，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cJun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JNK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cJun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cJun信号通路的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cJun(细胞原癌基因)信号通路，能够制备抑制由于cJun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cJun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’-UGCCGCCCUCUCGCUGCUCUAG-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-4632，可抑制血小板衍生生长因子BB(PDGFBB)对cJun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-4632可抑制血小板衍生生长因子BB(PDGFBB)激活cJun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’-CCGCTCGAGGACGAGCAGGACTGCGGA-3’；
所述SEQ ID NO.3所示的核苷酸序列如下：5’-CCGGAATTCCAAGGACCTGAGCCCCAC-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cJun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、肿瘤或肺动脉高压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的重组慢病毒；
优选地，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’-CCGCTCGAGGACGAGCAGGACTGCGGA-3’；
所述SEQ ID NO.3所示的核苷酸序列如下：5’-CCGGAATTCCAAGGACCTGAGCCCCAC-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cJun为靶点，通过抑制cJun分子的表达从而抑制该信号通路的活性，可作为一种新的cJun激酶抑制剂用于治疗由cJun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cJun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。
附图说明
图1A为miR-4632与cJ Jun的3’-UTR结合位点；
图1B为过表达miR-4632对cJun-UTR或cJun-UTR-mut荧光素酶活性的影响，其中，cJun-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-4632对人肺动脉平滑肌细胞cJun总蛋白表达影响，其中，Mimic Ctrl为化学合成的miR-4632类似物；
图1D为过表达miR-4632对PDGFBB刺激人肺动脉平滑肌细胞后，磷酸化激活状态cJun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-4632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-4632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-4632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-4632的制备
miR-4632的成熟序列为5’-UGCCGCCCUCUCGCUGCUCUAG-3’(22bp，SEQ ID NO.1)可通过以下方式制得：
1)化学合成
miR-4632的类似物(miR-4632mimic)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-4632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-4632的表达
(1)在miRBase(http：//www.mirbase.org/)数据库中得到miR-4632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’-CCGCTCGAGGACGAGC AGGACTGCGGA-3’(SEQ ID NO.2)；
下游引物的序列为5’-CCGGAATTCCAAGGACCTGAGCCC CAC-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-4632。
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-4632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-4632的细胞。
实施例2 miR-4632靶向抑制cJun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CCJUN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-4632过表达载体pLVX-CMV-miR-4632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-4632类似物(mimic)用于过表达或抑制miR-4632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CCJUN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-4632与cJun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-4632组的CJUN荧光素酶活性显著降低，说明miR-4632可能与cJun的3’-UTR能够靶向结合；基于结合位点构建3’-UTR的突变载体cJun-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，cJun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cJun蛋白抗体检测cJun表达，如图1C所示，与对照相比，过表达miR-4632可抑制人肺动脉平滑肌细胞内源性cJun的表达；利用cJun磷酸化抗体检测磷酸化cJun，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-4632可显著抑制cJun的磷酸化。
本实施例结果表明：miR-4632通过靶向CJUN的3’-UTR抑制CJUN的表达以及CJUN激酶信号通路。
实施例3 miR-4632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-4632对血小板衍生生长因子BB(PDGFBB)激活cJun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-4632的慢病毒感染来提高人肺动脉平滑肌细胞中的miR-4632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-4632分别使人肺动脉平滑肌细胞增殖下降32％；从图2C可见，过表达miR-4632还使人肺动脉平滑肌增殖标记蛋白PCNA表达下调31％；说明miR-4632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cJun为靶点，通过抑制cJun分子的表达从而抑制该信号通路的活性，可作为一种新的cJun激酶抑制剂用于治疗由cJun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cJun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cJun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、肿瘤或肺动脉高压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/或用于特异性检测所述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。
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