WO2019227687A1 - 一种HDAC4信号通路miRNA抑制剂及其过表达方法和应用 - Google Patents

一种HDAC4信号通路miRNA抑制剂及其过表达方法和应用 Download PDF

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WO2019227687A1
WO2019227687A1 PCT/CN2018/100938 CN2018100938W WO2019227687A1 WO 2019227687 A1 WO2019227687 A1 WO 2019227687A1 CN 2018100938 W CN2018100938 W CN 2018100938W WO 2019227687 A1 WO2019227687 A1 WO 2019227687A1
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hdac4
mir
inhibitor
expression vector
mirna
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French (fr)
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钱政江
李翔
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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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

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  • the present application belongs to the field of biomedical technology, and relates to an HDAC4 signal pathway miRNA inhibitor, and an overexpression method and application thereof.
  • Histone deacetylase 4 (histone deacetylase, HDAC4) is an important member of the family of histone deacetylases, which plays an important role in chromosome structural modification and gene expression regulation.
  • HDAC4 histone deacetylase 4
  • the acetylation of histones facilitates the dissociation of DNA from histone octamers and relaxes the structure of nucleosomes, thereby promoting the specific binding of various transcription factors and co-transcription factors to DNA binding sites and activating gene transcription.
  • histone acetylation and histone deacetylation are a dynamic equilibrium process, which is jointly regulated by histone acetyltransferase (HAT) and histone deacetylase (HDAC).
  • HAT transfers the acetyl group of acetyl-CoA to a specific lysine residue at the amino terminal end of histone.
  • HDAC deacetylates histone, tightly binds to negatively charged DNA, densely curls chromatin, and inhibits gene transcription.
  • HDAC4 gene is closely related to cell growth, differentiation and programmed death and angiogenesis.
  • Abnormal expression of HDAC4 signaling pathway is related to heart failure, myocardial infarction, myocardial hypertrophy caused by stress load, and myocardial protection induced by ischemic preconditioning.
  • cardiovascular diseases such as ischemia-reperfusion, hypertension, and angina pectoris are closely related, and they have great potential as new targets for the treatment of cardiovascular diseases.
  • HDAC4 gene abnormal activation is also related to the formation of gastrointestinal diseases, inflammatory diseases, leukemia, prostate, nervous system diseases and various tumors (such as liver cancer, gastric cancer, breast cancer, ovarian cancer).
  • the development of inhibitors of the HDAC4 kinase signaling pathway has become a research hotspot.
  • HDAC4 kinase signaling pathway inhibitors that are currently being studied include peptide and peptidomimetic inhibitors, natural product inhibitors, and small molecule inhibitors discovered through computer drug virtual screening technology.
  • peptide and peptidomimetic inhibitors are phosphopeptides designed based on the amino acid residue sequences of phosphorylation products of molecules related to the HDAC4 signaling pathway, which can block the transmission of the HDAC4 signaling pathway to achieve inhibitory effects, but such inhibitors Easily metabolized in vivo and low bioavailability;
  • natural product inhibitors mainly include natural active molecules of terpenes and flavonoids, but the extraction process of such compounds is complicated, the process is tedious, and the specificity is weak; through computer drug virtualization Small molecule inhibitors obtained by screening techniques are often difficult to synthesize and have weak biological inhibitory properties.
  • CN 106344923 A discloses the application of a histone deacetylase-4 inhibitor in the preparation of a drug for treating multiple myeloma diseases.
  • the inhibitor is an siRNA against the HDAC4 gene, which can inhibit the proliferation and induce the multiple myeloma cells. Apoptosis, autophagy occurs, and downstream transcription factors are up-regulated.
  • the above siRNAs are exogenous, have certain toxicity, the screening process is complicated, and gene mutation may affect the specificity of siRNA.
  • the present application provides an HDAC4 signaling pathway miRNA inhibitor and its overexpression method and application.
  • the inhibitor includes endogenous hsa-miR-1281, which solves the existing biological problems of HDAC4 signaling pathway inhibitors. Poor stability, low utilization, and cumbersome preparation processes.
  • the present application provides the use of a miRNA as an inhibitor of the HDAC4 signaling pathway.
  • miRNA is a kind of non-coding single-stranded small-molecule RNA with a length of about 22 bases. It can regulate gene expression after transcription. The results show that miRNA expression is closely related to cell function.
  • PDGFBB platelet-derived growth factor BB
  • HDAC4 is a potential target gene of miR-1281
  • overexpression of miR-1281 has an inhibitory effect on HDAC4, and HDAC4 is a target gene of miR-1281;
  • Overexpression of miR-1281 in human pulmonary artery smooth muscle cells can inhibit the activation of HDAC4 signaling pathway by PDGFBB, and abnormal proliferation of human pulmonary artery smooth muscle cells.
  • miR-1281 is used as a targeting molecule, which has a significant inhibitory effect on the HDAC4 signaling pathway.
  • the miRNA is miR-1281.
  • nucleic acid sequence of the miR-1281 is shown in SEQ ID NO.1;
  • SEQ ID NO.1 The nucleic acid sequence shown in SEQ ID NO.1 is as follows: 5'-TCGCCTCCTCCTCTCCC-3 '.
  • the present application provides an HDAC4 signaling pathway inhibitor, the inhibitor including miRNA.
  • the miRNA includes miR-1281.
  • nucleic acid sequence of the miR-1281 is as shown in SEQ ID NO.1.
  • the present application provides a method for overexpression of an inhibitor according to the second aspect, the method comprising liposome transfection and / or recombinant expression.
  • the liposome transfection includes encapsulating chemically synthesized miRNA with liposomes, transfecting the cells, and over-expressing the inhibitor.
  • chemical synthesis refers to the artificial synthesis of miRNA mimics by biological companies. All nucleic acid sequences in this application are synthesized by Guangzhou Ruibo Biotechnology Co., Ltd. Those skilled in the art can choose different organisms according to the actual situation. the company.
  • Lipofectamine 2000 and / or Lipofectamine 3000 are used as liposomes to transfect chemically synthesized miRNA.
  • the miRNA comprises miR-1281.
  • the recombinant expression includes the following steps:
  • step (2) (2) transferring the expression vector described in step (1) into competent cells, and selecting monoclonal cells for selection of the expression vector after culturing;
  • the selected expression vector is transfected into the host cell, and the inhibitor is overexpressed.
  • the constructing the expression vector in step (1) specifically includes:
  • step (1') The amplified product described in step (1') is inserted between the XhoI and EcoRI restriction sites of the plv4 / EGFP lentiviral vector.
  • the nucleic acid sequence of the PCR primers in step (1 ') is shown in SEQ ID Nos. 2 to 3;
  • the nucleic acid sequence shown in SEQ ID No. 2 is as follows:
  • the nucleic acid sequence shown in SEQ ID NO. 3 is as follows:
  • step (3) a step of packaging the screened expression vector to obtain a lentivirus is further included.
  • host cells are infected with the lentivirus obtained through packaging, and cells that stably overexpress miR-1281 can be obtained after screening.
  • the present application provides a method for overexpression of an inhibitor according to the second aspect, comprising the following steps:
  • step (3) transferring the expression vector described in step (2) into competent cells, and selecting monoclonal cells for selection of the expression vector after culturing;
  • the present application provides a pharmaceutical composition including the HDAC4 signaling pathway inhibitor according to the second aspect.
  • the pharmaceutical composition further includes any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient, or diluent.
  • the present application provides an application of the HDAC4 signaling pathway inhibitor according to the second aspect and / or the pharmaceutical composition according to the fourth aspect in the manufacture of a medicament for treating an abnormal disease of the HDAC4 gene.
  • the disease includes any one or a combination of at least two of cardio-cerebral vascular disease, gastrointestinal disease, inflammatory disease, hematological disease or neurological disease.
  • the miR-1281 of the present application uses HDAC4 as a target and inhibits the activity of the HDAC4 signaling pathway by inhibiting the expression of the HDAC4 gene, and has the function of an HDAC4 signaling pathway inhibitor;
  • the miR-1281 inhibitor of the present application is an endogenous miRNA in the human body, which is less toxic to the human body and can be better utilized by the human body. It can be introduced into the human body by intravenous injection and transported to a specific site through blood circulation. As a new HDAC4 inhibitor, it is used to prepare drugs for treating diseases caused by abnormal expression of HDAC4.
  • Figure 1A is the 3'-UTR binding site of miR-1281 and HDAC4
  • Figure 1B is the effect of overexpressing miR-1281 on the activity of wild-type HDAC4-UTR and mutant HDAC4-UTR luciferase
  • Figure 1C is the overexpression of miR -1281 on the expression of total HDAC4 protein in human pulmonary artery smooth muscle cells;
  • FIG. 2A is a typical fluorescent picture of 5-ethynyl-2′-deoxyuridine (EdU) detection of PDGFBB stimulated human pulmonary artery smooth muscle cells after overexpression of miR-1281 on cell proliferation.
  • Figure 2B is a statistical result of the effect of miR-1281 overexpression on cell proliferation after PDGFBB-stimulated human pulmonary artery smooth muscle cells were detected by EdU.
  • EdU 5-ethynyl-2′-deoxyuridine
  • the mature sequence of miR-1281 is shown in SEQ ID NO.1 and can be prepared by the following methods:
  • miR-1281 analog is a double-stranded RNA synthesized by Guangzhou Ruibo Biotechnology Co., Ltd., which mimics high-level expression of endogenous mature miR-1281; mimi-1281 is stored at -20 ° C. Environment, the 20 ⁇ M stock solution configured with RNase-free water-soluble dry powder was stored in -80 °C, and diluted to the required concentration before use.
  • the miR-1281 mature sequence and precursor sequence (pre-miRNA) information was obtained in the miRBase (http://www.mirbase.org/) database, and two approximately 300 bp flanking sequences of the mature sequence were found in the human genome database. , Design primers based on flanking sequences, and add XhoI and EcoRI restriction sites and protective bases, respectively, the primer sequences are as SEQ ID No. 2 to 3;
  • the designed primers were used for PCR amplification.
  • the PCR reaction conditions were denaturation at 95 ° C for 2 minutes, 95 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 40 seconds for 35 cycles, and 72 ° C extension for 3 minutes. ;
  • the lentiviral vector plv4 / EGFP was double-digested with restriction enzymes XhoI and EcoRI (NEB), and purified and used as a vector fragment;
  • the insert and the vector fragment were ligated with T4 ligase (Promega) at 16 ° C for 2h, and the ligated product was transformed into E. coli competent STBL3. After overnight culture at 37 ° C, a single colony that was well grown was picked in LB containing the corresponding antibiotic of the vector Amplified in the medium, and verified by sequencing after extracting the plasmid;
  • Cells were transfected with the validated expression vector pLVX-miR-1281, and miR-1281 was transiently expressed.
  • the prepared expression vector pLVX-miR-1281 was packaged with lentivirus.
  • the cell line used was HEK293T cell line.
  • the plasmid included the expression vector pLVX-miR-1281 and the lentivirus packaging plasmid. The specific steps are as follows:
  • Cells were transfected using the conventional calcium phosphate transfection method: After 2M CaCl 2 was mixed with the plasmid, 2 ⁇ HBS solution was added dropwise and mixed by gentle shaking. The mixed solution was slowly and uniformly added to the cell culture solution, and transfected for 12 hours. Change the culture medium afterwards;
  • the packaged lentivirus can be used to infect cells, and cells stably overexpressing miR-1281 can be obtained after screening by the antibiotic puromycin.
  • HEK293A cells purchased from ATCC, Manassas, VA
  • DMEN medium containing 10% fetal bovine serum
  • cells with good growth status were collected and seeded in 24-well plates at 6 ⁇ 10 4 / well, 37 ° C, 5% CO 2 culture for 24 hours;
  • Human pulmonary artery smooth muscle cells (purchased from Sciencell (San Diego, CA, USA)) were cultured in SMCM complete medium containing 5% fetal bovine serum, cells in good growth condition were collected, and centrifuged at 6 ⁇ 10 5 in a 60 mm culture dish Within 24 hours at 37 ° C and 5% CO 2 ;
  • HEK293A cells were seeded in a petri dish. When the cell density reached 70%, cells were transfected with calcium phosphate method and divided into 4 groups: 3'-UTR dual luciferase reporter vector + pLVX-CMV- control (control + HDAC4-UTR, WT), 3'-UTR dual luciferase reporter vector for HDAC4 + pLVX-CMV-miR-1281 (miR-1281 + HDAC4-UTRWT), 3'-UTR dual fluorescence for mutant HDAC4 Luciferase reporter vector + pLVX-CMV-control (control + HDAC4-UTR MUT), mutated HDAC4 3'-UTR dual luciferase reporter vector + pLVX-CMV-miR-1281 (miR-1281 + HDAC4-UTR mute ), Two days after transfection, remove the culture medium, wash the cells twice with PBS and lyse the cells with 50 ⁇ L lysis
  • Luciferase detection The Dual-Luciferase Reporter Assay System (E1810, Promega) kit was used to detect the luciferase activity. The firefly luciferase value was divided by the internal reference Renilla luciferase reading to obtain a corrected luciferase activity value. The luciferase activity value of the 3'-UTR vector of HDAC4 was compared with the control group.
  • Figure 1A shows the 3'-UTR binding site of miR-1281 and HDAC4;
  • Figure 1B shows the effect of miR-1281 overexpression on HDAC4-UTR WT or HDAC4-UTR MUT luciferase activity.
  • the HDAC4 luciferase activity in the miR-1281 group was significantly reduced, indicating that miR-1281 may target the 3'-UTR of HDAC4.
  • mutations in the HDAC4 binding site caused recovery of luciferase activity. .
  • Lipofectamine 2000 (Invitrogen) was used to transfect human pulmonary artery smooth muscle cells with miR-1281mimic, miR-1281 was overexpressed, the medium was changed 6 hours after transfection, and the culture was continued for 24 hours for proteomic detection.
  • Protein hybridization detection Collect total protein of the cells, quantify the protein, and perform polyacrylamide gel electrophoresis.
  • the HDAC4 protein antibody was used to detect the expression of HDAC4.
  • Example 5 miR-1281 inhibits PDGFBB-induced proliferation of human pulmonary artery smooth muscle cells
  • PDGFBB platelet-derived growth factor BB
  • the overexpression vector pLVX-CMV-miR-1281 was used to infect human pulmonary artery smooth muscle cells to increase the expression level of miR-1281 in human pulmonary artery smooth muscle cells, and the cell proliferation status was determined by detecting the expression level of cell proliferation marker protein EdU.
  • the main steps are:
  • the miR-1281 of the present application uses HDAC4 as a target, inhibits the activity of the HDAC4 signaling pathway by inhibiting the expression of the HDAC4 gene, and has a function as an inhibitor of the HDAC4 signaling pathway;
  • the miR-1281 inhibitor is a human body Derived miRNA is less toxic to the human body and can be better used by the human body. It can be introduced into the human body by intravenous injection and transported to specific sites through blood circulation. It can be used as a new HDAC4 inhibitor to prepare and treat HDAC4 abnormalities. Drugs that express the disease caused.

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Abstract

提供了一种miRNA作为HDAC4信号通路抑制剂的用途、包括该miRNA的HDAC4信号通路抑制剂及其过表达方法和应用，其中该miRNA包括miR-1281。该miRNA以HDAC4为靶点，通过抑制HDAC4基因表达从而抑制HDAC4信号通路的活性，具有作为HDAC4信号通路抑制剂的功能。

Description

一种HDAC4信号通路miRNA抑制剂及其过表达方法和应用 技术领域
本申请属于生物医学技术领域，涉及一种HDAC4信号通路miRNA抑制剂及其过表达方法和应用。
背景技术
组蛋白去乙酰化酶4(histone deacetylase，HDAC4)是组蛋白去乙酰化蛋白酶家族的重要成员，对染色体结构修饰和基因表达调控发挥着重要的作用。通常情况下，组蛋白的乙酰化有利于DNA与组蛋白八聚体解离，核小体结构松弛，从而促使各种转录因子和协同转录因子与DNA结合位点特异性结合，激活基因的转录。在细胞核内，组蛋白乙酰化与组蛋白去乙酰化是一个动态平衡过程，由组蛋白乙酰化转移酶(histone acetyltransferase，HAT)和组蛋白去乙酰化酶(HDAC)共同调控。HAT将乙酰辅酶A的乙酰基转移到组蛋白氨基末端特定的赖氨酸残基上，HDAC使组蛋白去乙酰化，与带负电荷的DNA紧密结合，染色质致密卷曲，抑制基因的转录。
大量研究结果表明，HDAC4基因与细胞生长、分化和程序性死亡及血管形成密切相关，HDAC4信号通路的异常表达与心力衰竭、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药物的新靶点。此外，HDAC4的基因异常激活还与胃肠道疾病、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)的形成相关。开发HDAC4激酶信号通路的抑制剂已经成为研究的热点。
目前研究较多的HDAC4激酶信号通路抑制剂主要包括肽和拟肽类抑制剂、 天然产物类抑制剂和通过计算机药物虚拟筛选技术发现的小分子抑制剂。其中，肽和拟肽类抑制剂是以HDAC4信号通路相关分子的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HDAC4信号通路的传递从而达到抑制效果，但此类抑制剂容易在体内代谢失活，生物利用度较低；天然产物类抑制剂主要包括萜类和黄酮类天然活性分子，但该类化合物的提取工艺复杂，流程繁琐，特异性较弱；通过计算机药物虚拟筛选技术获得的小分子抑制剂往往合成困难，生物抑制性较弱。
CN 106344923 A公开了组蛋白去乙酰化酶-4抑制剂在制备治疗多发性骨髓瘤疾病药物中的应用，所述抑制剂为针对HDAC4基因的siRNA，可以抑制多发性骨髓瘤细胞的增殖，诱导细胞凋亡，发生自噬，并导致下游转录因子上调，然而上述siRNA是外源性的，具有一定的毒性，筛选过程较繁琐，且基因的突变可能会影响siRNA的特异性。
因此，现有技术还有待于改进和发展，研发出效果更好的HDAC4抑制剂。
发明内容
针对现有技术的不足，本申请提供一种HDAC4信号通路miRNA抑制剂及其过表达方法和应用，所述抑制剂包括内源性hsa-miR-1281，解决了现有HDAC4信号通路抑制剂生物稳定性差、利用度低、制备工艺繁琐等问题。
为达此目的，本申请采用以下技术方案：
第一方面，本申请提供了一种miRNA作为HDAC4信号通路抑制剂的用途。
miRNA是一类长度约为22个碱基的非编码单链小分子RNA，具有转录后调节基因表达的作用。研究结果表明，miRNA表达与细胞功能密切相关。
本申请采用血小板衍生生长因子BB(platelet derived growth factor BB， PDGFBB)刺激人肺动脉平滑肌细胞，分析基因组范围内miRNA表达谱的变化，发现在检测的1098个miRNA中，有9个miRNA表达上调，5个miRNA表达下调，在表达下调的miRNA中miR-1281下调十分显著。
利用Targetscan等在线工具预测，发现HDAC4是miR-1281的潜在靶基因；利用3’-UTR实验及蛋白质水平检测，证明过表达miR-1281对HDAC4具有抑制作用，HDAC4是miR-1281的靶基因；通过在人肺动脉平滑肌细胞中过表达miR-1281，可抑制PDGFBB对HDAC4信号通路的激活，人肺动脉平滑肌细胞增殖异常。
本申请中，采用miR-1281作为靶向分子，对HDAC4信号通路具有显著的抑制作用。
进一步地，所述miRNA为miR-1281。
优选地，所述miR-1281的核酸序列如SEQ ID NO.1所示；
所述SEQ ID NO.1所示的核酸序列如下：5’-TCGCCTCCTCCTCTCCC-3’.
第二方面，本申请提供了一种HDAC4信号通路抑制剂，所述抑制剂包括miRNA。
进一步地，所述miRNA包括miR-1281。
优选地，所述miR-1281的核酸序列如SEQ ID NO.1所示。
第三方面，本申请提供了一种如第二方面所述抑制剂的过表达方法，所述方法包括脂质体转染和/或重组表达。
优选地，所述脂质体转染包括将化学合成的miRNA采用脂质体包裹后，转染细胞，进行抑制剂的过表达。
本申请中，化学合成指通过生物公司人工合成miRNA的类似物(miRNA  mimic)，本申请的所有核酸序列均由广州瑞博生物科技有限公司合成，本领域技术人员可根据实际情况选择不同的生物公司。
本申请中，采用Lipofectamine 2000和/或Lipofectamine 3000作为脂质体转染化学合成的miRNA。
优选地，所述miRNA包括miR-1281。
优选地，所述重组表达包括以下步骤：
(1)构建表达载体；
(2)将步骤(1)所述表达载体转入感受态细胞，培养后挑取单克隆细胞进行表达载体的筛选；
(3)将筛选后的表达载体转染宿主细胞，进行抑制剂的过表达。
优选地，步骤(1)所述构建表达载体具体包括：
(1’)以人基因组DNA为模板，PCR获得扩增产物；
(2’)将步骤(1’)所述扩增产物插入plv4/EGFP慢病毒载体的XhoI和EcoRI酶切位点之间。
优选地，步骤(1’)所述PCR的引物的核酸序列如SEQ ID NO.2～3所示；
所述SEQ ID NO.2所示的核酸序列如下：
5’-CCGCTCGAGAGCTCCGCTCCCGGCGTC-3’；
所述SEQ ID NO.3所示的核酸序列如下：
5’-CCGGAATTCAGAACTCGCGGCTCTAGA-3’.
优选地，在步骤(3)之后还包括将筛选后的表达载体包装得到慢病毒的步骤。
本申请采用包装得到的慢病毒感染宿主细胞，筛选后可以得到稳定过表达 miR-1281的细胞。
作为优选技术方案，本申请提供了一种如第二方面所述抑制剂的过表达方法，包括以下步骤：
(1)以人基因组DNA为模板，以SEQ ID NO.2～3所示的核酸序列为引物，PCR获得扩增产物；
(2)将步骤(1)所述扩增产物和慢病毒载体plv4/EGFP分别采用XhoI和EcoRI限制性内切酶双酶切，得到的酶切产物采用T4DNA连接酶连接为表达载体；
(3)将步骤(2)所述表达载体转入感受态细胞，培养后挑取单克隆细胞进行表达载体的筛选；
(4)将筛选后的表达载体转染宿主细胞，进行miR-1281抑制剂的表达
(5)将筛选后的表达载体包装得到慢病毒。
第四方面，本申请提供了一种药物组合物，所述药物组合物包括如第二方面所述HDAC4信号通路抑制剂。
进一步地，所述药物组合物还包括药学上可接受的载体、赋形剂或稀释剂中的任意一种或至少两种的组合。
第五方面，本申请提供了一种如第二方面所述HDAC4信号通路抑制剂和/或如第四方面所述药物组合物在制备治疗HDAC4基因异常疾病的药物中的应用。
优选地，所述疾病包括心脑血管疾病、胃肠道疾病、炎症性疾病、血液病或神经系统疾病中的任意一种或至少两种的组合。
与现有技术相比，本申请具有如下有益效果：
(1)本申请的miR-1281以HDAC4为靶点，通过抑制HDAC4基因表达从而抑制HDAC4信号通路的活性，具有作为HDAC4信号通路抑制剂的功能；
(2)本申请的miR-1281抑制剂是人体内源性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，可作为一种新的HDAC4抑制剂用于制备治疗HDAC4异常表达所引起的疾病的药物。
附图说明
图1A为miR-1281与HDAC4的3’-UTR结合位点，图1B为过表达miR-1281对野生型HDAC4-UTR和突变型HDAC4-UTR荧光素酶活性的影响，图1C为过表达miR-1281对人肺动脉平滑肌细胞HDAC4总蛋白表达的影响；
图2A为5-乙炔基-2’脱氧尿嘧啶核苷(5-ethynyl-2’-deoxyuridine，EdU)检测PDGFBB刺激人肺动脉平滑肌细胞后，过表达miR-1281对细胞增殖影响的典型荧光图片，图2B为EdU检测PDGFBB刺激人肺动脉平滑肌细胞后，过表达miR-1281对细胞增殖影响的统计结果。
具体实施方式
为进一步阐述本申请所采取的技术手段及其效果，以下结合实施例和附图对本申请作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本申请，而非对本申请的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
实施例1 miR-1281的制备
miR-1281的成熟序列如SEQ ID NO.1所示，可通过以下方式制备得到：
(1)化学合成
miR-1281类似物(miR-1281 mimic)由广州瑞博生物科技有限公司合成，为双链RNA，模拟内源性成熟miR-1281高水平表达；合成的miR-1281 mimic干粉保存于-20℃环境，采用无RNA酶水溶解干粉配置成的20μM储存液保存于-80℃环境，使用前稀释至所需浓度。
(2)重组表达
在miRBase(http：//www.mirbase.org/)数据库中得到miR-1281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的两个约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，引物序列如SEQ ID NO.2～3；
以人基因组DNA为模板，采用设计的引物进行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；
将扩增的PCR产物进行琼脂糖凝胶电泳后，采用凝胶回收试剂盒(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片段采用DNA纯化试剂盒(E.Z.N.A Cycle-Pure Kit，Omgega)回收后作为插入片段；
将慢病毒载体plv4/EGFP采用限制性内切酶XhoI和EcoRI(NEB)进行双酶切，回收纯化后作为载体片段；
将插入片段和载体片段利用T4连接酶(Promega)在16℃下连接2h，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；
采用验证的表达载体pLVX-miR-1281转染细胞，瞬时表达miR-1281。
实施例2 miR-1281过表达慢病毒的包装
将制得的表达载体pLVX-miR-1281进行慢病毒包装，采用的细胞系为HEK293T细胞系，质粒包括表达载体pLVX-miR-1281和慢病毒包装质粒，具体步骤如下：
细胞转染采用常规的磷酸钙转染法：采用2M CaCl 2与质粒混匀后，逐滴加入2×HBS溶液并轻摇混匀，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；
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；
包装好的慢病毒可用于感染细胞，通过抗生素嘌呤霉素筛选后获得可稳定过表达miR-1281的细胞。
实施例3 miR-1281的重组过表达
(1)细胞培养
HEK293A细胞(购自ATCC，Manassas，VA)采用含有10％胎牛血清的DMEN培养基培养，收集生长状态良好的细胞，以6×10 4/孔接种于24孔板内，37℃、5％CO 2培养24小时；
人肺动脉平滑肌细胞(购自Sciencell(San Diego，CA，USA))采用含有5％胎牛血清的SMCM完全培养基培养，收集生长状态良好的细胞，离心后以6×10 5接种于60mm培养皿内，37℃、5％CO 2培养24小时；
(2)细胞转染
将HEK293A细胞接种于培养皿中，待细胞密度达70％时采用磷酸钙法转染细胞，分为4组：分别共转染HDAC4的3’-UTR双荧光素酶报告载体+pLVX-CMV-control(对照+HDAC4-UTR WT)、HDAC4的3’-UTR双荧光素酶报告载体+pLVX-CMV-miR-1281(miR-1281+HDAC4-UTRWT)、突变的HDAC4的3’-UTR双荧光素酶报告载体+pLVX-CMV-control(对照+HDAC4-UTR MUT)、突变的HDAC4的3’-UTR双荧光素酶报告载体+pLVX-CMV-miR-1281(miR-1281+HDAC4-UTR MUT)，转染两天后，移去培养液，PBS清洗两次后采用50μL裂解缓冲液(1×Passive Lysis Buffer)裂解细胞；
荧光素酶检测：利用Dual-Luciferase ReporterAssay System(E1810，Promega)试剂盒进行荧光素酶活性检测，将萤火虫荧光素酶数值除以内参海肾荧光素酶读值，得到校正的荧光素酶活性数值，将HDAC4的3’-UTR载体的荧光素酶活性数值与对照组进行比较。
如图1A为miR-1281与HDAC4的3’-UTR结合位点；图1B为过表达miR-1281对HDAC4-UTR WT或HDAC4-UTR MUT荧光素酶活性的影响，与对照组相比，过表达miR-1281组的HDAC4荧光素酶活性显著降低，说明miR-1281可能与HDAC4的3’-UTR靶向结合，与野生型UTR相比，HDAC4结合位点的突变引起荧光素酶活性的恢复。
实施例4 miR-1281的瞬时转染过表达
采用Lipofectamine 2000(Invitrogen)向人肺动脉平滑肌细胞转染miR-1281mimic，过表达miR-1281，转染6小时后更换培养基，继续培养24小时进行蛋白杂交检测。
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HDAC4蛋白抗体检测HDAC4的表达。
如图1C所示，与对照组相比，过表达miR-1281可抑制人肺动脉平滑肌细胞内源性HDAC4的表达。
实施例3～4的结果表明：miR-1281通过靶向HDAC4的3’-UTR抑制HDAC4的表达。
实施例5 miR-1281抑制PDGFBB诱导人肺动脉平滑肌细胞增殖
本实施例探讨miR-1281对血小板衍生生长因子BB(PDGFBB)激活HDAC4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。
采用过表达载体pLVX-CMV-miR-1281感染人肺动脉平滑肌细胞，提高人肺动脉平滑肌细胞中miR-1281的表达水平，通过检测细胞增殖标志蛋白EdU的表达水平确定细胞的增殖状态。主要步骤为：
1)细胞转染：将1×10 4个细胞接种于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，随后加入PDGFBB和20μM EdU继续培养4小时；
2)细胞染色拍照：将细胞采用4％多聚甲醛室温固定30分钟，0.5％Triton X-100膜通透10分钟，PBS清洗后，每孔加入150μL 1×Apollo染色液孵育30分钟；
3)DNA采用1×Hochest(赫司特)(每孔150μL)染色5分钟，在荧光显微镜下拍照。
结果如图2A和图2B所示，在PDGFBB刺激下，与对照组相比，过表达miR-1281使人肺动脉平滑肌细胞增殖下降30％，说明miR-1281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，本申请的miR-1281以HDAC4为靶点，通过抑制HDAC4基因表达从而抑制HDAC4信号通路的活性，具有作为HDAC4信号通路抑制剂的功能；所述miR-1281抑制剂是人体内源性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，可作为一种新的HDAC4抑制剂用于制备治疗HDAC4异常表达所引起的疾病的药物。
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 (15)

  1. 一种miRNA作为HDAC4信号通路抑制剂的用途。
  2. 根据权利要求1所述的用途，其中，所述miRNA包括miR-1281；
    优选地，所述miR-1281的核酸序列如SEQ ID NO.1所示。
  3. 一种HDAC4信号通路抑制剂，其包括miRNA。
  4. 根据权利要求3所述的抑制剂，其中，所述miRNA包括miR-1281；
    优选地，所述miR-1281的核酸序列如SEQ ID NO.1所示。
  5. 一种如权利要求3或4所述抑制剂的过表达方法，其包括脂质体转染和/或重组表达。
  6. 根据权利要求5所述的方法，其中，所述脂质体转染包括将化学合成的miRNA采用脂质体包裹后，转染细胞，进行抑制剂的过表达；
    优选地，所述miRNA包括miR-1281。
  7. 根据权利要求5或6所述的方法，其中，所述重组表达包括以下步骤：
    (1)构建表达载体；
    (2)将步骤(1)所述表达载体转入感受态细胞，培养后挑取单克隆细胞进行表达载体的筛选；
    (3)将筛选后的表达载体转染宿主细胞，进行抑制剂的过表达。
  8. 根据权利要求5-7中任一项所述的方法，其中，步骤(1)所述构建表达载体具体包括：
    (1’)以人基因组DNA为模板，PCR获得扩增产物；
    (2’)将步骤(1’)所述扩增产物插入plv4/EGFP慢病毒载体的XhoI和EcoRI酶切位点之间。
  9. 根据权利要求8所述的方法，其中，步骤(1’)所述PCR的引物的核 酸序列如SEQ ID NO.2～3所示。
  10. 根据权利要求8所述的方法，其中，在步骤(3)之后还包括将筛选后的表达载体包装得到慢病毒的步骤。
  11. 根据权利要求5-10中任一项所述的方法，其中，所述重组表达包括以下步骤：
    (1)以人基因组DNA为模板，以SEQ ID NO.2～3所示的核酸序列为引物，PCR获得扩增产物；
    (2)将步骤(1)所述扩增产物和慢病毒载体plv4/EGFP分别采用XhoI和EcoRI限制性内切酶双酶切，得到的酶切产物采用T4DNA连接酶连接为表达载体；
    (3)将步骤(2)所述表达载体转入感受态细胞，培养后挑取单克隆细胞进行表达载体的筛选；
    (4)将筛选后的表达载体转染宿主细胞，进行miR-1281抑制剂的过表达；
    (5)将筛选后的表达载体包装得到慢病毒。
  12. 一种药物组合物，其包括如权利要求3或4所述HDAC4信号通路抑制剂。
  13. 根据权利要求12所述的药物组合物，其中，所述药物组合物还包括药学上可接受的载体、赋形剂或稀释剂中的任意一种或至少两种的组合。
  14. 一种如权利要求3或4所述HDAC4信号通路抑制剂和/或如权利要求12或13所述药物组合物在制备治疗HDAC4基因异常疾病的药物中的应用。
  15. 根据权利要求14所述的应用，其中，所述疾病包括心脑血管疾病、胃肠道疾病、炎症性疾病、血液病或神经系统疾病中的任意一种或至少两种的组合。
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WO2009117418A2 (en) * 2008-03-17 2009-09-24 The Board Of Regents Of The University Of Texas System Identification of micro-rnas involved in neuromuscular synapse maintenance and regeneration
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CN107312851A (zh) * 2017-07-19 2017-11-03 北京泱深生物信息技术有限公司 心肌梗死生物标志物miR‑1283

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