WO2018165929A1 - 一种双miRNA抑制表达载体及其构建方法和应用 - Google Patents

一种双miRNA抑制表达载体及其构建方法和应用 Download PDF

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WO2018165929A1
WO2018165929A1 PCT/CN2017/076860 CN2017076860W WO2018165929A1 WO 2018165929 A1 WO2018165929 A1 WO 2018165929A1 CN 2017076860 W CN2017076860 W CN 2017076860W WO 2018165929 A1 WO2018165929 A1 WO 2018165929A1
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mir
tud
sequence
mirna
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毛吉炎
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深圳市博奥康生物科技有限公司
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  • Double miRNA suppression expression vector construction method and application thereof
  • the present invention relates to the field of molecular biology, and in particular to a dual miRNA suppression expression vector and a construction method and application thereof.
  • RNA is an important substance in living organisms and plays various functions in life activities. RNA can be classified into messenger RNA (mRNA) and non-coding RNA (ncRNA) depending on whether the protein is encoded or not. Small RNA (small
  • RNA is an important class of ncRNAs. miRNAs are endogenous small RNAs in organisms that are typically 20-24 nt in length. The miRNA is part of the pri-miRNA (primary RNA) and is initially expressed in the nucleus by RNA polymerase ⁇ . The mature miRNA acts as a guiding molecule, binds to the target gene mRNA according to the principle of base pairing, and directs the silencing complex (RISC) to degrade mRNA or hinder its translation, thereby exerting a negative regulation effect on the expression of the target gene.
  • RISC silencing complex
  • miR-140 can inhibit the proliferation and invasion and metastasis of hepatocellular carcinoma by targeting TG FBR1 and other gene expression.
  • miR-140 is highly expressed in articular cartilage and plays a crucial role in the pathogenesis of osteoarthritis.
  • miR-424 is a miRNA discovered in recent years.
  • miR-424 acts on target genes in a variety of tumors and participates in the signal pathway of target gene regulation, thereby affecting the biological effects and development of tumor cells, playing a tumor-like, tumor-suppressing
  • the role of genes, or promote, inhibit tumor invasion and metastasis have shown that miR-424 is a multifunctional miRNA, which is associated with cell invasion and metastasis of cervical cancer, pancreatic cancer, etc.; it is associated with the expression of inflammatory factors such as IL-6 and TNF-oc; since the miR-424 promoter region has CpG islands It is also associated with methylation-induced gene silencing.
  • MiRNA functional studies usually require the use of miRNA silencing technology, mainly including anti-miR, antago miR, miRNA
  • Tough Decoy RNA is a novel miRNA-inhibiting miRNA that inhibits miRNA by introducing double-stranded RNA to target miRNAs. Because the inserted RNA is double-stranded and has a secondary structure of stem-loops, it is resistant to intracellular nuclease degradation and inhibits miRNAs in a long-term, stable, and efficient manner.
  • the technical problem to be solved by the present invention is to provide a dual miRNA suppression expression vector which is simple in structure, low in cost and simple in operation.
  • a Tud RNA targeting a dual miRNA the nucleotide sequence of which is shown in SEQ ID NO: 1 in the Sequence Listing.
  • a dual miRNA-inhibiting expression vector comprising the sequence of the invention SEQ ID NO: 1.
  • the double miRNA-inhibiting expression vector can inhibit the expression of miR-140 and miR-424 by transforming Hela cells.
  • a method for constructing a dual miRNA suppression expression vector according to the present invention comprises the following steps:
  • the synthesized sequence is two complementary single-stranded DNAs.
  • the two single-stranded DNAs were dissolved in ddH 2 0, mixed in an equimolar ratio, treated at 95 ° C for 5 min, and then allowed to cool to room temperature at room temperature.
  • the digested vector was recovered using the MinElute Reaction Cleanup Kit, and the TuD obtained in the previous step was further treated with T4 DNA ligase.
  • RNA sequence was ligated into the vector pLKO.l-puro to form the recombinant vector pLKO-TuD-140-424, and finally the ligation product was transformed into competent E. coli Stbl3 and applied to the ampicillin-containing LB medium. Plate, culture at 37 °C for 14 h. Single colonies were picked and sequenced. The bacteria with the correct sequencing were expanded and extracted with a small amount of extraction kit without endotoxin plasmid. The extracted plasmid was the plasmid of the homologous interference pLKO-T uD-140-424 required by the present invention.
  • the homologous interference miR-140 and miR-424 TuD RNA sequences designed by the present invention have a stem-loop structure and are not easily degraded, and the double-stranded Tud RNA has higher binding efficiency than the currently used single-stranded miRNA sponge. And the same target for two targets, can better achieve the interference of two miRNAs, improve the efficiency of miRNA function research.
  • Figure 1 shows the miRNA expression levels of 16HBE cells and TuD-140-424 cells, wherein a. miR-140 expression, b. miR-424 expression.
  • the lentiviral plasmid pLKO.l-puro vector used in the present invention was purchased from Addgene; the human bronchial epithelial cells (16HBE cell strain) used in the present invention were purchased from ATCC, USA.
  • TuD RNA design sequence Based on the TuD RNA design sequence and the sequence information of miR-140 and miR-424 provided in miRBase, a TuD RNA oligonucleotide sequence targeting miR-140 and miR-424 was designed, and its sequence is SEQ ID NO:
  • the synthesized sequence is two complementary single stranded DNAs.
  • the two single-stranded DNAs were dissolved in ddH 2 0, mixed in an equimolar ratio, treated at 95 ° C for 5 min, and allowed to stand at room temperature to allow them to naturally cool to room temperature.
  • Example 3 Construction of recombinant pLKO-Tud-140-424 lentiviral recombinant vector
  • the vector pLK0.1-puro was extracted and digested with Age I and Eco RI for 16 h, and the digested vector was recovered with MinElute Reaction Cleanup Kit, and then T4 DNA was used.
  • RNA sequence was ligated into the vector pLKO.l-puro to form the recombinant vector pLKO-Tud-140-424, and finally the ligation product was transformed into competent E. coli Stbl3 and plated onto a plate containing ampicillin LB medium. Incubate at 37 °C for 14 h. Single colonies were picked and sequenced. The correct sequencing bacteria were expanded and extracted with a small amount of endotoxin-free extraction kit. The extracted plasmid was the plasmid of the same interference interference pLKO-T uD-140-424 required by the present invention.
  • 16HBE cells were seeded in 6-well plates at 1,000,000 cells per well, and after 18 hours, the cell density was approximately 60%, using Lipfectamine.
  • the pLKO-TuD-140-424 plasmid was transduced into 16HBE cells and culture continued.
  • the cells were cultured for 3 days in DMEM medium containing 1.0 g/ml puromycin, and the cell line was screened as TuD-140-424 cell line.
  • the homologous interference miR-140 and miR-424 TuD RNA sequences designed by the present invention have a stem-loop structure and are not easily degraded, and the double-stranded Tud RNA has higher binding efficiency than the commonly used single-stranded miRNA sponge. And the same target for two targets, can better achieve the interference of two miRNAs, improve miRNA work

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Abstract

提供了一种双miRNA抑制表达载体及其构建方法和应用。该双miRNA抑制表达载体的构建方法是将靶向miR140和miR-424的Tud RNA连接到pLKO.1-puro克隆载体上，获得双miRNA抑制表达载体pLKO-Tud-140-424。所述双miRNA抑制表达载体起到抑制has-miR-140和has-miR-424活性的作用。

Description

一种双 miRNA抑制表达载体及其构建方法和应用 技术领域
[0001] 本发明涉及分子生物学领域， 特别是涉及一种双 miRNA抑制表达载体及其构建 方法和应用。
背景技术
RNA是生物体内一种重要物质， 在生命活动中发挥着各种各样的功能。 根据是 否编码蛋白质， RNA可分为信使 RNA (messagerRNA, mRNA) 和非编码 RNA (non-coding RNA, ncRNA) 。 小 RNA (small
RNA, smRNA) 是一类重要的 ncRNA。 miRNA是生物体内一种内源性小 RNA， 长度一般为 20-24nt。 miRNA是 pri-miRNA (primaryRNA) 的一部分， 最初在细 胞核中由 RNA聚合酶 Π转录表达。 成熟的 miRNA作为引导性分子， 根据碱基配对 原则与靶基因 mRNA结合， 引导沉默复合体 （RISC) 降解 mRNA或阻碍其翻译 ， 从而发挥对靶标基因表达的负调控作用。
miR- 140与多种疾病的发生发展密切相关， 如骨、 关节疾病， 肝脏疾病， 垂体 腺瘤， 睾丸发育， 头颈部肿瘤， 卵巢与乳腺疾病等。 miR-140能通过靶向调节 TG FBR1等基因表达抑制肝细胞癌增殖和侵袭转移， miR-140特异性高表达于关节软 骨中， 并在骨关节炎的发病机制中发挥至关重要的作用； miR-424是近年来发现 的一个 miRNA, 其在多种肿瘤中通过作用于靶基因， 参与靶基因调控的信号通 路， 从而影响肿瘤细胞生物学效应和发生发展， 发挥类似于癌基因、 抑癌基因 的作用， 或促进、 抑制肿瘤的侵袭转移。 有研究表明 miR-424是多功能 miRNA, 它与宫颈癌， 胰腺癌等细胞侵袭转移相关； 与炎性因子如 IL-6、 TNF-oc的表达相 关； 由于 miR-424启动子区域具有 CpG岛， 它与甲基化诱导的基因沉默也相关。 通过控制 miR-140和 miR-424的表达， 同吋与其他药物协同作用， 能为治疗癌症 提供新的表观遗传思路。
技术问题
[0004] MiRNA的功能研究通常需要用到 miRNA沉默技术， 主要包括 anti-miR， antago miR, miRNA
sponge等， 这些技术均属于瞬吋转染技术， 无法保持对目的 miRNA的长期稳定 沉默， 沉默效果远未达到最优。
[0005] Tough Decoy RNA (Tud RNA) 是一种新幵发出的 miRNA抑制手段， 其通过引 入双链 RNA对目标 miRNA进行吸附， 达到抑制 miRNA的目的。 由于弓 |入的 RNA 为双链并且带有茎环的二级结构， 因此其够抵抗胞内核酸酶的降解， 能长期、 稳定和高效地抑制 miRNA。
问题的解决方案
技术解决方案
[0006] 本发明要解决的技术问题是提供一种结构简单、 成本低、 操作简便的双 miRNA 抑制表达载体。
[0007] 一种靶向双 miRNA的 Tud RNA, 其核苷酸序列如序列表中 SEQ ID NO: 1所示。
[0008] 一种双 miRNA抑制表达载体， 其包括本发明所述的序列 SEQ ID N0:1。
[0009] 本发明所述双 miRNA抑制表达载体在肿瘤治疗研究中的应用， 所述双 miRNA 抑制表达载体转化 Hela细胞后能同吋抑制 miR-140和 miR-424的表达。
[0010] 一种本发明所述的双 miRNA抑制表达载体的构建方法， 包括如下步骤：
[0011] (1) 根据 miR- 140和 miR-424序列以及 Tud RNA技术原理， 设计权利要求 1所 述的靶向双 miRNA的 Tud RNA Seql， 所述 miR- 140和 miR-424序列的核苷酸序列 分别如序列表中 SEQ ID N0:2和 SEQ ID N0:3所示， 所述 Seql序列的核苷酸序 列如序列表 SEQ ID NO: 1所示， 并委托上海生工进行合成。
[0012] (2) 合成好的序列是两条互补的单链 DNA。 将两条单链 DNA溶解于 ddH 20中 ， 按照等摩尔比混合后， 95°C处理 5 min， 再将其置于室温使其自然冷却至室温
[0013] (3) 提取载体 pLK0.1-puro， 使用 Age I和 Eco RI酶双酶切处理 1
h后， 用 MinElute Reaction Cleanup Kit回收酶切后的载体， 再用 T4 DNA连接酶将 上一步得到的 TuD
RNA序列连接到载体 pLKO.l-puro中， 形成重组载体 pLKO-TuD- 140-424， 最后将 连接产物转化到感受态大肠杆菌 Stbl3中， 并涂布到含氨苄青霉素 LB培养基的平 板上， 37 °C培养 14 h。 挑取单菌落并进行测序。 取测序正确的菌扩大培养并用无 内毒素质粒小量提取试剂盒提取， 提取的质粒为本发明所需的同吋干扰 pLKO-T uD- 140-424的质粒。
发明的有益效果
有益效果
[0014] 本发明设计的同吋干扰 miR-140和 miR-424 TuD RNA序列带有茎环结构， 不容 易降解， 双链的 Tud RNA相对目前常用的单链的 miRNA sponge, 其结合效率更 高， 并且同吋针对两个靶点， 能较好地实现两个 miRNA的干扰， 提高 miRNA功 能研究的效率。
对附图的简要说明
附图说明
[0015] 图 1 16HBE细胞与 TuD-140-424细胞的 miRNA表达水平情况， 其中， a. miR-140 的表达情况， b. miR-424的表达情况。
实施该发明的最佳实施例
本发明的最佳实施方式
[0016] 根据下述实施例， 可以更好地理解本发明。 然而， 本领域的技术人员容易理解
， 实施例所描述的具体的物料配比、 工艺条件及其结果仅用于说明本发明， 而 不应当也不会限制权利要求书中所详细描述的本发明。
[0017] 本发明所使用的慢病毒质粒 pLKO.l-puro载体购自 Addgene; 本发明所使用的人 支气管上皮细胞 （16HBE细胞株） 购自美国 ATCC。
[0018] 实施例一靶向 miR-140和 miR-424a的 TuD RNA的设计与合成
[0019] 根据 TuD RNA设计序列和 miRBase中提供的 miR-140和 miR-424的序列信息， 设 计出同吋针对 miR- 140和 miR-424的 TuD RNA寡核苷酸序列， 其序列如 SEQ ID
ΝΟ:1所示， 委托上海生工以基因合成的方式合成。
[0020] 实施例二序列的退火
[0021] 合成好的序列是两条互补的单链 DNA。 将两条单链 DNA溶解于 ddH 20中， 按 照等摩尔比混合后， 95°C处理 5 min， 再将其置于室温使其自然冷却至室温。 [0022] 实施例三重组 pLKO-Tud-140-424慢病毒重组载体的构建
[0023] 提取载体 pLK0.1-puro， 使用 Age I和 Eco RI酶双酶切处理 16 h后， 用 MinElute Reaction Cleanup Kit回收酶切后的载体， 再用 T4 DNA
连接酶将上一步得到的 TuD
RNA序列连接到载体 pLKO.l-puro中， 形成重组载体 pLKO-Tud- 140-424， 最后将 连接产物转化到感受态大肠杆菌 Stbl3中， 并涂布到含氨苄青霉素 LB培养基的平 板上， 37 °C培养 14 h。 挑取单菌落并进行测序。 取测序正确的菌扩大培养并用无 内毒素质粒小量提取试剂盒提取， 提取的质粒为本发明所需的同吋干扰 pLKO-T uD- 140-424的质粒。
[0024] 实施例四 pLKO-TuD-140-424的质粒 16HBE细胞
[0025] 接种 16HBE细胞于 6孔板中， 每孔 1000000个细胞， 18h后细胞密度约为 60% ， 用 Lipfectamine
2000将 pLKO-TuD-140-424质粒转导至 16HBE细胞中， 继续培养 48
h后， 更换含 1.0 g/ml嘌呤霉素的 DMEM培养基筛选培养 3 d， 筛选获得的细胞株 命名为 TuD- 140-424细胞株。
[0026] 实施例五荧光定量 PCR检测 miRNA的表达水平变化
[0027] 分别接种正常 16HBE细胞、 TuD- 140-424细胞至 6孔板， 培养细胞约 24 h后至融 合度 80%。 用 miRcute miRNA提取分离试剂盒提取这些细胞的 miRNA, 逆转录得 到相应的 cDNA。 取 2种细胞的 cDNA各 2
为模板， 荧光定量 PCR分别检测 miR-140和 miR-424表达水平的变化， 实验重 复 3次， 每孔设置 3个平行样，以 snord
44作为内参。 结果如图 1所示， 可以看到与 TuD-140-424细胞的 miR-140的表达水 平比 16HBE细胞低 57%， miR-424的表达水平比 16HBE细胞低 62%， 差异有统计 学意义 （ρ<0.01) ， 说明 TuD- 140-424细胞株构建成功。
工业实用性
[0028] 本发明设计的同吋干扰 miR-140和 miR-424 TuD RNA序列带有茎环结构， 不容 易降解， 双链的 Tud RNA相对目前常用的单链的 miRNA sponge, 其结合效率更 高， 并且同吋针对两个靶点， 能较好地实现两个 miRNA的干扰， 提高 miRNA功
Figure imgf000006_0001

Claims

权利要求书
[权利要求 1] 一种靶向双 miRNA的 Tud RNA, 其特征在于： 其核苷酸序列如序列 表 SEQ ID NO:l所示。
[权利要求 2] —种双 miRNA抑制表达载体， 其特征在于： 其包括权利要求 1所述的 序列。
[权利要求 3] 权利要求 2所述双 miRNA抑制表达载体在肿瘤治疗研究中的应用， 所 述双 miRNA抑制表达载体同吋抑制人源 miR-140和 miR-424的表达。
[权利要求 4] 一种权利要求 2所述的双 miRNA抑制表达载体的构建方法， 其特征在 于： 包括如下步骤：
( 1 ) 根据 miR- 140和 miR-424序列以及 Tud RNA技术原理， 设计权利 要求 1所述的靶向双 miRNA的 Tud RNA Seql， 所述 miR- 140和 miR-424序列的核苷酸序列分别如序列表中 SEQ ID NO:2 和 SEQ ID NO:3所示， 所述 Seql序列的核苷酸序列如序列表 SEQ ID ΝΟ:1所示， 并委托上海生工进行合成。
(2) 合成好的序列是两条互补的单链 DNA。 将两条单链 DNA溶解于 ddH 20中， 按照等摩尔比混合后， 95°C处理 5 min， 再将其置于室温 使其自然冷却至室温。
(3) 提取载体 pLK0.1-puro， 使用 Age I和 Eco RI酶双酶切处理 1 h后
， 回收酶切后的载体， 再用 T4 DNA连接酶将上一步得到的 TuD RNA 序列连接到载体 pLKO.l-puro中， 形成重组载体 pLKO-TuD- 140-424， 最后将连接产物转化到感受态大肠杆菌 Stbl3中， 并涂布到含氨苄青 霉素 LB培养基的平板上， 37 °C培养 14 h。 挑取单菌落并进行测序。 取测序正确的菌扩大培养并用无内毒素质粒小量提取试剂盒提取， 提 取的质粒为本发明所需的同吋干扰 pLKO-TuD-140-424的质粒。
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