WO2019036871A1 - 特异敲低人miR-148a、miR-185和miR-424表达的Tud RNA及其应用 - Google Patents

特异敲低人miR-148a、miR-185和miR-424表达的Tud RNA及其应用 Download PDF

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WO2019036871A1
WO2019036871A1 PCT/CN2017/098369 CN2017098369W WO2019036871A1 WO 2019036871 A1 WO2019036871 A1 WO 2019036871A1 CN 2017098369 W CN2017098369 W CN 2017098369W WO 2019036871 A1 WO2019036871 A1 WO 2019036871A1
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tud
vector
rna
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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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  • the present invention relates to a Tud
  • RNA in particular, relates to a Tud RNA that specifically knocks down the expression of human miR-148a, miR-185 and miR-424 and its use.
  • MicroRNAs are a class of endogenous, non-coding RNAs found in eukaryotes, typically between 22 and 25 nt in size. miRNAs are widely distributed in plants, animals, and multicellular organisms, and can Play an important regulatory role, and in the study of human miRNAs, it is found that the expression of miRNA in normal tissues and tumor tissues is significantly different, some miRNAs are lowly expressed in tumor tissues, and some are highly expressed in tumor tissues. This suggests that miRNAs play a crucial role in tumorigenesis.
  • miR-148a is a microRNA that has been studied more in recent years. It is reported that miR-148a is closely related to exogenous substance metabolism, apoptosis, occurrence, development and epigenetics of various cancers; miR-1 85 is a 22 nt miRNA located at human chromosome 22ql L.21, plays an important role as a tumor suppressor gene in the development and invasion of tumors such as colon cancer, gastric cancer, esophageal cancer, lung cancer, liver cancer, etc.
  • DNMT1 methylation-related tumor suppressor miRNA that can be directly Targeting the expression of DNMT1 affects the methylation level of the whole genome, and then regulates the methylation status of certain genes, affecting gene expression; miR-424 is a miRNA discovered in recent years, which plays a role in various tumors. Target genes, involved in the signal pathway of target gene regulation, thereby affecting the biological effects and development of tumor cells, playing a role similar to oncogenes, tumor suppressor genes, or promoting or inhibiting the invasion and metastasis of tumors.
  • 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- ⁇ ; since the miR-424 promoter region has CpG Island, which is also associated with methylation-induced gene-specific knockdown.
  • the synergy with other drugs can provide new epigenetic ideas for the treatment of cancer.
  • 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 primary object of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a specific knockdown of miR-
  • Tud RNA expressed by 148a, miR-185 and miR-424 Tud RNA expressed by 148a, miR-185 and miR-424.
  • Another object of the present invention is to provide the above-described use of Tud RNA which specifically knocks down the expression of human miR-148a, miR-185 and miR-424.
  • a further object of the present invention is to provide a recombinant vector comprising the above Tud RNA.
  • a Tud RNA specifically knockdown expression of human miR-148a, miR-185 and miR-424, the DNA sequence encoding the Tud RNA which inhibits expression of human miR-148a, miR-185 and miR-424 is as follows Shown as follows:
  • the recombinant vector of RNA acts on WRL-68 cells to inhibit the expression of miR-148a, miR-185 and miR-424;
  • the lentiviral vector is a pGLV3/Hl/GFP+Puro (pGLV3) lentiviral shuttle vector;
  • the preparation method of the recombinant vector containing the Tud RNA comprises the following steps:
  • Tud-148a-185-424 antisense strand [0020] Tud-148a-185-424 antisense strand:
  • the 5' end of the sense strand template is added with GATCC, which is complementary to the sticky end formed by BamHI digestion; the AATTC is added to the 5' end of the antisense strand template, which is complementary to the sticky end formed by EcoRI digestion;
  • Recombinant vector pGLV3-Tud-148a-185-424 and packaging vectors pGag/Pol, pRev, pVSV- G is co-transfected into 293T cells, and the culture supernatant is collected to obtain virus particles containing the desired Tud RNA, which can be used for transfection of the target cells.
  • the Tud RNA of the present invention which inhibits the expression of human miR-148a, miR-185 and miR-424 specifically knocks down the expression of human miR-148a, miR-185 and miR-424.
  • the Tud RNA which inhibits the expression of human miR-148a, miR-185 and mi R-424 is constructed on a lentiviral vector, and is effective not only for dividing and non-dividing cells, but also for in vivo and in vitro studies.
  • FIG. 1 is a structural diagram of a pGLV3 vector
  • FIG. 2 is a miRNA expression level of each group of cells, wherein, a.
  • TuD RNA oligonucleosides targeting miR-148a, miR-185 and miR-424 were designed based on the sequence information of miR-148a, miR-185 and miR-424 provided in the TuD RNA design sequence and miRBase.
  • Tud-148a-185-424 antisense strand [0035]
  • the 5' end of the sense strand template is added with GATCC, which is complementary to the sticky end formed by BamHI digestion; the AATTC is added to the 5' end of the antisense strand template, which is complementary to the sticky end formed by EcoRI digestion.
  • the pGLV3 vector (Gimma Gene Co., Ltd.) was digested with BamH I and EcoR I, and the vector was linearized.
  • the conditions of the digestion were as follows: pGLV3 vector (10 ⁇ , BamH I) Dicer (5 L, Fermentas), EcoR I endonuclease (5 L, Fermentas), FastDigest buffer (8 (VL, TOYOBO) were mixed, placed at 37 ° C for 1 hour, using a gel back kit ( Axygen) recovered the linear vector fragment and diluted its concentration to 50 ng ⁇ L.
  • the grown monoclonal was selected in LB containing 100 ⁇ ⁇ / ⁇ 1 ampicillin Incubate overnight at 30 ° C in liquid medium, using plasmid extraction kit (Omega) Bio-tek) The plasmid was purified and verified by sequencing to prepare a recombinant plasmid pGLV3-Tud-148a-185-424.
  • G is co-transfected into 293T cells, and the culture supernatant is collected to obtain virus particles containing the target Tud RNA, which can be used for transfection of the target cells.
  • WRL-68 cells were inoculated into 6-well plates at 1000000 cells per well. After 12 hours, the cell density was about 50%. The virus solution was taken separately. The virus was diluted 10 times with DMEM complete medium, and then polyglycolamine was added.
  • the medium in the 6-well plate was removed, and the virus-containing DMEM complete medium (containing 10% fetal bovine serum) was added. After 24 hours, the virus-containing DMEM complete medium was discarded, and the fresh DMEM complete medium was replaced. After 24 hours, 0.5 was used.
  • the cells were screened at a g/ml concentration of puromycin. After 10 days of screening, the medium was changed once every 3 days, and the concentration of puromycin was continuously increased to 1.0 g/ml.
  • the cell line obtained by screening was designated as TuD-148a-185-424 cell line.
  • the set kit reverse-transcribes and tails the miRNA to obtain the corresponding cDNA.
  • the cDNA of each of the two cells was used as a template, and the expression levels of miR-148a, miR-185 and miR-424 were detected by real-time PCR.
  • the experiment was repeated 3 times, and 3 parallel samples were set per well, with snord 44 as the internal reference. .
  • Fig. 2 it was found that the expression level of miR-148a with TuD-148a-185-424 cells was 57 ⁇ 3 ⁇ 4 lower than that of WRL-68 cells, and the expression level of m iR-185 was lower than that of WRL-68 cells.
  • the expression level of miR-424 was 49% lower than that of WRL-68 cells. The difference was statistically significant (/? ⁇ 0.01), indicating that the TuD-148a-185-424 cell line was successfully constructed.
  • the Tud RNA of the present invention which inhibits the expression of human miR-148a, miR-185 and miR-424 specifically knocks down the expression of human miR-148a, miR-185 and miR-424.
  • the Tud RNA which inhibits the expression of human miR-148a, miR-185 and mi R-424 is constructed on a lentiviral vector, and is effective not only in splitting and non-dividing Cells are also suitable for in vivo and in vitro studies.

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Abstract

提供了一种特异敲低人mir-148a、mir-185和miR-424表达的Tud RNA及其应用。编码该Tud RNA的DNA序列，如下所示：5'-ggcgctaggatcatcaacagtcacgtgatgatcttcttgaaacacaatccccgaccgaaatctaggagaccagcaagttttgcactcatctcgcgacgatacaagtattctggtcacagaatacaacagtcacgtgatgatcttcttgaaacacaatccccgaccgaaatctaggagaccagcaagttttgcactcatctcgcgacgatacaagatgatcctagcgccaccttttt-3'。该Tud RNA的应用包括将该Tud RNA构建于慢病毒载体上，得到含有该Tud RNA的重组载体，将含有该Tud RNA的重组载体作用于人源细胞，达到抑制人mir-148a、miR-185和miR-424的目的。

Description

特异敲氐人 miR-148a、 miR-185和 miR-424表达的 Tud
RNA及其应用
技术领域
[0001] 本发明涉及一种 Tud
RNA, 特别涉及一种特异敲低人 miR- 148a、 miR-185和 miR-424表达的 Tud RNA 及其应用。
背景技术
[0002] MicroRNA (miRNA) 是在真核生物中发现的一类内源性的非编码 RNA， 大小 一般在 22-25 nt之间， miRNA广泛分布于植物、 动物和多细胞生物中， 并且能发 挥重要的调节作用， 而在人类 miRNA的研究中， 发现 miRNA在正常组织和肿瘤 组织中的表达有着显著差异， 有些 miRNA会在肿瘤组织中有低表达， 有些则在 肿瘤组织中有高表达， 这说明 miRNA在肿瘤发生过程中起了至关重要的作用。
[0003] miR- 148a是近几年研究得较多的一种 microRNA。 据报道， miR-148a与外源性 物质代谢、 细胞凋亡、 多种癌症的发生、 发展和表观遗传等都密切有关； miR-1 85是一个长度为 22nt的 miRNA, 定位于人染色体 22ql l.21， 在结肠癌、 胃癌、 食 管癌、 肺癌、 肝癌等肿瘤的发生和侵袭等方面作为一个抑癌基因发挥重要作用 ， 另外它一种甲基化相关的抑瘤性 miRNA, 可通过直接靶向 DNMT1的表达而影 响全基因组的甲基化水平， 进而调控某些基因的甲基化修饰状态， 影响基因表 达； 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 m iR-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-α的表达相关； 由于 miR-424启动子区域具有 CpG岛， 它与甲基化 诱导的基因特异敲低也相关。 通过控制 miR-148a、 miR- 185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的功能研究主要通过 miRNA干扰和过表达技术完成。 现有 miRNA干扰 技术中， anti-miR和 antagomiR为瞬吋转染技术， 其干扰效果不能稳定保持， 而 m iRNA sponge效果远未达到最优， 现有技术缺乏一种干扰效果好且能实现长期稳 定干扰的技术。
[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] 本发明的首要目的在于克服现有技术的缺点与不足， 提供一种特异敲低人 miR-
148a、 miR-185和 miR-424表达的 Tud RNA。
[0007] 本发明的另一目的在于提供上述特异敲低人 miR-148a、 miR-185和 miR-424表达 的 Tud RNA的应用。
[0008] 本发明的再一目的在于提供一种含有上述 Tud RNA的重组载体。
[0009] 本发明的目的通过下述技术方案实现：
[0010] 一种特异敲低人 miR-148a、 miR-185和 miR-424表达的 Tud RNA， 编码所述的抑 制人 miR-148a、 miR-185和 miR-424表达的 Tud RNA的 DNA序列如下所示：
[0011] 5'- ggcgctaggatcatcaacagtcacgtgatgatcttcttgaaacacaatccccgaccgaaatctaggagaccagcaagttttgc actcatctcgcgacgatacaagtattctggtcacagaatacaacagtcacgtgatgatcttcttgaaacacaatccccgaccga aatctaggagaccagcaagttttgcactcatctcgcgacgatacaagatgatcctagcgccaccttttt -3 ' °
[0012] 所述的特异敲低人 miR- 148a、 miR-185和 miR-424表达的 Tud RNA的应用， 该应 用包括将所述的 Tud RNA构建于慢病毒载体上， 得到含有所述 Tud RNA的重组载 体； 将含有所述 Tud
RNA的重组载体作用于 WRL-68细胞， 达到抑制 miR-148a、 miR- 185和 miR-424表 达的目的；
[0013] 所述慢病毒载体为 pGLV3/Hl/GFP+Puro (pGLV3) 慢病毒穿梭载体； [0014] 所述的含有所述 Tud RNA的重组载体的制备方法， 包含以下步骤：
[0015] ( 1) 设计含有抑制人 miR-148a、 miR-185和 miR-424表达的 Tud RNA的 DNA序 列， 如下所示：
[0016]
ggcgctaggatcatcaacagtcacgtgatgatcttcttgaaacacaatccccgaccgaaatctaggagaccagcaagttttgc actcatctcgcgacgatacaagtattctggtcacagaatacaacagtcacgtgatgatcttcttgaaacacaatccccgaccga aatctaggagaccagcaagttttgcactcatctcgcgacgatacaagatgatcctagcgccaccttttt -3 '；
[0017] 为将其克隆到 PGLV3慢病毒穿梭载体上， 合成以下正义链 （F) 及反义链 （R
DNA序歹 'J :
[0018] Tud- 148a- 185-424正义链：
[0019] 5，-
GATCCggcgctaggatcatcaacagtcacgtgatgatcttcttgaaacacaatccccgaccgaaatctaggagaccagc aagttttgcactcatctcgcgacgatacaagtattctggtcacagaatacaacagtcacgtgatgatcttcttgaaacacaatcc ccgaccgaaatctaggagaccagcaagttttgcactcatctcgcgacgatacaagatgatcctagcgccacctttttG -3'
[0020] Tud- 148a- 185-424反义链：
[0021]
AATTCaaaaaggtggcgctaggatcatcttgtatcgtcgcgagatgagtgcaaaacttgctggtctcctagatttcggtcg gggattgtgtttcaagaagatcatcacgtgactgttgtattctgtgaccagaatacttgtatcgtcgcgagatgagtgcaaaactt gctggtctcctagatttcggtcggggattgtgtttcaagaagatcatcacgtgactgttgatgatcctagcgccG - 3，。
[0022] 上述正义链模板的 5'端添加了 GATCC , 与 BamHI酶切后形成的粘端互补； 反义 链模板的 5'端添加了 AATTC, 与 EcoRI酶切后形成的粘端互补；
[0023] (2) 将等量的正义链和反义链 DNA混合， 进行退火， 以形成 DNA双链， 得到 Tud RNA模板；
[0024] (3) 将 pGLV3载体用 BamHI和 EcoRI双酶切， 得到线性化的 pGLV3载体；
[0025] (4) 将步骤 (2)中的 Tud RNA模版和步骤 (3)中线性化的 pGLV3载体进行连接， 得到重组载体 pGLV3-Tud- 148a- 185-424；
[0026] (5) 将重组载体 pGLV3-Tud-148a-185-424与包装载体 pGag/Pol、 pRev、 pVSV- G共转染至 293T细胞， 收集培养上清获得含有目的 Tud RNA的病毒颗粒， 可用于 转染目的细胞。
发明的有益效果
有益效果
[0027] 本发明所述抑制人 miR-148a、 miR-185和 miR-424表达的 Tud RNA能特异敲低 人 miR-148a、 miR-185和 miR-424的表达。 将所述抑制人 miR-148a、 miR-185和 mi R-424表达的 Tud RNA构建于慢病毒载体上， 不仅可有效作用于分裂及非分裂状 态的细胞， 也适用于体内及体外研究。
对附图的简要说明
附图说明
[0028] 图 1为 pGLV3载体的结构图； 图 2各组细胞的 miRNA表达水平， 其中， a.
miR- 148a的表达情况， b. miR-185的表达情况， c. 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
[0029] 下面结合实施例及附图对本发明作进一步详细的描述， 但本发明的实施方式不 限于此。
[0030] 实施例一特异敲氐人 miR-148a、 miR-185和 miR-424表达的 Tud RNA的设计与 合成
[0031] 根据 TuD RNA设计序列和 miRBase中提供的 miR-148a、 miR- 185和 miR-424的序 列信息， 设计出同吋针对 miR-148a、 miR- 185和 miR-424的 TuD RNA寡核苷酸序 歹 |J， 其序列为 5' - ggcgctaggatcatcaacagtcacgtgatgatcttcttgaaacacaatccccgaccgaaatctaggagaccagcaagttttgc actcatctcgcgacgatacaagtattctggtcacagaatacaacagtcacgtgatgatcttcttgaaacacaatccccgaccga aatctaggagaccagcaagttttgcactcatctcgcgacgatacaagatgatcctagcgccaccttttt -3' ° 委托上海 生工合成。
[0032] 为将其克隆到 pGLV3慢病毒穿梭载体上， 设计以下正义链 （F) 及反义链 （R ) DNA序歹 IJ : [0033] Tud- 148a- 185-424正义链：
[0034] 5'-
GATCCggcgctaggatcatcaacagtcacgtgatgatcttcttgaaacacaatccccgaccgaaatctaggagaccagc aagttttgcactcatctcgcgacgatacaagtattctggtcacagaatacaacagtcacgtgatgatcttcttgaaacacaatcc ccgaccgaaatctaggagaccagcaagttttgcactcatctcgcgacgatacaagatgatcctagcgccacctttttG -3'
[0035] Tud- 148a- 185-424反义链：
[0036] 5'-
AATTCaaaaaggtggcgctaggatcatcttgtatcgtcgcgagatgagtgcaaaacttgctggtctcctagatttcggtcg gggattgtgtttcaagaagatcatcacgtgactgttgtattctgtgaccagaatacttgtatcgtcgcgagatgagtgcaaaactt gctggtctcctagatttcggtcggggattgtgtttcaagaagatcatcacgtgactgttgatgatcctagcgccG -3'°
[0037] 上述正义链模板的 5'端添加了 GATCC , 与 BamHI酶切后形成的粘端互补； 反义 链模板的 5'端添加了 AATTC, 与 EcoRI酶切后形成的粘端互补。
[0038] (2) 将等量的正义链 （10 μΜ， 5 μΐ) 和反义链 （10 μΜ， 5 μΐ) DNA混合， 加 入 90 μ1 (1(1Η2Ο， 在 PCR仪上按照如下程序进行退火处理： 95°C 5 min， 然后以每 秒下降 O.rC的速率降至 25°C进行退火， 最后 4°C保存备用。 将退火后所得 Tud RNA模板溶液稀释 5倍， 使其终浓度为 200 nM， 用于连接反应。
[0039] (3) 将 pGLV3载体（吉玛基因股份有限公司）用 BamH I和 EcoR I双酶切， 进 行载体的线性化处理， 酶切条件如下所述： 将 pGLV3载体（10μ 、 BamH I内 切酶（5 L， Fermentas)、 EcoR I内切酶（5 L， Fermentas)、 FastDigest buffer(8(VL ， TOYOBO)混匀， 置于 37°C反应 1小吋， 用凝胶回试剂盒（Axygen)回收线性 载体片段， 将其浓度稀释至 50ng^L。
[0040] (4) 将线性化的 pGLV3载体 1 μ1、 经退火处理的 Tud RNA模版 1μ1、 Τ4 DNA 连接酶 （NEB) 、 1μ1、 10xT4连接缓冲液 （NEB) 2μ1、 去离子水 15 μΓ混匀， 于 4°C连接过夜。 取连接产物 5 μ1转化大肠杆菌 ToplO感受态细胞， 在含有 10(Vg/ml氨苄青霉素的 LB平板上于 30°C培养过夜。 挑选长出的单克隆在含有 100 μ§/ηι1氨苄青霉素的 LB液体培养基中于 30°C培养过夜， 用质粒抽提试剂盒 (Omega bio-tek) 纯化质粒， 经测序验证后大量制备重组质粒 pGLV3-Tud-148a-185-424。
[0041] (5) 将重组载体 pGLV3-Tud-148a-185-424与包装载体 pGag/Pol、 pRev、 pVSV-
G共转染至 293T细胞， 收集培养上清获得含有目的 Tud RNA的病毒颗粒， 可用于 转染目的细胞。
[0042] 实施例二慢病毒转导 WRL-68细胞
[0043] 接种 WRL-68细胞于 6孔板中， 每孔 1000000个细胞， 12h后细胞密度约为 50 % , 分别取病毒液， 用 DMEM完全培养基 10倍稀释病毒， 再加入聚凝胺
(polybrene)至终浓度为 8 g/mL。 去除 6孔板中的培养基， 加入含病毒的 DMEM 完全培养基（含 10%胎牛血清)， 24h后弃去含病毒的 DMEM完全培养基， 更换新 鲜的 DMEM完全培养基， 24h后用 0.5 g/ml浓度的嘌呤霉素筛选细胞。 筛选 10d, 每隔 3d更换培养基一次， 并不断的增加嘌呤霉素的浓度至 1.0 g/ml。 筛 选获得的细胞株命名为 TuD- 148a- 185-424细胞株。
[0044] 实施例三荧光定量 PCR检测 miRNA的表达水平变化
[0045] 分别接种正常 WRL-68细胞、 TuD- 148a- 185-424细胞至 6孔板 （每孔约 300000个 ) ， 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， 然后用 S-Poly(T) hsa-miR-148a qPCR-assay primer
set、 S-Poly(T) hsa-miR-185 qPCR-assay primer set和 S-Poly(T) hsa-miR-424 qPCR-assay primer
set试剂盒对 miRNA进行逆转录和加尾， 得到相应的 cDNA。 取 2种细胞的 cDNA 各 2 μί为模板， 荧光定量 PCR检测 miR-148a、 miR-185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 2所示， 可 以看到与 TuD-148a-185-424细胞的 miR-148a的表达水平比 WRL-68细胞低 57<¾， m iR-185的表达水平比 WRL-68细胞低 54%， miR-424的表达水平比 WRL-68细胞低 4 9%。 差异有统计学意义 （/?<0.01) ， 说明 TuD-148a-185-424细胞株构建成功。 工业实用性
[0046] 本发明所述抑制人 miR-148a、 miR-185和 miR-424表达的 Tud RNA能特异敲低 人 miR-148a、 miR-185和 miR-424的表达。 将所述抑制人 miR-148a、 miR-185和 mi R-424表达的 Tud RNA构建于慢病毒载体上， 不仅可有效作用于分裂及非分裂状 态的细胞， 也适用于体内及体外研究。

Claims

权利要求书 一种特异敲低人 miR-148a、 miR-185和 miR-424表达的 Tud RNA， 其特 征在于： 编码所述 Tud RNA的 DNA序列如下所示： 5'- ggcgctaggatcatcaacagtcacgtgatgatcttcttgaaacacaatccccgaccgaaatctaggagacc agcaagttttgcactcatctcgcgacgatacaagtattctggtcacagaatacaacagtcacgtgatgatctt cttgaaacacaatccccgaccgaaatctaggagaccagcaagttttgcactcatctcgcgacgatacaaga tgatcctagcgccaccttttt -3，。 权利要求 1所述的 Tud RNA在制备 miR-148a、 miR-185和 miR-424功能 研究的产品以及在制备 miR-148a、 miR-185和 miR-424有关疾病的靶 向治疗药物中的应用。 根据权利要求 2所述的 Tud RNA在制备 miR-148a、 miR-185和 miR-424 功能研究的产品以及在制备 miR-148a、 miR-185和 miR-424有关疾病 的靶向治疗药物中的应用， 其特征在于该应用包括将权利要求 1中所 述的 DNA序列构建于慢病毒载体上， 得到含有权利要求 1中所述 DNA 序列的重组载体。 根据权利要求 3所述的 Tud RNA在制备 miR-148a、 miR-185和 miR-424 功能研究的产品以及在制备 miR-148a、 miR-185和 miR-424有关疾病 的靶向治疗药物中的应用， 其特征在于： 所述慢病毒载体为 PGLV3慢 病毒穿梭载体。 根据权利要求 4所述的 Tud RNA在制备 miR-148a、 miR-185和 miR-424 功能研究的产品以及在制备 miR-148a、 miR-185和 miR-424有关疾病 的靶向治疗药物中的应用， 其特征在于： 所述的含有权利要求 1中所述 DNA序列的重组载体的制备方法， 包含 以下步骤：
( 1) 设计含有抑制人 miR-148a、 miR-185和 miR-424表达的 Tud RNA 的 DNA序歹 ij， 如下所示：
5，- ggcgctaggatcatcaacagtcacgtgatgatcttcttgaaacacaatccccgaccgaaatctaggagacc agcaagttttgcactcatctcgcgacgatacaagtattctggtcacagaatacaacagtcacgtgatgatctt cttgaaacacaatccccgaccgaaatctaggagaccagcaagttttgcactcatctcgcgacgatacaaga tgatcctagcgccaccttttt -3，；
为将其克隆到 pGLV3慢病毒穿梭载体上， 合成以下正义链及反义链 D NA序列：
Tud-148a- 185-424正义链：
5，-
GATCCggcgctaggatcatcaacagtcacgtgatgatcttcttgaaacacaatccccgaccgaaatct aggagaccagcaagttttgcactcatctcgcgacgatacaagtattctggtcacagaatacaacagtcacg tgatgatcttcttgaaacacaatccccgaccgaaatctaggagaccagcaagttttgcactcatctcgcgac gatacaagatgatcctagcgccacctttttG -3'；
Tud-148a- 185-424反义链：
5，-
AATTCaaaaaggtggcgctaggatcatcttgtgtttcaagaagatcatcacgtgactttgggttcaaga cagatagtacgtgactttgtatcgtcgcgagatgagtgcaaaacgttgtattctgtgaccagaatacttgtgtt tcaagaagatcatcacgtgactttgggttcaagacagatagtacgtgactttgtatcgtcgcgagatgagtg caaaacgttgatgatcctagcgccG -3'°
上述正义链模板的 5'端添加了 GATCC, 与 BamHI酶切后形成的粘端 互补； 反义链模板的 5'端添加了 AATTC, 与 EcoRI酶切后形成的粘端 互补；
(2) 将等量的正义链和反义链 DNA混合， 进行退火， 以形成 DNA双 链， 得到 Tud RNA模板；
(3) 将 pGLV3载体用 BamHI和 EcoRI双酶切， 得到线性化的 pGLV3 载体；
(4) 将步骤 （2) 中的 Tud RNA模版和步骤 (3)中线性化的 pGLV3载 体进行连接， 得到重组载体 pGLV3-Tud-148a-185-424。
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EP2363467A1 (en) * 2008-10-23 2011-09-07 The University of Tokyo Method for inhibiting function of micro-rna

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EP2363467A1 (en) * 2008-10-23 2011-09-07 The University of Tokyo Method for inhibiting function of micro-rna

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XIE, XING ET AL.: "Construction of a Human Bronchial Epithelial Hsa-miR-148a-3p Knockdown Cell Line", CARCINOGENESIS, TERATOGENESIS & MUTAGENESIS, vol. 26, no. 3, 30 May 2014 (2014-05-30), pages 204 - 208, XP055447857, ISSN: 1004-616X, DOI: 10.3969/j.issn.1004-616x.2014.03.010 *

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