WO2018170753A1 - 一种敲减 miRNA-29a 、 miRNA-152 和 miRNA-185 的 Tud RNA 及其应用 - Google Patents
一种敲减 miRNA-29a 、 miRNA-152 和 miRNA-185 的 Tud RNA 及其应用 Download PDFInfo
- Publication number
- WO2018170753A1 WO2018170753A1 PCT/CN2017/077594 CN2017077594W WO2018170753A1 WO 2018170753 A1 WO2018170753 A1 WO 2018170753A1 CN 2017077594 W CN2017077594 W CN 2017077594W WO 2018170753 A1 WO2018170753 A1 WO 2018170753A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- mirna
- mir
- tud
- rna
- tud rna
- Prior art date
Links
Definitions
- the present invention relates to a Tud RNA which knocks down miRNA-29a, miRNA-152 and miRNA-185 and an application thereof, and belongs to the field of genetic engineering technology.
- 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-29a is a small RNA closely related to cell proliferation. It is involved in many diseases and can act as a tumor suppressor gene in a variety of tumors. It has the ability to grow and invade cells such as human gastric cancer and bladder cancer.
- miR-152 is a kind of Multi-functional miRNA, the study found that miR-152 is related to methylation, such as the methyltransferase DNMT1 content and enzyme activity, miR-152 can be methylated by endometrial cancer DNA into a silent gene, and its It is involved in the development of various cancers. It is a tumor suppressor microRNA, which is associated with many diseases such as preeclampsia, trophoblastic tumor, bladder cancer, gastrointestinal cancer, ovarian cancer, etc.
- miR-185 is a 22 nt miRNA. , located in human chromosome 22ql l.21, plays an important role as a tumor suppressor gene in the occurrence and invasion of colon cancer, gastric cancer, esophageal cancer, lung cancer, liver cancer and other tumors. Further it is one methylation-related inhibition of miRNA, may affect the level of the whole genome methylation by direct targeted expression of DNMT1, further regulation of the methylation status of certain modification genes, gene expression. By controlling the expression of miR-2 9a, miR-152 and miR-185, the synergy with other drugs can provide new epigenetic ideas for the treatment of cancer.
- MiRNA functional studies often require miRNA silencing techniques, 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 can inhibit miRNAs in a long-term, stable and efficient manner.
- the present invention provides a knockdown of miRNA-29a, miRNA-152 and miRNA-185 of Tud RNA, the nucleotide sequence of which is shown in SEQ ID NO: 1, mainly in inhibiting human miR-29a, miR-152 And miR-185 expression products are used.
- the present invention also provides a method for preparing a shRNA interference carrier, and the steps are as follows:
- the interference fragment obtained in the step (1) was ligated to a plasmid vector linearized by BamHI and Hindlll to obtain a Tud RNA vector.
- the interference fragment obtained in the step (1) was ligated to the p-Ge nesil 1.0 plasmid vector linearized by BamHI and Hindlll to obtain the Tud RNA vector p-Genesil-Tud-29a-152-185.
- RNA vector preparation in inhibiting human miR-29a, miR-152 and miR-185 expression products
- Tud RNA sequences were designed by comparative analysis of human miR-29a, miR-152 and miR-185 sequences.
- human miR-29a, 1 ⁇ 11-152 and 1 ⁇ 11-185 sequences single-stranded Tud RNA fragments with BamHI and Hindlll cleavage sites were synthesized, renatured into double strands, ligated to The p-Genesill.O vector linearized by BamHI and Hindlll. The effective interference fragment constructed and screened is Tud-29a-152-185.
- the same design of the present invention interferes with miR-29a, !
- the ⁇ 11-152 and 1 ⁇ 11-185 TuD RNA sequences have a stem-loop structure and are not easily degraded.
- the double-stranded Tud RNA has higher binding efficiency than the currently used single-stranded miRNA sponge, and its Target, can better achieve the interference of three miRNAs, improve the efficiency of mi RNA function research.
- FIG. 1 miRNA expression levels of each group of cells, wherein, a. miR-29a expression, b.
- pGenesill.O vector was purchased from Biovector Plasmid Vector Culture Cell Genetic Collection Center; Reverse Transcription Kit
- SYBR fluorescence real-time quantitative PCR kits were purchased from Takara Reagent Co., Ltd.; endotoxin plasmid extraction kits were purchased from Tiangen Biochemical.
- TuD designed for miR-29a, miR-152 and miR-185 was designed.
- RNA oligonucleotide sequence ⁇ lJTud-29a-152-185 whose sequence ⁇ ij is shown in SEQ ID NO 1, was commissioned by Shanghai Biotech to synthesize by gene synthesis.
- the Tud-29a-152-185 sequence and p-Genesill.0 were separately treated with BamHI and Hindlll enzymes, respectively, and electrophoresed and recovered, and then ligated with T4 DNA ligase at 4 ° C overnight, and then transformed into competent E. coli ToplO, Single colonies were cultured and sent to Shanghai Biotech for sequencing.
- the correctly sequenced p-Genesil-Tud-29a-152-185 vector was constructed.
- the p-Genesil-Tud-29a-152-185 vector was extracted using an endotoxin plasmid extraction kit.
- T24 cells were seeded in 6-well plates at 1000000 cells per well, and the cell density was about 60% after 18 h.
- the p-Genesil-Tud-29a-152-185 vector was transduced into T24 cells using jetPrime transfection reagent. After 4 h of reaction, the medium was changed to fresh DMEM complete medium and culture was continued for 24 h.
- the miRNA extraction and isolation kit extracts the miRNAs of these cells, and then uses S-Poly(T) hsa-miR-29a qPCR-assay primer set, S-Poly(T) hsa-miR-152 qPCR-assay primer set and S-Poly (T) hsa-miR-185 qPCR-assay primer set kit (both purchased from Shenzhen Anran Bio) Reverse transcription and tailing of miRNA to obtain the corresponding cDNA. Take 2 kinds of cells of cDNA 2
- the expression levels of miR-29a, miR-152 and miR-185 were detected by real-time PCR, and the experiment was repeated 3 times, and 3 parallel samples were set per well, and snord 44 was used as an internal reference.
- the results are shown in Figure 1. It can be seen that the expression level of miR-29a in TuD-29a-152-185 cells is 53 ⁇ 3 ⁇ 4 lower than that in T24 cells, and the expression level of miR-152 is 69% lower than that in T24 cells, miR-185 The expression level is 62% lower than that of T24 cells. The difference is statistical Significance ( ⁇ 0.01), indicating that the TuD-29a-152-185 cell line was successfully constructed.
- the present invention is designed to interfere with the miR-29a, 1 ⁇ &-152 and 1 ⁇ !-185 TuD RNA sequences with a stem-loop structure, which is not easily degraded, and the double-stranded Tud RNA is relatively common with the currently used single-stranded miRNA sponge.
- the binding efficiency is higher, and at the same time, the interference of the three miRNAs can be well achieved for the three targets, and the efficiency of the miRNA function research is improved.
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
提供了一种敲减miRNA-29a、miRNA-152和miRNA-424的Tud RNA，其编码核苷酸序列如SEQ ID NO.1所示。还提供了一种含有所述Tud RNA编码核苷酸序列的干扰载体的制备方法。
Description
发明名称：一种敲减 miRNA-29a、 miRNA-152和 miRNA-185的 Tud
RNA及其应用
技术领域
[0001] 本发明涉及一种敲减 miRNA-29a、 miRNA- 152和 miRNA- 185的 Tud RNA及其应 用， 属于基因工程技术领域。
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-29a是一个与细胞增殖紧密相关的小分子 RNA， 参与多种疾病， 可在多种 肿瘤中起到抑癌基因作用， 它与人胃癌和膀胱癌等细胞的生长和侵袭能力相关 ， 其表达水平是评价胶质瘤良恶性的重要参考指标， 还与动脉粥样硬化肝纤维 化等疾病相关， 对多种肿瘤的治疗具有重要的潜在应用价值； miR-152是一种具 有多功能的 miRNA, 研究发现 miR-152与甲基化相关， 如与甲基转移酶 DNMT1 含量和酶活性相关， miR-152可被子宫内膜癌 DNA甲基化变为沉默基因， 并且其 与多种癌症的发生发展相关， 它是一种肿瘤抑制 microRNA, 与子痫前期、 滋养 细胞肿瘤、 膀胱癌、 胃肠癌、 卵巢癌等诸多疾病相关； miR-185是一个长度为 22 nt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。 通过控制 miR-2 9a、 miR-152和 miR-185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] 本发明提供一种敲减 miRNA-29a、 miRNA- 152和 miRNA- 185的 Tud RNA， 其核 苷酸序列如 SEQ ID N0 1所示， 主要在抑制人源 miR-29a、 miR- 152和 miR- 185表 达制品中应用。
[0007] 本发明还提供了一种 shRNA干扰载体的制备方法， 步骤如下：
[0008] (1) 在 SEQ ID N0.1所示的核苷酸序列两端加入合成带有 BamHI和 Hindlll酶 切位点， 获得 Tud RNA片段；
[0009] (2) 将步骤 （1) 中获得的干扰片段连接到经过 BamHI和 Hindlll线性化的质粒 载体上， 获得 Tud RNA载体。
[0010] 具体步骤如下：
[0011] (1) 在 SEQ ID NO 1所示的核苷酸序列两端加入合成带有 BamHI和 Hindlll酶 切位点， 获得 Tud RNA片段；
[0012] (2) 将步骤 （1) 中获得的干扰片段连接到经过 BamHI和 Hindlll线性化的 p-Ge nesil 1.0质粒载体上， 获得 Tud RNA载体 p-Genesil-Tud-29a-152-185。
[0013] 本发明提供的 Tud
RNA载体的制备方法在抑制人源 miR-29a、 miR- 152和 miR- 185表达制品中的应用
[0014] 具体而言， 经对比分析后的人源 miR-29a、 miR- 152和 miR- 185序列， 设计 Tud RNA序列。
[0015] 根据人源 miR-29a、 1^11-152和1^11-185序列， 分别合成带有 BamHI和 Hindlll酶 切位点的单链 Tud RNA片段， 复性成双链后， 连接到经过 BamHI和 Hindlll线性化 的 p-Genesill.O载体。 构建并筛选的有效干扰片段为 Tud-29a-152-185。
发明的有益效果
有益效果
[0016] 本发明设计的同吋干扰 miR-29a、 ！^11-152和1^11-185 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， 并且同吋针对三个靶点， 能较好地实现三个 miRNA的干扰， 提高 m iRNA功能研究的效率。
对附图的简要说明
附图说明
[0017] 图 1各组细胞的 miRNA表达水平情况， 其中， a. miR-29a的表达情况， b.
miR-152的表达情况， c. miR-185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
[0018] 以下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。 下述实施例中的实验方法， 如无特殊说明， 均为常规方法。 下述实施 例中所用的试验材料， 如无特别说明， 均为购自市场。
[0019] pGenesill.O载体购自 Biovector质粒载体菌种细胞基因保藏中心； 反转录试剂盒
、 SYBR荧光实吋定量 PCR试剂盒均购自 Takara试剂公司； 去内毒素质粒提取试 剂盒购自天根生化。
[0020] SEQ ID NO 1： 5'-
AGCCAATCAAGATGATCCTAGCGCCACCTTTTT -3' =
[0021] 实施例 1 :
[0022] 靶向 miR-29a、 miR-152和 miR-185的 Tud RNA的设计与合成
[0023] 根据 TuD RNA设计序列和 miRBase中提供的 miR-29a、 miR-152和 miR-185的序 列信息， 设计出同吋针对 miR-29a、 miR-152和 miR-185的 TuD
RNA寡核苷酸序歹lJTud-29a-152-185， 其序歹 ij如 SEQ ID NO 1所示， 委托上海生工 以基因合成的方式合成。
[0024] 实施例 2:
[0025] p-Genesil-Tud-29a- 152- 185载体的构建
[0026] 用 BamHI和 Hindlll酶分别处理 Tud-29a-152-185序列和 p-Genesill.0， 分别电泳回 收后， 用 T4 DNA连接酶 4°C连接过夜， 然后转化感受态大肠杆菌 ToplO, 取单菌 落培养并送至上海生工测序。 测序正确的即为成功构建的 p-Genesil-Tud-29a-152- 185载体。 用去内毒素质粒提取试剂盒提取 p-Genesil-Tud-29a- 152- 185载体。
[0027] 实施例 3:
[0028] -06^8 丁11(1-29&-152-185载体转导丁24细胞
[0029] 接种 T24细胞于 6孔板中， 每孔 1000000个细胞， 18 h后细胞密度约为 60%， 用 jetPrime转染试剂将 p-Genesil-Tud-29a-152-185载体转导 T24细胞中， 反应 4 h后 ， 将培养基换成新鲜的 DMEM完全培养基， 然后继续培养 24 h。
[0030] 实施例 4:
[0031] miR-29a、 miR-152和 miR-185表达水平的检测
[0032] 取 T24细胞和转导 p-Genesil-Tud-29a- 152- 185载体的 T24细胞， 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-29a qPCR-assay primer set、 S-Poly(T) hsa-miR-152 qPCR-assay primer set和 S-Poly(T) hsa-miR-185 qPCR-assay primer set试剂盒 （均购自深圳盎然生物） 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
为模板， 荧光定量 PCR检测 miR-29a、 miR-152和 miR-185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-29a-152-185细胞的 miR-29a的表达水平比 T24细胞低 53<¾， miR-152的表 达水平比 T24细胞低 69%， miR-185的表达水平比 T24细胞低 62%。 差异有统计学
意义 （ρ<0.01) ， 说明 TuD-29a-152-185细胞株构建成功。
工业实用性
本发明设计的同时干扰 miR-29a、 1^&-152和1^! -185 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， 并且同时针对三个靶点， 能较好地实现三个 miRNA的干扰， 提高 m iRNA功能研究的效率。
Claims
(1)在 SEQ ID NO 1所示的核苷酸序列两端加入 BamHI和 Hindlll酶切位 点， 得到 Tud RNA片段；
(2)将步骤 (1)中获得的干扰片段连接到经过 BamHI和 Hindlll线性化的 质粒载体上， 获得干扰载体。
[权利要求 3] 根据权利要求 2所述的方法， 其特征在于， 具体步骤如下：
(1)在 SEQ ID NO 1所示的核苷酸序列两端加入 BamHI和 Hindlll酶切位 点， 得到 Tud RNA片段；
(2)将步骤 (1)中获得的 Tud RNA片段连接到经过 BamHI和 Hindlll线性 化的 p-Genesill.O质粒载体上， 获得干扰载体 p-Genesil-Tud-29a-152-18
5。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2017/077594 WO2018170753A1 (zh) | 2017-03-21 | 2017-03-21 | 一种敲减 miRNA-29a 、 miRNA-152 和 miRNA-185 的 Tud RNA 及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2017/077594 WO2018170753A1 (zh) | 2017-03-21 | 2017-03-21 | 一种敲减 miRNA-29a 、 miRNA-152 和 miRNA-185 的 Tud RNA 及其应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2018170753A1 true WO2018170753A1 (zh) | 2018-09-27 |
Family
ID=63584873
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2017/077594 WO2018170753A1 (zh) | 2017-03-21 | 2017-03-21 | 一种敲减 miRNA-29a 、 miRNA-152 和 miRNA-185 的 Tud RNA 及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO2018170753A1 (zh) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010138263A2 (en) * | 2009-05-28 | 2010-12-02 | University Of Massachusetts | Novel aav 's and uses thereof |
-
2017
- 2017-03-21 WO PCT/CN2017/077594 patent/WO2018170753A1/zh active Application Filing
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010138263A2 (en) * | 2009-05-28 | 2010-12-02 | University Of Massachusetts | Novel aav 's and uses thereof |
Non-Patent Citations (4)
| Title |
|---|
| DATABASE Genbank [O] 11 December 2014 (2014-12-11), YU , M.: "Homo sapiens microRNA 185 (MIR185), microRNA", XP055540744, Database accession no. NR_029706 * |
| DATABASE Genbank [O] 21 May 2015 (2015-05-21), SERAFIN, A.: "Homo sapiens microRNA 29a (MIR29A), microRNA", XP055540730, Database accession no. NR_029503 * |
| DATABASE Genbank [O] 21 May 2015 (2015-05-21), YAO, Y.: "Homo sapiens microRNA 152 (MIR152), microRNA", XP055540739, Database accession no. NR_029687 * |
| ZHU, LINWENSI ET AL.: "The Structure and Clinical Roles of MicroRNA in Colorectal Cancer", GASTROENTEROLOGY RESEARCH AND PRACTICE, vol. 2016, 31 December 2016 (2016-12-31), pages 1 - 6, XP055538564, ISSN: 1687-630X * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Xu et al. | MALAT-1: a long non-coding RNA and its important 3'end functional motif in colorectal cancer metastasis | |
| Ahmed | Role of miRNA in carcinogenesis and biomarker selection: a methodological view | |
| Price et al. | Matrigel basement membrane matrix influences expression of microRNAs in cancer cell lines | |
| Pan et al. | Role of long non-coding RNAs in gene regulation and oncogenesis | |
| EP3778892A1 (en) | Novel small activating rna | |
| AU2020291467A1 (en) | Novel crispr DNA targeting enzymes and systems | |
| Wulf et al. | Chemical capping improves template switching and enhances sequencing of small RNAs | |
| WO2023283600A1 (en) | Method for analyzing an ability of target nucleic acid sequences to impact gene expression | |
| WO2018170753A1 (zh) | 一种敲减 miRNA-29a 、 miRNA-152 和 miRNA-185 的 Tud RNA 及其应用 | |
| WO2018165929A1 (zh) | 一种双miRNA抑制表达载体及其构建方法和应用 | |
| CN111411155B (zh) | lncRNA IGFL2-AS1作为结肠癌诊断标志物的应用 | |
| WO2018170751A1 (zh) | 拮抗 miRNA 表达的 Tud RNA 及其应用 | |
| WO2018170750A1 (zh) | 小 RNA-29a 、 148a 和 152 的表达抑制的 Tud RNA 及其应用 | |
| Patutina et al. | Search for oligonucleotides selectively binding oncogenic miR-21 | |
| Hill et al. | Unravelling the Impact of miR-21 Overexpression on the MicroRNA Network and Cancer Pathways | |
| WO2018170752A1 (zh) | 一种敲减 miRNA-29a 、 miRNA-148a 和 miRNA-424 的 Tud RNA 及其应用 | |
| WO2018170622A1 (zh) | 一种同步下调微小rna-152和微小rna-185表达的方法 | |
| Florescu et al. | Study of MIR-200c and MIR-9 methylation on patients with breast cancer | |
| WO2017214949A1 (zh) | 一种抑制 miRNA-29a 表达的慢病毒载体的构建及其应用 | |
| WO2018170623A1 (zh) | 降低微小rna-152和微小rna-424表达的方法 | |
| WO2018170620A1 (zh) | 一种同时敲减两种miRNA表达的载体 | |
| WO2017219166A1 (zh) | 一种同时抑制双 miRNA 表达的慢病毒载体及其应用 | |
| WO2018170761A1 (zh) | 一种敲低 miR-140 、 miR-152 和 miR-185 表达的重组腺相关病毒 | |
| WO2018170619A1 (zh) | 抑制双miRNA表达的载体及其应用 | |
| WO2017219168A1 (zh) | 一种 miRNA-29a 和 miR-152 表达敲低的慢病毒载体及其应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 17902101 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 17902101 Country of ref document: EP Kind code of ref document: A1 |