WO2018170751A1 - 拮抗 miRNA 表达的 Tud RNA 及其应用 - Google Patents
拮抗 miRNA 表达的 Tud RNA 及其应用 Download PDFInfo
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- WO2018170751A1 WO2018170751A1 PCT/CN2017/077592 CN2017077592W WO2018170751A1 WO 2018170751 A1 WO2018170751 A1 WO 2018170751A1 CN 2017077592 W CN2017077592 W CN 2017077592W WO 2018170751 A1 WO2018170751 A1 WO 2018170751A1
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- the present invention relates to a Tud RNA which antagonizes the expression inhibition of miR-29a, miR-148a and miR-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. Correlation, its expression level is an important reference for evaluating the benign and malignant glioma, and it is also associated with diseases such as atherosclerosis liver fibrosis, and has important potential application value for the treatment of various tumors; miR-148a is in recent years A more studied micr 0 RNA.
- miR-148a is closely related to exogenous substance metabolism, apoptosis, occurrence, development and epigenetics of various cancers; miR-185 is a 22 nt miRNA, located on 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, which in turn regulates the methylation status of certain genes and affects gene expression.
- 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 inhibits miRNAs in a long-term, stable, and efficient manner.
- the present invention provides a Tud RNA which antagonizes miRNA expression, the nucleotide sequence of which is shown in SEQ ID NO: 1, and is mainly used for inhibiting expression products of human miR-29a, miR-148a and miR-185.
- 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-148a-185.
- a method of preparing an RNA vector for use in inhibiting expression of human miR-29a, miR-148a and miR-185 expression products is provided.
- Tud RNA sequences were designed by comparative analysis of human miR-29a, miR-148a and miR-185 sequences.
- the homologous interference miR-29a, miR-148a and miR-185 TuD RNA sequences designed by the present invention have a stem-loop structure and are 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 the same target for three targets can better achieve the interference of three miRNAs and improve the efficiency of miRNA 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.
- the TuD RNA design sequence and the sequence of miR-29a, miR-148a and miR-185 provided in miRBase In the column information, the TuD RNA oligonucleotide sequence ⁇ ijTud-29a-148a-185 targeting miR-29a, miR-148a and miR-185 was designed, and its sequence ⁇ ij is shown in SEQ ID NO 1.
- the raw workers are synthesized by means of gene synthesis.
- the Tud-29a-148a-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 pG eneS il-T U d-29a-14 8a-185 vector was constructed.
- the p-Genesil-Tud-29a-148a-185 vector was extracted using an endotoxin plasmid extraction kit.
- 16HBE 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-148a-185 vector was transduced with 16HBE 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.
- 16HBE cells and 16HBE cells transduced with p-Genesil-Tud-29a-148a-185 vector were used to extract miRNAs from these cells using the miRcute miRNA extraction and isolation kit, followed by S-Poly(T) hsa-miR- 29a qPCR-assay primer set, S-Poly(T) hsa-miR-148a qPCR-assay primer set and S-Poly(T) hsa-miR-185 qPCR-assay primer set kit (both purchased from Shenzhen Anran Bio) The miRNA is reverse transcribed and tailed to obtain the corresponding cDNA. Take 2 kinds of cells of cDNA 2
- the expression levels of miR-29a, miR-148a and miR-185 were detected by real-time PCR, and the experiment was repeated 3 times with 3 parallel samples per well, with snord 44 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-148a-185 cells is 52 ⁇ 3 ⁇ 4 lower than that in 16HBE cells, and the expression level of miR-1 48a is 57% lower than that in 16HBE cells, miR- The expression level of 185 was 56 ⁇ 3 ⁇ 4 lower than that of 16HBE cells. The difference was statistically significant (/? ⁇ 0.01), indicating that the TuD-29a-148a-185 cell line was successfully constructed.
- the homology of the present invention interferes with miR-29a, miR-148a and miR-185 TuD RNA sequences with stem loops
- the structure is not easily degraded.
- the double-stranded Tud RNA has higher binding efficiency than the commonly used single-stranded miRNA sponge, and the same target can better interfere with the three miRNAs and enhance the miRNA function. The efficiency of the research.
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Abstract
提供了一种拮抗miRNA表达的Tud RNA，其核苷酸序列如SEQ ID NO.1。还提供了一种抑制小RNA-29a、148a和185表达的Tud RNA干扰载体，其通过以下方法构建：根据经对比分析后的人源miR-29a、miR-148a和miR-185序列，合成带有BamHI和HindIII酶切位点的片段，并将该片段连接到经过BamHI和HindIII线性化的质粒载体上，经筛选后获得。
Description
拮抗 miRNA表达的 Tud RNA及其应用 技术领域
[0001] 本发明涉及一种拮抗 miR-29a、 miR-148a和 miR-185的表达抑制的 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-148a是近几 年研究得较多的一种 micr0RNA。 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-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-148a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] 本发明提供一种拮抗 miRNA表达的的 Tud RNA, 其核苷酸序列如 SEQ ID NO 1 所示， 主要在抑制人源 miR-29a、 miR-148a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-148a-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- 148a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- 148a和 miR- 185序列， 设计 Tud RNA序列。
[0015]
根据人源 miR-29a、 miR- 148a和 miR- 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-148a-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、 miR-148a和 miR-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 miRNA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- 148a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'-
TAGCCAATCAAGATGATCCTAGCGCCACCTTTTT -3，。
[0021] 实施例 1 :
[0022] 靶向 miR-29a、 miR-148a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- 148a和 miR- 185的序
列信息， 设计出同吋针对 miR-29a、 miR-148a和 miR-185的 TuD RNA寡核苷酸序 歹 ijTud-29a-148a-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-148a- 185载体的构建
[0026] 用 BamHI和 Hindlll酶分别处理 Tud-29a-148a-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-14 8a- 185载体。 用去内毒素质粒提取试剂盒提取 p-Genesil-Tud-29a- 148a- 185载体。
[0027] 实施例 3:
[0028] p-Genesil-Tud-29a-148a-185载体转导 16HBE细胞
[0029] 接种 16HBE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-148a-185载体转导 16HBE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- 148a和 miR-185表达水平的检测
[0032] 取 16HBE细胞和转导 p-Genesil-Tud-29a- 148a- 185载体的 16HBE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-148a 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- 148a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-148a-185细胞的 miR-29a的表达水平比 16HBE细胞低 52<¾， miR-1 48a的表达水平比 16HBE细胞低 57%， miR- 185的表达水平比 16HBE细胞低 56<¾。 差异有统计学意义 （/?<0.01) ， 说明 TuD-29a-148a-185细胞株构建成功。
工业实用性
[0033] 本发明设计的同吋干扰 miR-29a、 miR- 148a和 miR- 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 miRNA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- 148a- 1
85。
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| WO2010138263A2 (en) * | 2009-05-28 | 2010-12-02 | University Of Massachusetts | Novel aav 's and uses thereof |
| WO2015164786A1 (en) * | 2014-04-25 | 2015-10-29 | University Of Massachusetts | Recombinant aav vectors useful for reducing immunity against transgene products |
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Patent Citations (2)
| 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 |
| WO2015164786A1 (en) * | 2014-04-25 | 2015-10-29 | University Of Massachusetts | Recombinant aav vectors useful for reducing immunity against transgene products |
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| LI, QUANQIU ET AL.: "Research Progress on the Relationship between miR-185 and Tumor", JOURNAL OF MODERN MEDICINE & HEALTH, vol. 31, no. 3, 15 February 2015 (2015-02-15), pages 380 - 382, ISSN: 1009-5519 * |
| LINWENSI ZHU 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 - 7, XP055538564, ISSN: 1687-630X * |
| XIE , XING ET AL.: "Construction of a Human Bronchial Epithelial Hsa-miR-148a-3p Knockdown Cell Line", CARCINOGENESIS, TERATOGENESIS & MUTAGENESIS, vol. 26, no. 3, 14 May 2014 (2014-05-14), pages 204 - 208, 212, XP055447857, ISSN: 1004-616X, DOI: doi:10.3969/j.issn.1004-616x.2014.03.010 * |
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