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WO2018170623A1 - 降低微小rna-152和微小rna-424表达的方法 - Google Patents

降低微小rna-152和微小rna-424表达的方法 Download PDF

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WO2018170623A1
WO2018170623A1 PCT/CN2017/077173 CN2017077173W WO2018170623A1 WO 2018170623 A1 WO2018170623 A1 WO 2018170623A1 CN 2017077173 W CN2017077173 W CN 2017077173W WO 2018170623 A1 WO2018170623 A1 WO 2018170623A1
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mir
pri
expression
microrna
vector
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毛吉炎
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Shenzhen Biocan Technologies Co Ltd
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Shenzhen Biocan Technologies Co Ltd
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  • the present invention relates to the field of life sciences, and more particularly to a method of reducing the expression of microRNA-152 and microRNA-424.
  • MicroRNA-152 (miR-152) is a multifunctional miRNA. It was found that miR-152 is related to methylation, such as methyltransferase DNMT1 content and enzyme activity, miR-152 can be endometrium. Cancer DNA methylation becomes a silent gene, and it is associated with the development of various cancers. It is a tumor suppressing microRNA, and many diseases such as preeclampsia, trophoblastic tumor, bladder cancer, gastrointestinal cancer, ovarian cancer, etc. Related; microRNA-424 (miR-424) is a miRNA discovered in recent years, which affects the biological effects and development of tumor cells by acting on target genes in various tumors and participating in the signal pathway of target gene regulation.
  • 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 islands It is also associated with methylation-induced gene silencing.
  • miRNA function research is generally achieved by miRNA overexpression and silencing.
  • miRNA silencing is the presentation of artificially synthesized oligonucleotide small molecules into cells, forming heteroduplexes with endogenous miRNAs, and reducing miRNAs to target genes. Inhibition to achieve regulation of gene function. Silencing of miRNAs, especially long-term stable silencing, is currently more difficult to achieve.
  • Commonly used methods for silencing miRNA mainly include anti-miR, antagomiR, miRNA sponge, etc. These methods have short acting time, poor stability, complex construction, high instrumental requirements and complicated technical operation, which is difficult to achieve convenient, simple and effective. The effect of miR-152 and miR-424 expression was reduced.
  • the object of the present invention is to provide a method for reducing the expression of miR-152 and miR-424, which is simple in design, easy to operate, and can easily and effectively down-regulate the expression of miR-152 and miR-424 to overcome the existing The lack of technology.
  • the present invention is a method for down-regulating the expression of miR-152 and miR-424, and constructing a eukaryotic expression vector pRI- targeting miR-152 and miR-424 on the eukaryotic vector pRI-GFP/Neo containing the H1 promoter. 152-424, the expression of miR-152 and miR-424 in eukaryotic cells was down-regulated; after transfecting the vector into eukaryotic cells in vitro, the expression levels of miR-152 and miR-424 were detected by a real-time PCR instrument.
  • pRI-152-424 specifically, the sequence design of Tud-152-424 was performed by software; the backbone vectors pRI-GFP/Neo and Tud-152-424 sequences were double-digested with Xho I and Bgl II. The recovered fragments were purified and then ligated overnight at 4 °C; after transformation, 6 colonies were picked and inoculated into LB medium containing 10 ⁇ g/ml kanamycin. After 12 hours, the bacterial liquid was extracted and sent to Shanghai Yingjun. Sequencing. The correctly constructed vector was the successfully constructed recombinant plasmid pRI-152-424.
  • the present invention utilizes a molecular biology technique to construct a eukaryotic expression vector containing Tud-152-424 on the basis of a eukaryotic vector containing a CMV promoter, thereby down-regulating the expression of miR-152 and miR-424; After transfecting eukaryotic cells in vitro, the expression of miR-152 and miR-424 was detected by real-time PCR.
  • the invention is not only suitable for transfection of cells, but also for the production of whole horizontal transgenic animals.
  • Figure 1 is a schematic diagram showing the structure of the pRI-GFP/Neo vector
  • Figure 2 is a quantitative PCR detection of miRNA expression levels, wherein a. miR-152 expression, b. miR-424 expression.
  • Tud RNA targeting hsa-miR-152 and hsa-miR-424 was designed according to the design principle of Tough Decoy RNA (Tud RNA).
  • Tud RNA targeting hsa-miR-152 and hsa-miR-424 was designed according to the design principle of Tough Decoy RNA (Tud RNA).
  • Xho I and Bgl II restriction sites were added to the 5' and 3' ends of the Tud RNA to obtain the desired Tud-152-424 sequence.
  • the nucleotide sequence of Tud-152-424 is shown in SEQ ID No. 1.
  • the recombinant plasmid pRI-152-424 was extracted using an endotoxin-free plasmid extraction kit (Tiangen Biochemical).
  • the present invention utilizes molecular biology techniques to construct a eukaryotic expression vector containing Tud-152-424 on the basis of a eukaryotic vector containing a CMV promoter, thereby down-regulating the expression of miR-152 and miR-424; After transfecting eukaryotic cells in vitro, the expression of miR-152 and miR-424 was detected by real-time PCR.
  • the invention is not only suitable for transfection of cells, but also for the production of whole horizontal transgenic animals.

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Abstract

提供了一种降低微小RNA-152和微小RNA-424表达的方法，包括在含H1启动子的真核载体pRI-GFP/Neo上构建靶向miR-152和miR-424的真核表达载体pRI-152-424，实现下调miR-152和miR-424的表达。

Description

降低微小RNA-152和微小RNA-424表达的方法 技术领域
本发明涉及生命科学领域，尤其是一种降低微小RNA-152和微小RNA-424表达的方法。
背景技术
微小RNA-152(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；微小RNA-424(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-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沉默也相关。通过控制miR-152和miR-424的表达，同时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
目前miRNA功能研究一般通过miRNA过表达和沉默来实现，miRNA沉默是把人工合成的寡核苷酸小分子提呈到细胞内，与内源性miRNA形成异源双链，使miRNA降低对靶基因的抑制作用，以实现对基因功能的调控。miRNA沉默，特别是长期稳定沉默目前较难以实现。常用的沉默miRNA的方法主要有anti-miR，antagomiR，miRNA sponge等，这些方法存在作用时间短、稳定性差、构建复杂、仪器条件要求高且技术操作复杂的不足，难以达到方便、简单而有效地降低miR-152和miR-424表达的效果。
问题的解决方案
技术解决方案
本发明的目的是：提供一种降低miR-152和miR-424表达的方法，它方法设计简单，操作容易，能简单而有效地下调miR-152和miR-424表达的效果，以克服现有技术的不足。
本发明的实现方法：下调miR-152和miR-424表达的方法，在含H1启动子的真核载体pRI-GFP/Neo上构建靶向miR-152和miR-424的真核表达载体pRI-152-424，以下调miR-152和miR-424在真核细胞中的表达；将该载体体外转染真核细胞后，利用荧光定量PCR仪检测miR-152和miR-424的表达水平。
所述的pRI-152-424的构建，具体是，利用软件完成Tud-152-424的序列设计；用Xho I、Bgl II双酶切骨架载体pRI-GFP/Neo和Tud-152-424序列，回收片段纯化，再4℃的条件下过夜连接；连接后转化，挑取6个菌落，接种到含10μg/ml卡那霉素的LB培养基中，12小时后提取菌液并送上海英骏测序。测序正确的载体即为成功构建的重组质粒pRI-152-424。
发明的有益效果
有益效果
本发明利用分子生物学技术，在含CMV启动子的真核载体基础上，构建含Tud-152-424的真核表达载体，实现下调miR-152和miR-424的表达；并通过将该载体体外转染真核细胞后，利用荧光定量PCR仪检测miR-152和miR-424表达情况。
本发明不仅适于细胞的转染，也可用于整体水平转基因动物的制作。
对附图的简要说明
附图说明
图1为所述pRI-GFP/Neo载体的结构示意图；图2为定量PCR检测miRNA表达水平情况，其中，a.miR-152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
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。
实施例下调miR-152和miR-424表达的方法
①首先在miRBase数据库中检索出hsa-miR-152和hsa-miR-424的序列，根据Tough Decoy RNA(Tud RNA)的设计原则设计靶向hsa-miR-152和hsa-miR-424的Tud RNA，最后在Tud RNA的5’端和3’端加上Xho I和Bgl II酶切位点，获得所需的Tud-152-424序列。所述Tud-152-424的核苷酸序列如SEQ ID No 1所示。
②用Xho I和Bgl II双酶切骨架载体pRI-GFP/Neo和Tud-152-424序列，回收片段纯化，再4℃的条件下过夜连接；连接后转化，挑取6个菌落，接种到含10μg/ml卡那霉素的LB培养基中，12小时后提取菌液并送上海英骏测序。测序正确的载体即为成功构建的重组质粒pRI-152-424。
③应用无内毒素质粒提取试剂盒(天根生化)提取重组质粒pRI-152-424。
④按3万/ml的密度将Hela细胞接种于6孔板中，将10ug质粒pRI-152-424利用Lip ofectamine 2000瞬时转染细胞，继续在5％CO2、37℃条件下培养；48小时后，抽取各组细胞总RNA，利用Realtime PCR分别检测miR-152和miR-424表达情况，结果显示，pRI-152-424转染组中，miR-152和miR-424表达均水平显著下调(图2)，提示该载体可以用于降低miR-152和miR-424的表达。
工业实用性
本发明利用分子生物学技术，在含CMV启动子的真核载体基础上，构建含Tud-152-424的真核表达载体，实现下调miR-152和miR-424的表达；并通过将该载体体外转染真核细胞后，利用荧光定量PCR仪检测miR-152和miR-424表达情况。本发明不仅适于细胞的转染，也可用于整体水平转基因动物的制作。

Claims (2)

  1. 一种降低微小RNA-152和微小RNA-424的方法，其特征在于：在含H1启动子的真核载体pRI-GFP/Neo上构建靶向miR-152和miR-424的真核表达载体pRI-152-424，以下调miR-152和miR-424在真核细胞中的表达；将该载体体外转染真核细胞后，利用荧光定量PCR仪检测miR-152和miR-424的表达水平。
  2. 根据权利要求1所述的降低微小RNA-152和微小RNA-424表达的方法，其特征在于：所述的pRI-152-424的构建，具体是，利用软件完成Tud-152-424的序列设计；用Xho I、Bgl II双酶切骨架载体pRI-GFP/Neo和Tud-152-424序列，回收片段纯化，再4℃的条件下过夜连接；连接后转化，挑取6个菌落，接种到含10μg/ml卡那霉素的LB培养基中，12小时后提取菌液并送上海英骏测序。测序正确的载体即为成功构建的重组质粒pRI-152-424。
PCT/CN2017/077173 2017-03-18 2017-03-18 降低微小rna-152和微小rna-424表达的方法 Ceased WO2018170623A1 (zh)

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