WO2019227254A1 - miRNA-25过表达载体的构建及其应用 - Google Patents

miRNA-25过表达载体的构建及其应用 Download PDF

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WO2019227254A1
WO2019227254A1 PCT/CN2018/088529 CN2018088529W WO2019227254A1 WO 2019227254 A1 WO2019227254 A1 WO 2019227254A1 CN 2018088529 W CN2018088529 W CN 2018088529W WO 2019227254 A1 WO2019227254 A1 WO 2019227254A1
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mir
overexpression
mirna
construction
expression vector
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PCT/CN2018/088529
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English (en)
French (fr)
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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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Publication of WO2019227254A1 publication Critical patent/WO2019227254A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease

Definitions

  • the invention belongs to the field of genetic engineering, and particularly relates to the construction and application of a miRNA-25 overexpression vector.
  • MicroRNA is a type of small single-stranded short RNA with a length of about 22 to 25 nucleotides. Although it does not encode a protein, it can bind to homologous mRNA in an incomplete base-pairing manner and inhibit it. The expression of mRNA may induce its degradation, thereby negatively regulating mRNA and affecting the production of related proteins.
  • miRNAs are regulated by miRNAs, and they are almost involved in a series of physiological and biochemical processes such as cell growth, differentiation, apoptosis, glucose metabolism, fat metabolism, insulin secretion, brain formation, cardiac development, and stem cell differentiation. The occurrence of diseases, including tumors, is closely related.
  • colorectal cancer is in the third place of malignant tumors. With the change of people's diet and structure and the aging of the population, its incidence and mortality are on the rise.
  • the occurrence of colorectal cancer is related to the abnormal expression of multiple genes. Regulating the expression of certain key genes in the process of tumorigenesis to block malignant transformation is one of the effective methods to control tumors.
  • miRNA-25 is upregulated in a variety of human malignant solid tumors such as gastric cancer, esophageal squamous cell carcinoma, liver cancer and pancreatic cancer. It also plays an important role in the pathogenesis of colorectal cancer, but the related mechanism has not been thoroughly explored in the existing research, and there are few reports on related research.
  • the present invention studies the role of miR-25 in tumors by constructing a miR-25 overexpression recombinant vector and overexpressing it in HCT116 cells.
  • the experimental results showed that the recombinant vector was successfully and efficiently expressed at the cell level, and the expression level of miR-25 was significantly increased.
  • An object of the present invention is to provide a miR-25 overexpression recombinant expression vector, which contains the sequence shown in SEQ ID No.1.
  • the vector is pcDNA3.1.
  • Another object of the present invention is to provide a method for constructing the miR-25 overexpression recombinant expression vector, which method comprises: a. Designing primers and PCR-amplifying the miR-25 gene; b. The amplified gene and expression vector enzyme Cut, ligate the gene of interest and the expression vector; c. Transform the ligated product into E. coli and culture; d. Extract the recombinant plasmid after identification.
  • upstream primer 5'-GGCCAGTGTTGAGAGGCG-3 '
  • downstream primer 5'-GCCGGCACTGTCAGACCGAG-3'.
  • the miR-25 overexpression recombinant vector provided by the present invention can greatly increase the expression level of miR-25 in tumor cells, and provides a new technical means for studying the role of miR-25 in tumorigenesis and development.
  • Figure 1 shows the miR-25 expression levels of HCT116 cells in the control and experimental groups.
  • the human miR-25 gene sequence (accession number: AC073842.5) was found from Genbank. Primer 5 software was used to select the partial sequences at both ends of each CDS for analysis, determine the upstream and downstream primer sequences, and commission Shanghai Inventec Jieji Company to synthesize primers. .
  • the newly synthesized primer was dissolved in TE buffer and adjusted to a final concentration of 10 ⁇ mol / L.
  • primers were added, and Premix PrimeSTAR HS was used for amplification.
  • the amplification conditions were as follows: 98 ° C 5 min, 1 Cycles: 98 ° C 5 s, 56 ° C 5 s, 72 ° C 10 s, 30 cycles.
  • the PCR product was electrophoresed on a 1% agarose gel, and then the gel was recovered to purify the purified PCR product.
  • HCT116 cells were seeded into six-well plates at a seeding density of 50%.
  • Transfection Dissolve 1 ⁇ g pcDNA-miR25 recombinant plasmid and 3 ⁇ l PEI (1 ⁇ g / ⁇ l) in 100 ⁇ l Opti-MEM medium, and mix by vortexing. The medium in the six-well plate was changed to serum-free medium, 1.4 ml per well, and 600 ⁇ l of the transfection mixture was added, and replaced with DMEM medium preheated with 10% fetal bovine serum at 37 ° C. for 5 h. Incubate at 37 ° C for 5% CO2, 90% humidity for 48 h, and collect cells.
  • Example 2 The total RNA of the cells collected in Example 2 was quantified by the probe method Hairpin-it miRNAs quantification and U6 calibration qRT-PCR kit (GenePharma) for fluorescent quantitative PCR. For specific methods, refer to the kit instructions. The results are shown in Figure 1.
  • HCT116 cells (experimental group) transfected with pcDNA-miR25 recombinant plasmid were detected by fluorescent quantitative PCR, and the miR-25 expression level was 104 times higher than that of normal HCT116 cells (control group), indicating that the pcDNA-miR25 recombinant plasmid Efficient expression of miR-25.
  • the miR-25 overexpression recombinant vector provided by the present invention can greatly increase the expression level of miR-25 in tumor cells, and provides a new technical means for studying the role of miR-25 in tumorigenesis and development.

Abstract

提供了一种miRNA-25过表达重组载体的构建及应用。通过构建miR-25过表达重组载体并在HCT116细胞中过表达，研究miR-25在肿瘤中的作用。实验结果显示，该重组载体在细胞水平成功高效表达，miR-25表达水平大幅提升。

Description

miRNA-25过表达载体的构建及其应用 技术领域
本发明属于基因工程领域，尤其涉及一种miRNA-25过表达载体的构建及其应用。
 背景技术
MicroRNA（miRNA）是一类长度约为22～25个核苷酸的单链短序列小RNA，尽管其不编码蛋白质，但却能以非完全性碱基配对的方式结合于同源mRNA，抑制mRNA的表达或是诱导其降解，从而对mRNA进行负性调控，影响相关蛋白的生成。人类有三分之一的基因受miRNA调控，几乎参与了细胞生长、分化、凋亡、糖代谢、脂肪代谢、胰岛素分泌、大脑形成、心脏发生、干细胞分化等一系列生理生化进程，并与许多疾病（包括肿瘤）的发生密切相关。
 技术问题
结直肠癌的发病率处于恶性肿瘤的第三位，随着人们饮食习惯和结构的改变及人口的老龄化，其发病率和死亡率呈上升趋势。结直肠癌的发生与多基因异常表达有关，调节肿瘤发生过程中某些关键基因的表达以阻断恶性转化，是控制肿瘤的有效手段之一。miRNA-25在多种人类恶性实体瘤，如胃癌、食管鳞状细胞癌、肝癌和胰腺癌中表达上调。其在结直肠癌的发病过程中也起到了重要作用，但现有研究尚未对其相关机制进行深入的探讨，相关研究的报道也较少。
 技术解决方案
本发明通过构建miR-25过表达重组载体并在HCT116细胞中过表达，研究miR-25在肿瘤中的作用。实验结果显示，该重组载体在细胞水平成功高效表达，miR-25表达水平大幅提升。
本发明的目的是提供miR-25过表达重组表达载体，其含有如SEQ ID No.1所示的序列。
所述载体为pcDNA3.1。
本发明的另一个目的是提供构建所述miR-25过表达重组表达载体的方法，该方法包括：a. 设计引物，PCR扩增miR-25基因；b. 将扩增的基因和表达载体酶切，连接目的基因和表达载体；c. 将连接产物转化大肠杆菌，培养；d. 鉴定后提取重组质粒。
所述引物序列：上游引物：5’-GGCCAGTGTTGAGAGGCG-3’；下游引物：5’-GCCGGCACTGTCAGACCGAG-3’。
 有益效果
本发明提供的miR-25过表达重组载体可大幅提升miR-25在肿瘤细胞中的表达水平，为研究miR-25在肿瘤发生发展中的作用提供了新的技术手段。
 附图说明
图1为对照组和实验组HCT116细胞的miR-25表达水平。
 本发明的实施方式
下面结合附图与具体实施例对本发明做进一步的说明。
实施例一 miR-25 过表达载体构建
1、序列获得和引物设计
从Genbank中找到人miR-25基因序列（登录号：AC073842.5），利用Primer 5软件分别选取各个CDS两端的部分序列进行分析，确定上下游引物序列，并委托上海英潍捷基公司合成引物。所述引物序列：上游引物：5’-GGCCAGTGTTGAGAGGCG-3’；下游引物：5’-GCCGGCACTGTCAGACCGAG-3’。
2、目的基因序列的获得
将新合成的引物加入TE buffer溶解，调整引物的最终浓度为10 μmol/L，以HCT116细胞基因组DNA为模板，加入引物，Premix PrimeSTAR HS进行扩增，扩增条件如下：98℃ 5 min，1个循环；98℃ 5 s，56℃ 5 s，72℃ 10 s，30个循环。PCR产物通过1%琼脂糖凝胶电泳，然后切胶回收纯化PCR产物。
Bam HI和Xho I分别双酶切pcDNA3.1质粒和PCR产物，回收后按pcDNA3.1质粒：PCR产物为1:5混合后，T4 DNA连接酶16℃连接过夜。转化大肠杆菌Top 10，氨苄青霉素筛选培养并挑单克隆菌株，测序鉴定，获得pcDNA-miR25质粒。
实施例二 HCT116 细胞转染
用含100 μg/ml氨苄青霉素的LB培养基，37℃大量培养测序正确的大肠杆菌，无内毒素提取pcDNA-miR25重组质粒。
转染前一天将HCT116细胞接种至六孔板，接种密度50%。转染：取1 μg pcDNA-miR25重组质粒和3 μl PEI（1 μg/μl）溶于100 μl Opti-MEM培养基，涡旋混匀。将六孔板内培养基换成无血清培养基，每孔1.4 ml，并加入600 μl转染混合液，5 h换成37℃预热有10% 胎牛血清的DMEM培养基。置37℃ 5% CO2，90% 湿度培养48 h，收集细胞。
实施例三   miR-25 表达水平的鉴定
提取实施例二中收集细胞的总RNA，探针法Hairpin-it miRNAs定量和U6校准qRT-PCR试剂盒(GenePharma)进行荧光定量PCR，具体方法参照试剂盒说明书，结果见图1。
如图1所示，荧光定量PCR检测转染了pcDNA-miR25重组质粒的HCT116细胞（实验组），其miR-25表达水平较正常HCT116细胞（对照组）高104倍，说明pcDNA-miR25重组质粒可高效表达miR-25。
 工业实用性
本发明提供的miR-25过表达重组载体可大幅提升miR-25在肿瘤细胞中的表达水平，为研究miR-25在肿瘤发生发展中的作用提供了新的技术手段。

Claims (3)

  1. 一种miR-25过表达重组表达载体，其含有如SEQ ID No.1所示的序列。
  2. 构建权利要求1所述的miR-25过表达重组表达载体的方法，该方法包括：a. 设计引物，PCR扩增miR-25基因；b. 将扩增的基因和表达载体酶切，连接目的基因和表达载体；c. 将连接产物转化大肠杆菌，培养；d. 鉴定后提取重组质粒。
  3. 根据权利要求2所述的方法，其中所述引物为：
    上游引物：5’-GGCCAGTGTTGAGAGGCG-3’
    下游引物：5’-GCCGGCACTGTCAGACCGAG-3’
PCT/CN2018/088529 2018-05-26 2018-05-26 miRNA-25过表达载体的构建及其应用 WO2019227254A1 (zh)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105902559A (zh) * 2005-08-01 2016-08-31 俄亥俄州立大学研究基金会 用于乳腺癌的诊断、预后和治疗的基于MicroRNA的方法和组合物
JP2016192918A (ja) * 2015-03-31 2016-11-17 公立大学法人大阪市立大学 ｐｒｅ−ｍｉＲＮＡ又はｓｈＲＮＡの活性を抑制するための核酸分子
US20170218364A1 (en) * 2014-10-08 2017-08-03 Teikyo University miR-96-5p INHIBITOR AND A SCREENING METHOD FOR THE INHIBITOR

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105902559A (zh) * 2005-08-01 2016-08-31 俄亥俄州立大学研究基金会 用于乳腺癌的诊断、预后和治疗的基于MicroRNA的方法和组合物
US20170218364A1 (en) * 2014-10-08 2017-08-03 Teikyo University miR-96-5p INHIBITOR AND A SCREENING METHOD FOR THE INHIBITOR
JP2016192918A (ja) * 2015-03-31 2016-11-17 公立大学法人大阪市立大学 ｐｒｅ−ｍｉＲＮＡ又はｓｈＲＮＡの活性を抑制するための核酸分子

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