WO2019227257A1 - miRNA-210过表达载体的构建及其应用 - Google Patents

miRNA-210过表达载体的构建及其应用 Download PDF

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WO2019227257A1
WO2019227257A1 PCT/CN2018/088532 CN2018088532W WO2019227257A1 WO 2019227257 A1 WO2019227257 A1 WO 2019227257A1 CN 2018088532 W CN2018088532 W CN 2018088532W WO 2019227257 A1 WO2019227257 A1 WO 2019227257A1
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mir
mirna
overexpression
construction
expression vector
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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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    • 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

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  • the invention belongs to the field of genetic engineering, and particularly relates to the construction and application of a miRNA-210 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.
  • miRNA-210 is abundantly expressed in human placental tissue. Accumulated studies have shown that it can directly or indirectly regulate many cell life processes, including cell cycle, cell development, membrane transport, cell differentiation, migration and adhesion, binding, oxyacid Catabolism and processing. Further research found that miRNA-210 is associated with hypoxia-related diseases and regulates the pathological conditions of these diseases.
  • miRNA-210 can be induced by hypoxic or hypoxic conditions, and malignant tumors are closely related to hypoxic and hypoxic conditions, it is likely to participate in the development of malignant tumors.
  • a large number of studies have shown that the expression of malignant tumors in a variety of solid malignancies, including breast cancer, pancreatic cancer, and non-small cell lung cancer, is up-regulated, and the up-regulation in these solid tumors has adversely affected the treatment of malignant tumors .
  • the above research shows that miRNA-210 plays an important role in the occurrence and development of many malignant tumors, but its role in the occurrence and development of gliomas and its related mechanisms are unclear, and there are fewer reports on related studies. .
  • the present invention studies the role of miR-210 in tumors by constructing miR-210 overexpression recombinant vectors and overexpressing them in LN-18 cells.
  • the experimental results showed that the recombinant vector was successfully and efficiently expressed at the cell level, and the expression level of miR-210 was significantly increased.
  • the object of the present invention is to provide a miR-210 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-210 overexpression recombinant expression vector, which method comprises: a. Designing primers and PCR amplifying the miR-210 gene; b. Combining 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'-ACCCGGCAGTGCCTCCAG-3 '
  • downstream primer 5'-TCGCGCTGCCCAGGCACAG-3'.
  • the miR-210 overexpression recombinant vector provided by the present invention can greatly increase the expression level of miR-210 in tumor cells, and provides a new technical means for studying the role of miR-210 in tumorigenesis and development.
  • Figure 1 shows miR-210 expression levels of LN-18 cells in the control and experimental groups.
  • a 100 ⁇ g / ml ampicillin-containing LB medium was used to incubate the correctly sequenced E. coli at 37 ° C.
  • the pcDNA-miR210 recombinant plasmid was extracted without endotoxin.
  • LN-18 cells were seeded into six-well plates at a seeding density of 50%.
  • Transfection Take 1 ⁇ g pcDNA-miR210 recombinant plasmid and 3 ⁇ l PEI (1 ⁇ g / ⁇ l) in 100 ⁇ l Opti-MEM medium, and vortex to mix. 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.
  • LN-18 cells (experimental group) transfected with pcDNA-miR210 recombinant plasmid were detected by fluorescent quantitative PCR.
  • the miR-210 expression level was 237.1 times higher than that of normal LN-18 cells (control group), indicating that pcDNA -miR210 recombinant plasmid can efficiently express miR-210.
  • the miR-210 overexpression recombinant vector provided by the present invention can greatly increase the expression level of miR-210 in tumor cells, and provides a new technical means for studying the role of miR-210 in tumorigenesis and development.

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Abstract

提供一种miRNA-210过表达重组载体的构建及应用。通过构建miR-210过表达重组载体并在LN-18细胞中过表达，研究miR-210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-210表达水平大幅提升。

Description

miRNA-210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-210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
miRNA-210在人胎盘组织中大量表达，积累的研究表明，能够直接或者间接的调节许多细胞生命过程，包括细胞周期、细胞发育、膜运输、细胞分化、迁移和粘附、结合、氧基酸分解代谢和的加工等过程。进一步的研究发现，miRNA-210跟缺氧相关疾病相关，并调节了这些疾病的病理情形。
由于miRNA-210能够被低氧或者缺氧条件诱导，而恶性肿瘤与低氧和缺氧条件密切相关，因此极可能参与恶性肿瘤的发生发展。事实上，大量的研究表明，在多种实体恶性肿瘤包括乳腺癌、胰腺癌、非小细胞肺癌等恶性肿瘤表达上调，并且在这些实体瘤中的上调都对恶性肿瘤的治疗产生了恶劣的影响。以上研究说明miRNA-210在多种恶性肿瘤的发生发展中都扮演重要的作用，但其在神经胶质瘤的发生发展中所起的作用及相关机制尚不清楚，相关研究的报道也较少。
 技术解决方案
本发明通过构建miR-210过表达重组载体并在LN-18细胞中过表达，研究miR-210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-210表达水平大幅提升。
本发明的目的是提供miR-210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-210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-210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’-ACCCGGCAGTGCCTCCAG-3’；下游引物：5’-TCGCGCTGCCCAGGCACAG-3’。
 有益效果
本发明提供的miR-210过表达重组载体可大幅提升miR-210在肿瘤细胞中的表达水平，为研究miR-210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LN-18细胞的miR-210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-210 过表达载体构建
1、序列获得和引物设计
从Genbank中找到人miR-210基因序列（登录号：AC138374.2），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’-ACCCGGCAGTGCCTCCAG-3’；下游引物：5’-TCGCGCTGCCCAGGCACAG-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LN-18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-miR210质粒。
实施例二 LN-18 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-miR210重组质粒。
转染前一天将LN-18细胞接种至六孔板，接种密度50%。转染：取1 μg pcDNA-miR210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-210 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-miR210重组质粒的LN-18细胞（实验组），其miR-210表达水平较正常LN-18细胞（对照组）高237.1倍，说明pcDNA-miR210重组质粒可高效表达miR-210。
 工业实用性
本发明提供的miR-210过表达重组载体可大幅提升miR-210在肿瘤细胞中的表达水平，为研究miR-210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-210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-210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-210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’-ACCCGGCAGTGCCTCCAG-3’
    下游引物：5’-TCGCGCTGCCCAGGCACAG-3’
PCT/CN2018/088532 2018-05-26 2018-05-26 miRNA-210过表达载体的构建及其应用 WO2019227257A1 (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
KR20170127089A (ko) * 2016-05-10 2017-11-21 조선대학교산학협력단 알츠하이머 질병의 진단을 위한 바이오마커
US20170335375A1 (en) * 2015-02-09 2017-11-23 Heimbiotek Inc. Kit and method for detection of microrna

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
US20170335375A1 (en) * 2015-02-09 2017-11-23 Heimbiotek Inc. Kit and method for detection of microrna
KR20170127089A (ko) * 2016-05-10 2017-11-21 조선대학교산학협력단 알츠하이머 질병의 진단을 위한 바이오마커

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