WO2019227259A1 - 一种基于哺乳动物病毒的介导miRNA过表达方法 - Google Patents

一种基于哺乳动物病毒的介导miRNA过表达方法 Download PDF

Info

Publication number
WO2019227259A1
WO2019227259A1 PCT/CN2018/088534 CN2018088534W WO2019227259A1 WO 2019227259 A1 WO2019227259 A1 WO 2019227259A1 CN 2018088534 W CN2018088534 W CN 2018088534W WO 2019227259 A1 WO2019227259 A1 WO 2019227259A1
Authority
WO
WIPO (PCT)
Prior art keywords
mir
cells
mammalian virus
overexpression
virus
Prior art date
Application number
PCT/CN2018/088534
Other languages
English (en)
French (fr)
Inventor
毛吉炎
Original Assignee
深圳市博奥康生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳市博奥康生物科技有限公司 filed Critical 深圳市博奥康生物科技有限公司
Priority to PCT/CN2018/088534 priority Critical patent/WO2019227259A1/zh
Publication of WO2019227259A1 publication Critical patent/WO2019227259A1/zh

Links

Classifications

    • 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
    • C12N15/67—General methods for enhancing the expression

Definitions

  • the invention relates to a mammalian virus-based method for mediating miRNA overexpression, and the method can be used to study the role played by miRNA in tumorigenesis and development.
  • Lung cancer is a serious threat to human health and life.
  • lung cancer has significantly increased its morbidity and mortality, ranking first among all tumors.
  • the clinical treatment of lung cancer is mainly multidisciplinary comprehensive treatment such as surgery, chemoradiotherapy, biology, and traditional Chinese medicine treatment, but the 5-year survival rate of patients has not improved significantly.
  • Lung cancer is still the first place in the world to cause cancer death.
  • the cause of lung cancer is not completely clear so far, and a lot of data show that it is closely related to many factors such as smoking, asbestos, arsenic, ionizing radiation, family genetics, decreased immune function, and endocrine dysfunction.
  • MicroRNA is a class of endogenous non-coding genes widely found in animal and plant cells, with a size of about 21-25 nt. It is highly conserved in the evolution of different species and plays an important role in regulating post-transcriptional gene expression. After miRNA is transcribed by polymerase, it forms a primary nucleotide product and is cut by the endonuclease Drosha to form a hairpin precursor. After being transported into the cytoplasm and then cleaved by enzymes, mature miRNA is finally formed. The mature miRNA, in the form of a RISC-miRNA complex, regulates the expression of the target gene by binding to the corresponding region of the 3 'untranslated region of the target gene.
  • miR-143 is localized at chromosome 5q32, and its expression is reduced in a variety of tumors, such as osteosarcoma, breast cancer, colon cancer, rectal cancer, and prostate cancer.
  • miR-143 is involved in tumorigenesis by regulating classic oncogenes or tumor suppressor genes. For example, in colon cancer, the expression level of miR-143 is very low. After its expression is restored, it can significantly inhibit the proliferation of colon cancer cells. Further molecular mechanism studies have found that mi R-143 inhibits the translation of tyrosine kinase ERK5 and reduces ERK5 Expression, thereby attenuating the proliferative signal of MAPK-ERK.
  • miR-143 In rectal cancer, exogenously overexpressed miR-143 can bind to 3'UTR of K-ras and inhibit its translation, thereby inhibiting cell proliferation. In T lymphoma, miR-143 overexpression can also inhibit cell proliferation and promote apoptosis induced by chemotherapy drugs. In addition, miR-143 also plays a role in suppressing cancer by regulating a series of molecules closely related to cell proliferation, differentiation, and apoptosis, such as type III fibronectin FNDC3B, DNA methyltransferase DNMT3A and inflammatory factor NF-kB. At present, there are few studies on miR-143 in lung cancer. There are reports in the literature that miR-143 expression is reduced in non-small cell lung cancer, but the specific mechanism and function are still unclear. 143 Functional studies of lentivirus-mediated overexpression methods.
  • the object of the present invention is to provide a method for mediating miR-143 overexpression based on mammalian viruses.
  • the present invention adopts the following technical steps:
  • SK-MES-1 cells were infected with lentivirus, and cells overexpressing miR-143 were selected by puromycin.
  • the mammalian virus-based miR-143 overexpression method provided by the present invention can greatly increase the expression level of miR-143 in tumor cells, and provides a new technical means for studying the role of miR-143 in tumorigenesis and development.
  • Figure 1 shows miR-143 expression levels of SK-MES-1 cells in the control and experimental groups.
  • Embodiment one miR-143 Construction of an overexpression lentiviral vector
  • Age I and EcoR I enzymes were used to double digest the plasmid containing the synthetic sequence and the pLKO.1-puro vector, respectively, and then recovered and purified.
  • the recovered miR-143 sequence was mixed 1: 6 with pLKO.1-puro vector, and then ligated with NEB T4 DNA ligase.
  • the ligated product was transformed into competent E. coli DH5 ⁇ , and then expanded and sequenced to screen out bacteria that completely matched the expected results. Then expand the culture, and use the endotoxin-free plasmid extraction kit to extract the recombinant plasmid in E. coli and name it pLKO-miR143.
  • Example 2 Packaging of lentivirus
  • 293T cells were cultured, and well-growth cells were inoculated into six wells. Each well had 1,000,000 cells.
  • Recombinant plasmids pLKO-miR143 and pCMV-dR8.91 and pCMV-VSV-G were cotransformed with 1 ⁇ g each of the auxiliary plasmids using Lipofectamine 2000 After staining to 293T cells, the virus-containing supernatant medium was collected 48 hours later, and the virus solution was filtered through a 0.45 ⁇ m sieve to infect SK-MES-1 cells.
  • Example 3 Lentivirus infection SK-MES-1 cell
  • SK-MES-1 cells were seeded in a six-well plate with 1,000,000 cells per well, and the cell density was about 50% after 12 hours.
  • the virus solution obtained in Example 2 was taken, and the virus was diluted 10-fold with DMEM complete medium, and then added Polybrene to a final concentration of 8 ⁇ g / mL.
  • Remove the medium in the six-well plate add virus-containing DMEM complete medium (containing 10% fetal calf serum), discard the virus-containing DMEM complete medium after 24 hours, and replace with fresh DMEM complete medium (containing 1 ⁇ g / mL puromycin) for cell selection.
  • the screening time was 7 days, and the solution was changed (containing 1 ⁇ g / mL puromycin) every other day to eliminate the effect of dead cells on surviving cells and maintain the screening pressure. After screening, a large number of surviving cells were cultured.
  • the mammalian virus-based miR-143 overexpression method provided by the present invention can greatly increase the expression level of miR-143 in tumor cells, and provides a new technical means for studying the role of miR-143 in tumorigenesis and development.

Landscapes

  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

提供了一种基于哺乳动物病毒的介导miRNA过表达方法，该方法通过用携带miR-143前体片段的哺乳动物病毒感染肿瘤细胞，实现肿瘤细胞miR-143的过表达。

Description

一种基于哺乳动物病毒的介导miRNA过表达方法 技术领域
本发明涉及一种基于哺乳动物病毒的介导miRNA过表达方法，利用该方法能够研究miRNA在肿瘤发生发展中所起的作用。
 背景技术
肺癌严重威胁人类的健康和生命，近年来其发病率和病死率都明显升高，上升幅度居各肿瘤之首。肺癌的临床治疗以手术、放化疗、生物及中医治疗等多学科综合治疗为主，但患者的5年生存率并没有明显的改善，肺癌依旧位于目前全世界癌症死因的第一名。肺癌的病因至今尚不完全明确，大量资料表明与吸烟、石绵、砷、电离辐射以及家族遗传、免疫机能降低、内分泌功能失调等多种因素密切相关。随着分子生物学研究的深入，发现肺癌的发生是由于细胞出现了异常的增殖、分化和凋亡所致，而这些异常的生物学行为与体内一系列相关的分子异常表达、活化和突变相关。
MicroRNA（miRNA）是一类广泛存在动植物细胞内的内源性非编码基因，大小约21~25 nt。它在不同物种进化中高度保守，对转录后的基因表达有重要的调控作用；miRNA经聚合酶转录后，形成核苷酸初级产物，并被内切酶Drosha切割，形成发夹状前体在转运入细胞质后，再经酶切割后，最终形成成熟miRNA。成熟的miRNA以RISC-miRNA复合物的形式，通过结合靶基因3’非翻译区相应区域，调节目的基因的表达。
 技术问题
miR-143定位于染色体5q32处，在多种肿瘤，如骨肉瘤、乳腺癌，结肠癌、直肠癌、前列腺癌中表达均降低，参与肿瘤的增殖、侵袭、转移和药物抵抗等。miR-143通过调控经典的癌基因或抑癌基因，参与肿瘤的发生。如在结肠癌中，miR-143 的表达水平非常低，恢复其表达后能够明显抑制结肠癌细胞的增殖，进一步的分子机制研究发现mi R-143 抑制酪氨酸激酶ERK5的翻译而减少ERK5的表达，从而减弱MAPK-ERK的促增殖信号。在直肠癌中，外源过表达miR-143能够结合于K-ras的3’UTR上抑制其翻译，从而抑制细胞增殖。在T淋巴瘤中，过表达miR-143也能够抑制细胞增殖，并促进化疗药物诱导的细胞凋亡。此外，miR-143还通过调控Ⅲ型纤连蛋白FNDC3B，DNA甲基转移酶DNMT3A以及炎症因子NF-kB等一系列与细胞增殖、分化、凋亡密切相关的分子来发挥抑癌的功能。目前有关miR-143在肺癌中的研究还很少，有文献报道miR-143在非小细胞肺癌中表达降低，但具体的机制及其功能尚不清楚，现有技术中也缺乏可用于miR-143功能研究的慢病毒介导的过表达方法。
 技术解决方案
本发明的目的在于，提供一种基于哺乳动物病毒的介导miR-143过表达方法。
为了实现上述目的，本发明采取如下的技术步骤：
a. 构建携人源miR-143前体片段的重组慢病毒载体，并将其包装成慢病毒；
b. 慢病毒感染SK-MES-1细胞，并通过嘌呤霉素进行筛选出过表达miR-143的细胞。
 有益效果
本发明提供的基于哺乳动物病毒的介导miR-143过表达方法可大幅提升miR-143在肿瘤细胞中的表达水平，为研究miR-143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SK-MES-1细胞的miR-143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-143 过表达慢病毒载体的构建
根据Genbank中人miR-143的基因序列（登录号：AC131025.1），并且在其5’端加上Age I酶切位点，3’端加上EcoR I酶切位点。委托上海生工按照基因合成的方式合成该序列。
使用Age I和EcoR I酶分别对含有合成序列的质粒和pLKO.1-puro载体进行双酶切，然后进行回收纯化。回收后的miR-143序列与pLKO.1-puro载体按1:6混匀后，用NEB T4 DNA连接酶进行连接。
连接产物转化感受态大肠杆菌DH5α，扩大培养后测序，筛选出测序结果与预期完全相符的菌。再扩大培养，并应用无内毒素质粒提取试剂盒提取大肠杆菌中的重组质粒，命名为pLKO-miR143。
实施例二：慢病毒的包装
培养293T细胞，取生长状态良好的细胞接种到六孔中，每孔1000000个细胞，用Lipofectamine 2000将重组质粒pLKO-miR143和pCMV-dR8.91、pCMV-VSV-G各1 μg辅助质粒共转染至293T细胞，48h后收集含病毒的上清培养基，用0.45 μm的筛子过滤病毒液，用于感染SK-MES-1细胞。
实施例三：慢病毒感染 SK-MES-1 细胞
接种SK-MES-1细胞于六孔板中，每孔1000000个细胞，12h后细胞密度约为50%，取实施例二中获得的病毒液，用DMEM完全培养基10倍稀释病毒，再加入polybrene至终浓度为8 μg/mL。去除六孔板中的培养基，加入含病毒的DMEM完全培养基（含10%胎牛血清），24h后弃去含病毒的DMEM完全培养基，更换新鲜的DMEM完全培养基（含1 μg/mL嘌呤霉素）进行细胞筛选。筛选时间为7d，隔天换液（含1 μg/mL嘌呤霉素）一次，以消除死细胞对存活细胞的影响，并保持筛选压力。筛选结束后，大量培养存活细胞。
实施例四：荧光定量 PCR 检测 miR-143 表达水平
分别接种正常SK-MES-1细胞（对照组）、经筛选后获得的miR-143过表达细胞（实验组）至六孔板。细胞密度达到80%-90%时，用RNeasy Mini Kit提取各组细胞的总RNA，利用PrimeScrip RT reagent Kit将mRNA逆转录为cDNA，-20℃保存。
取各组细胞的cDNA 1 μL为模板，以U6为内参，实时荧光定量PCR检测miR-143的相对表达水平，设置反应条件：95℃ 30s，1循环；60℃ 30s 40循环；95℃ 5s，60℃ 1min，95℃ 15s。结果如图1所示，可以看到，实验组细胞的miR-143表达水平较对照组细胞有60倍以上的升高，说明本发明提供的miRNA过表达方法能特异、持续、高效、稳定地促进miR-143基因高表达。
 工业实用性
本发明提供的基于哺乳动物病毒的介导miR-143过表达方法可大幅提升miR-143在肿瘤细胞中的表达水平，为研究miR-143在肿瘤发生发展中的作用提供了新的技术手段。

Claims (3)

  1. 一种基于哺乳动物病毒的介导miRNA过表达方法，其特征在于，所述哺乳动物病毒携带人源miR-143前体片段。
  2. 根据一种基于哺乳动物病毒的介导miRNA过表达方法，其特征在于，所述哺乳动物病毒为慢病毒。
  3. 根据一种基于哺乳动物病毒的介导miRNA过表达方法，其特征在于，具体按下列步骤进行：
    a. 构建携人源miR-143前体片段的重组慢病毒载体，并将其包装成慢病毒；
    b. 慢病毒感染SK-MES-1细胞，并通过嘌呤霉素进行筛选出过表达miR-143的细胞。
PCT/CN2018/088534 2018-05-26 2018-05-26 一种基于哺乳动物病毒的介导miRNA过表达方法 WO2019227259A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/088534 WO2019227259A1 (zh) 2018-05-26 2018-05-26 一种基于哺乳动物病毒的介导miRNA过表达方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/088534 WO2019227259A1 (zh) 2018-05-26 2018-05-26 一种基于哺乳动物病毒的介导miRNA过表达方法

Publications (1)

Publication Number Publication Date
WO2019227259A1 true WO2019227259A1 (zh) 2019-12-05

Family

ID=68697360

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/088534 WO2019227259A1 (zh) 2018-05-26 2018-05-26 一种基于哺乳动物病毒的介导miRNA过表达方法

Country Status (1)

Country Link
WO (1) WO2019227259A1 (zh)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103751801A (zh) * 2013-09-10 2014-04-30 上海大学 非小细胞肺癌中miR-143基因的应用
WO2014159633A1 (en) * 2013-03-14 2014-10-02 Medimmune, Llc Recombinant polypeptide production

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014159633A1 (en) * 2013-03-14 2014-10-02 Medimmune, Llc Recombinant polypeptide production
CN103751801A (zh) * 2013-09-10 2014-04-30 上海大学 非小细胞肺癌中miR-143基因的应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LI, YONG ET AL.: "Effect of miR-143 Overexpression on the Invasion and Migration of Osteosarcoma 143B Cells", GUANGDONG MEDICAL JOURNAL, vol. 38, no. 12, 25 June 2017 (2017-06-25), pages 1809 - 1813, ISSN: 1001-9448 *
SHEN, JIANZHEN ET AL.: "Overexpression of MicroRNA-143 Inhibits Growth and Induces Apoptosis in Human Leukemia Cells", ONCOLOGY REPORTS, vol. 31, no. 5, 31 May 2014 (2014-05-31), pages 2035 - 2042, XP055659388, ISSN: 1021-335X *

Similar Documents

Publication Publication Date Title
Li et al. MicroRNA-19 triggers epithelial–mesenchymal transition of lung cancer cells accompanied by growth inhibition
Xu et al. Down-regulation of ribosomal protein S15A mRNA with a short hairpin RNA inhibits human hepatic cancer cell growth in vitro
US10323244B2 (en) LncRNA and oncolytic adenovirus, and application thereof
Wang et al. Differential expression of microRNAs in avian leukosis virus subgroup J-induced tumors
Chang et al. Identification and characterization of microRNAs in the white‐backed planthopper, Sogatella furcifera
CN104946646B (zh) 用于预防和/或治疗埃博拉病毒性出血热的小核酸分子、dna分子、蛋白及应用
Shao et al. milR4 and milR16 mediated fruiting body development in the medicinal fungus Cordyceps militaris
CN109182562A (zh) 与蛋鸭卵泡发育相关的miRNA apla-mir-25-42及其检测引物、抑制物和应用
CN105132424A (zh) microRNA抑制剂、microRNA抑制剂表达载体及其构建方法和应用
CN102766654A (zh) 特异促进肝细胞cyp1a2基因高表达的慢病毒表达载体及其构建方法与应用
CN105861551A (zh) 联合表达microRNAs抑制乳腺癌细胞增殖的载体及其构建方法和应用
CN109402118A (zh) 与蛋鸭卵泡发育相关的miRNA apla-mir-145-4及其检测引物、抑制物和应用
Sui et al. Integrated miRNA and mRNA expression profiles reveal differentially expressed miR-222a as an antiviral factor against duck hepatitis A virus type 1 infection
CN106350519A (zh) 一种调节鹿茸软骨快速生长的microRNA及其应用
WO2019227259A1 (zh) 一种基于哺乳动物病毒的介导miRNA过表达方法
Li et al. miR-218 affects the invasion and metastasis of cervical cancer cells by inhibiting the expression of SFMBT1 and DCUNIDI
WO2019227260A1 (zh) 一种基于哺乳动物病毒的介导miRNA过表达方法
Nakaya et al. Enrichment of stem-like cell population comprises transformation ability of Epstein–Barr virus latent membrane protein 2A for non-transformed cells
CN108588221B (zh) Stil基因的用途及其相关药物
WO2019227258A1 (zh) 一种基于哺乳动物病毒的介导miRNA过表达方法
CN108517335B (zh) 一种肝细胞miR-199b低表达的慢病毒表达载体及其构建方法
CN115820638B (zh) 一种抑制水禽源禽呼肠孤病毒复制的外源性人工miRNA及其应用
WO2019227261A1 (zh) 一种基于哺乳动物病毒的介导miRNA过表达方法
CN117568347B (zh) Ppef1作为神经母细胞瘤药物靶点的应用
CN116832162A (zh) Cd244在作为抗非洲猪瘟的基因编辑靶点中的应用

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: 18920913

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: 18920913

Country of ref document: EP

Kind code of ref document: A1