WO2017214950A1 - Construction et application d'un vecteur lentiviral pour l'inhibition de l'expression de l'arnmi-140 humain - Google Patents

Construction et application d'un vecteur lentiviral pour l'inhibition de l'expression de l'arnmi-140 humain Download PDF

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Publication number
WO2017214950A1
WO2017214950A1 PCT/CN2016/086077 CN2016086077W WO2017214950A1 WO 2017214950 A1 WO2017214950 A1 WO 2017214950A1 CN 2016086077 W CN2016086077 W CN 2016086077W WO 2017214950 A1 WO2017214950 A1 WO 2017214950A1
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sequence
medium
cells
vector
mir
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PCT/CN2016/086077
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English (en)
Chinese (zh)
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毛侃琅
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毛侃琅
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Priority to PCT/CN2016/086077 priority Critical patent/WO2017214950A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors

Definitions

  • the present invention relates to the field of gene editing and epigenetics, and in particular to the construction and application of a lentiviral vector which knocks down human miRNA-140 expression.
  • 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-140 is closely related to the development of various diseases, such as bone and joint diseases, liver diseases, pituitary adenomas, testicular development, head and neck tumors, ovarian and breast diseases. miR-140 can inhibit the proliferation and invasion and metastasis of hepatocellular carcinoma by targeting TG FBR1 and other gene expression. miR-140 is highly expressed in articular cartilage and plays a vital role in the pathogenesis of osteoarthritis. Under the control of multiple mechanisms, m iR-140 plays an oncogene role in some tumors, and plays a tumor suppressor role in other tumors, and is associated with multiple tumor chemotherapy resistance. By controlling the expression of miR-140, peers and other drugs can provide new epigenetic ideas for the treatment of cancer.
  • diseases such as bone and joint diseases, liver diseases, pituitary adenomas, testicular development, head and neck tumors, ovarian and breast diseases.
  • miR-140 can inhibit the proliferation and invasion and metastasis of hepato
  • miRNA silencing is the presentation of synthetic oligonucleotides into cells, with endogenous miRNAs.
  • a heteroduplex allows the miRNA to reduce the inhibition of the target gene to achieve regulation of gene function.
  • Silencing of miRNAs is currently difficult to achieve.
  • Commonly used methods for silencing miRNA are anti-miR, antagomiR, miRNA sponge, etc.
  • anti-miR and antagomiR are transient transfection techniques, and the interference effect cannot be stably maintained, while the miRNA sponge effect is far from optimal. There is no report on optimizing the performance of miR-140 interference.
  • Tough Decoy RNA is a novel miRNA suppression method that The double-stranded RNA is adsorbed to the target miRNA to achieve the purpose of inhibiting the miRNA. Since the inserted RNA is double-stranded and has a secondary structure of a stem loop, it is resistant to intracellular nuclease degradation and can inhibit miRNA for a long time, stably and efficiently.
  • the object of the present invention is to provide a Tumor RNA for constructing miR-140 interference and to construct a lentiviral vector capable of stably maintaining interference effects, and to apply it to the field of gene editing, in view of the deficiencies in the prior art.
  • the gene interference sequence of the corresponding TuD RNA of miR-140 was designed and synthesized, and the nucleotide sequence thereof is shown in SEQ ID ⁇ .: 1. This sequence was ligated to the lentiviral vector pLKO.l-puro to obtain a lentiviral vector pLKO-Tud-140 lentiviral vector capable of stably maintaining the interference effect, the nucleotide sequence of which is shown in SEQ ID NO.: 2
  • the present invention designs and synthesizes miR-140 corresponding TuD
  • RNA interference sequence of RNA is linked to the lentiviral vector pLKO.l-puro, and the resulting vector has the function of interfering with miR-140.
  • the specific integration steps are as follows:
  • RNA oligonucleotide sequence which has the sequence shown in SEQ ID NO.: 1, was commissioned by Shanghai Biotech to synthesize the sequence as a primer.
  • S20 The synthesized sequence is two complementary single-stranded DNAs. Dissolve two single-stranded DNA in ddH20
  • the digested vector was recovered using the MinElute Reaction Cleanup Kit, and the TuD obtained in the previous step was further treated with T4 DNA ligase.
  • RNA sequence was ligated into the vector pLKO.l-puro to form the recombinant vector pLKO-TuD-140, and finally the ligation product was transformed into competent E. coli ToplO and plated onto a plate containing ampicillin LB medium. On, culture at 37 °C for 14 h. Five single colonies were picked from the plates and added to 5 tubes of ampicillin-containing liquid LB medium for 8 hours at 37 °C. The bacteria were sent to Shanghai Biotech for sequencing. The correct sequencing strain was taken and extracted with a small amount of endotoxin-free extraction kit, and the extracted plasmid was the plasmid for interfering with miR-140 required by the present invention.
  • the miR-140 interference designed by the present invention is TuD
  • FIG. 1 is a schematic view showing the structure of a pLKO-TuD-140 lentiviral expression vector according to an embodiment
  • FIG. 2 is a flow diagram showing the steps required to transform the lentiviral expression vector shown in FIG. 1 into the lentiviral vector of the present invention
  • Example 3 is the expression level of miR-140 of 16HBE cells and TuD-140 cells in Example 6.
  • the lentiviral plasmid pLKO. l-puro vector used in the present invention was purchased from Addgene; the human bronchial epithelial cells (16HBE cell strain) used in the present invention were purchased from the United States ATCC; S-Poly(T) hsa-miR- 140 qPCR-assay primer set miRNA reverse transcription and fluorescence quantification kit was purchased from Shenzhen Anran Biotechnology Co., Ltd.
  • RNA design sequence and the sequence information of miR-140 provided in miRBase were designed to design a T uD RNA oligonucleotide sequence targeting miR-140, the sequence of which is shown in SEQ ID NO.: l, commissioned by Shanghai Biotech to synthesize the gene. The way to synthesize.
  • the synthesized sequence is two complementary single-stranded DNA.
  • the two single-stranded DNAs were dissolved in ddH20, mixed at an equimolar ratio, treated at 95 ° C for 5 min, and allowed to cool to room temperature by allowing them to stand at room temperature.
  • the vector pLK0.1-puro was extracted and digested with Age I and Eco RI for 16 h, and the digested vector was recovered with MinElute Reaction Cleanup Kit, and then T4 DNA was used.
  • RNA sequence was ligated into the vector pLKO.l-puro to form the recombinant vector pLKO-Tud-140, and finally the ligation product was transformed into competent E. coli ToplO and plated onto a plate containing ampicillin LB medium, 37 ° C culture 14
  • the bacterial solution was sent to Shanghai Biotech for sequencing, and the sequencing result was completely correct, which was pLKO-Tud-140 lentivirus recombinant vector.
  • the dilution ratio of the solution of the recombinant lentivirus is 1, 10, 100, 1000, 10000, 1
  • the solution of the recombinant lentivirus was serially diluted with a medium, and then 100 gradient-diluted solutions of the recombinant lentivirus and 10 (L perforated plate in a cell culture medium in a multiwell plate) Mixed transfection in different wells, 24h after transfection, aspirate the medium and change 50 (VL containing 5 U DNasel fresh medium, cultured at 37 ° C for 30 min to remove residual plasmid DNA that may adhere to the cell surface, then the medium was changed to 1 mL of normal medium, continue to culture for 48 h;
  • the medium in each well of the multiwell plate was aspirated, 50 (L-trypsin-EDTA solution was added to digest the cells, reacted at 37 ° C for 1 minute, and then the medium was added to terminate the digestion reaction. After the cells are purged, the cells of each well are collected by centrifugation, the total RNA of each well is extracted, and then the total cDNA of each well is reverse-transcribed; and the total cDNA of each of the obtained cells is separately fluorescent.
  • Quantitative PCR was performed to obtain the ct value of each well of the cells, and the experimental group with the smallest difference from the ⁇ value of the control group but exceeding 2 was selected to obtain the dilution factor, and the lentivirus titer was calculated according to the following formula:
  • T 20000 x R, where T is the lentivirus titer, T is in units of TU/mL, and R is the dilution factor.
  • the lentivirus titer of the package is greater than 10000000 TU/mL, indicating that the packaging of the lentivirus is successful.
  • 16HBE cells were seeded in 6-well plates, 1000000 cells per well, and the cell density was about 50% after 12 hours.
  • the virus solution was taken separately, and the virus was diluted 10 times with DMEM complete medium, and then polyglycolamine was added.
  • the medium in the 6-well plate was removed, and the virus-containing DMEM complete medium (containing 10% fetal bovine serum) was added. After 24 hours, the virus-containing DMEM complete medium was discarded, and the fresh DMEM complete medium was replaced. After 24 hours, 0.5 was used.
  • the cells were screened at a g/ml concentration of puromycin. After 10 days of screening, the medium was changed once every 3 days, and the concentration of puromycin was continuously increased to 1.0 g/ml.
  • the cell line obtained by screening was named TuD-140 cell line.
  • the miRNA extraction and isolation kit extracts the miRNAs of these cells, and then uses S-Poly(T) hsa-miR-140 qPCR-assay primer
  • the set kit reverse-transcribes and tails the miRNA to obtain the corresponding cDNA.
  • the cDNA of each of the two cells was used as a template, and the expression level of hsa-miR-140 was detected by real-time PCR.
  • the experiment was repeated three times, and three parallel samples were set in each well, and snord 44 was used as an internal reference.
  • the results are shown in Figure 3. It can be seen that the expression level of miR-140 in TuD-140 cells is 72% lower than that in 16HBE cells, and the difference is statistically significant (p ⁇ 0.01). , indicating that the TuD-140 cell line was successfully constructed.
  • the miR-140 interference designed by the present invention is TuD
  • the RNA sequence has a stem-loop structure and is not easily degraded.
  • the double-stranded Tud RNA is relative to the currently used single-stranded miRNA.

Abstract

L'invention concerne la construction et l'application d'un vecteur lentiviral pour inhiber spécifiquement l'expression de l'ARNmi-140 humain. Le vecteur lentiviral comprend une séquence fondamentale, une séquence de gène de résistance, une séquence de sites multiples de clonage, une séquence de promoteur et une séquence oligonucléotidique ciblant l'ARNmi-140 d'un vecteur d'expression de pLKO.1-puro. Un site multiple de clonage comprend un site de découpe d'enzyme Age I et un site de découpe d'enzyme EcoR I, la séquence oligonucléotidique ciblant l'ARNmi-140 étant insérée vers l'avant dans le site multiple de clonage.
PCT/CN2016/086077 2016-06-16 2016-06-16 Construction et application d'un vecteur lentiviral pour l'inhibition de l'expression de l'arnmi-140 humain WO2017214950A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108517335A (zh) * 2018-04-20 2018-09-11 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所） 一种肝细胞miR-199b低表达的慢病毒表达载体及其构建方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101954077A (zh) * 2010-09-14 2011-01-26 中国人民解放军第二军医大学 一种用于增强肿瘤化疗药物化疗效果的表达质粒佐剂及其制备方法
CN102264898A (zh) * 2008-10-23 2011-11-30 国立大学法人东京大学 微小rna的功能抑制方法
WO2012149646A1 (fr) * 2011-05-05 2012-11-08 Sunnybrook Research Institute Inhibiteurs d'arnmi et leurs utilisations
CN105132424A (zh) * 2015-08-17 2015-12-09 深圳大学 microRNA抑制剂、microRNA抑制剂表达载体及其构建方法和应用
WO2016040347A2 (fr) * 2014-09-08 2016-03-17 University Of Iowa Research Foundation Composés inhibiteurs de micro-arn et procédés d'utilisation de ceux-ci

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102264898A (zh) * 2008-10-23 2011-11-30 国立大学法人东京大学 微小rna的功能抑制方法
CN101954077A (zh) * 2010-09-14 2011-01-26 中国人民解放军第二军医大学 一种用于增强肿瘤化疗药物化疗效果的表达质粒佐剂及其制备方法
WO2012149646A1 (fr) * 2011-05-05 2012-11-08 Sunnybrook Research Institute Inhibiteurs d'arnmi et leurs utilisations
WO2016040347A2 (fr) * 2014-09-08 2016-03-17 University Of Iowa Research Foundation Composés inhibiteurs de micro-arn et procédés d'utilisation de ceux-ci
CN105132424A (zh) * 2015-08-17 2015-12-09 深圳大学 microRNA抑制剂、microRNA抑制剂表达载体及其构建方法和应用

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108517335A (zh) * 2018-04-20 2018-09-11 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所） 一种肝细胞miR-199b低表达的慢病毒表达载体及其构建方法
CN108517335B (zh) * 2018-04-20 2019-11-26 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所） 一种肝细胞miR-199b低表达的慢病毒表达载体及其构建方法

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