WO2017214951A1 - Construction et application d'un vecteur lentiviral pour l'inhibition de l'expression de l'arnmi-152 humain - Google Patents

Construction et application d'un vecteur lentiviral pour l'inhibition de l'expression de l'arnmi-152 humain Download PDF

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WO2017214951A1
WO2017214951A1 PCT/CN2016/086078 CN2016086078W WO2017214951A1 WO 2017214951 A1 WO2017214951 A1 WO 2017214951A1 CN 2016086078 W CN2016086078 W CN 2016086078W WO 2017214951 A1 WO2017214951 A1 WO 2017214951A1
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sequence
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cells
vector
mir
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PCT/CN2016/086078
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Chinese (zh)
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毛侃琅
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毛侃琅
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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
    • 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 inhibits the expression of human miRNA-152.
  • 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-152 is a multifunctional miRNA. It was found that miR-152 is associated with methylation, such as methyltransferase DNMT1 content and enzyme activity, miR-152 can be endometrial cancer DNA A The basalization becomes a silent gene, and it is associated with the development of various cancers. It is a tumor suppressing microRNA, which is associated with many diseases such as preeclampsia, trophoblastic tumor, bladder cancer, gastrointestinal cancer, ovarian cancer, etc. Controlling the high expression and low expression of miR-152, synergistic with other drugs, can provide new epigenetic ideas for the treatment of cancer.
  • 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, and currently There have been reports of optimization of miR-152 interference to improve its effectiveness.
  • Tough Decoy RNA is a newly developed miRNA suppression method
  • the double-stranded RNA is adsorbed to the target miRNA to achieve the purpose of inhibiting the miRNA. Since the introduced 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-term, stable, and efficient manner.
  • the object of the present invention is to provide a Tumor RNA for constructing miR-152 interference and 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 miR-152 corresponding TuD RNA was designed and synthesized, and the nucleotide sequence thereof is shown in SEQ ID NO.: 1. This sequence was ligated to the lentiviral vector pLKO.l-puro to obtain a lentiviral vector pLKO-Tud-152 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-152 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-152.
  • 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-152, and finally the ligation product was transformed into competent E. coli ToplO and applied to 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-152 required by the present invention.
  • FIG. 1 is a schematic view showing the structure of a pLKO-TuD-152 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-152 of 16HBE cells and TuD-152 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- 152 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-152 provided in miRBase were designed to design a T uD RNA oligonucleotide sequence targeting miR-152, 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-152.
  • the ligation product was finally 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-152 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 dilutions of the recombinant lentivirus solution and the cell culture medium in the ⁇ porous plate were respectively different in the multiwell plate.
  • 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 ⁇ 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 ⁇ /ml.
  • the cell line obtained by screening was named TuD-152 cell line.
  • the miRNA extraction and isolation kit extracts the miRNAs of these cells, and then uses S-Poly(T) hsa-miR-152 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-152 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-152 in TuD-152 cells is 5793 ⁇ 4 lower than that in 16HBE cells, and the difference is statistically significant (p ⁇ 0.01). , indicating that the TuD-152 cell line was successfully constructed.
  • the miR-152 designed by the present invention interferes with 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.

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Abstract

L'invention concerne la construction et l'application d'un vecteur lentiviral pour inhiber spécifiquement l'expression de l'ARNmi-152 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-152 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-152 étant insérée vers l'avant dans le site multiple de clonage. Le vecteur d'expression lentiviral pLKO-Tud-152 présente les avantages d'une efficacité de transfection élevée et d'une faible quantité requise, il est capable d'inhiber, de manière spécifique, en continu, avec efficacité et stabilité l'expression de l'ARNmi-152 humain, et peut servir d'outil puissant pour la préparation de médicaments destinés à traiter les maladies associées à l'expression anormale de l'ARNmi-152.
PCT/CN2016/086078 2016-06-16 2016-06-16 Construction et application d'un vecteur lentiviral pour l'inhibition de l'expression de l'arnmi-152 humain WO2017214951A1 (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

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WO2010130351A1 (fr) * 2009-05-15 2010-11-18 Bayer Schering Pharma Ag Micro-arn comme biomarqueurs et cibles thérapeutiques pour l'insuffisance cardiaque
CN102703507A (zh) * 2012-05-18 2012-10-03 深圳市疾病预防控制中心 特异抑制肝细胞CYP2E1基因表达的shRNA慢病毒表达载体及其构建方法与应用
WO2013174692A1 (fr) * 2012-05-22 2013-11-28 Ruprecht-Karls-Universität Heidelberg Cibles thérapeutiques micro arn dans des maladies pulmonaires chroniques
WO2015164786A1 (fr) * 2014-04-25 2015-10-29 University Of Massachusetts Vecteurs de virus adéno-associés recombinants utiles pour réduire une immunité contre des produits transgéniques

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010130351A1 (fr) * 2009-05-15 2010-11-18 Bayer Schering Pharma Ag Micro-arn comme biomarqueurs et cibles thérapeutiques pour l'insuffisance cardiaque
CN102703507A (zh) * 2012-05-18 2012-10-03 深圳市疾病预防控制中心 特异抑制肝细胞CYP2E1基因表达的shRNA慢病毒表达载体及其构建方法与应用
WO2013174692A1 (fr) * 2012-05-22 2013-11-28 Ruprecht-Karls-Universität Heidelberg Cibles thérapeutiques micro arn dans des maladies pulmonaires chroniques
WO2015164786A1 (fr) * 2014-04-25 2015-10-29 University Of Massachusetts Vecteurs de virus adéno-associés recombinants utiles pour réduire une immunité contre des produits transgéniques

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HARAGUCHI, T. ET AL.: "Vectors expressing efficient RNA decoys achieve the long-term suppression of specific microRNA activity in mammalian cells", NUCLEIC ACIDS RESEARCH, vol. 37, no. 6, 17 February 2009 (2009-02-17), pages 1 - 13, XP002656483 *
XIE, XING ET AL.: "Construction of a human bronchial epithelial hsa-miR-148a-3p knockdown cell line", CARCINOGENESIS,TERATOGENESIS & MUTAGENESIS, vol. 26, no. 3, 31 May 2014 (2014-05-31), pages 204 - 208 and 212, XP055447857, DOI: doi:10.3969/j.issn.1004-616x.2014.03.010 *

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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