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WO2019000147A1 - Arn tud inhibant efficacement l'expression des mir-148a, mir-152 et mir-185 humains et son utilisation - Google Patents

Arn tud inhibant efficacement l'expression des mir-148a, mir-152 et mir-185 humains et son utilisation Download PDF

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Publication number
WO2019000147A1
WO2019000147A1 PCT/CN2017/089927 CN2017089927W WO2019000147A1 WO 2019000147 A1 WO2019000147 A1 WO 2019000147A1 CN 2017089927 W CN2017089927 W CN 2017089927W WO 2019000147 A1 WO2019000147 A1 WO 2019000147A1
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WIPO (PCT)
Prior art keywords
mir
tud
vector
pglv3
preparation
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Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/CN2017/089927
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English (en)
Chinese (zh)
Inventor
毛吉炎
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Shenzhen Biocan Technologies Co Ltd
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Shenzhen Biocan Technologies Co Ltd
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Priority to PCT/CN2017/089927 priority Critical patent/WO2019000147A1/fr
Publication of WO2019000147A1 publication Critical patent/WO2019000147A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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 present invention relates to a Tud
  • RNA in particular, relates to a Tud RNA effective for inhibiting the expression of human miR-148a, miR-152 and miR-185 and its use.
  • 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-148a is a microRNA that has been studied more in recent years. It is reported that miR-148a is closely related to exogenous substance metabolism, apoptosis, occurrence, development and epigenetics of various cancers; miR-1 52 is a multifunctional miRNA, and the study found that miR- 152 is associated with methylation, such as methyltransferase DNMT1 content and enzyme activity, miR-152 can be methylated by endometrial cancer DNA into a silent gene, and it is associated with the development of various cancers, it It is a tumor suppressor microRNA, which is associated with many diseases such as preeclampsia, trophoblastic tumor, bladder cancer, gastrointestinal cancer, ovarian cancer, etc.
  • miR-185 is a 22 nt miRNA located at human chromosome 22ql l.21, It plays an important role as a tumor suppressor gene in the development and invasion of tumors such as colon cancer, gastric cancer, esophageal cancer, lung cancer, and liver cancer. In addition, it is a methylation-related tumor suppressor miRNA that can directly target DNMT1. Expression affects the level of methylation in the whole genome, which in turn regulates the methylation status of certain genes and affects gene expression. By controlling the expression of miR-148a, miR-152 and miR-185, the synergy with other drugs can provide new epigenetic ideas for the treatment of cancer.
  • Tough Decoy RNA is a novel miRNA-inhibiting miRNA that inhibits miRNA by introducing double-stranded RNA to target miRNAs. Because the inserted RNA is double-stranded and has a secondary structure of stem-loops, it is resistant to intracellular nuclease degradation and inhibits miRNAs in a long-term, stable, and efficient manner.
  • the primary object of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide an effective suppression of human miR-
  • Tud RNA expressed by 148a, miR-152 and miR-185 Tud RNA expressed by 148a, miR-152 and miR-185.
  • Another object of the present invention is to provide the above-described use of Tud RNA which effectively inhibits the expression of human miR-148a, miR-152 and miR-185.
  • a further object of the present invention is to provide a recombinant vector comprising the above Tud RNA.
  • a Tud RNA which effectively inhibits the expression of human miR-148a, miR-152 and miR-185, and the DNA sequence encoding the Tud RNA which inhibits the expression of human miR-148a, miR-152 and miR-185 is as follows Show:
  • Tud RNA which is effective for inhibiting the expression of human miR-148a, miR-152 and miR-185
  • the application comprising constructing the Tud RNA on a lentiviral vector to obtain the Tud RNA containing the Tud RNA Recombinant vector; will contain the Tud
  • the recombinant vector of RNA acts on 16HBE cells to inhibit the expression of miR-148a, miR-152 and miR-185;
  • the lentiviral vector is a pGLV3/Hl/GFP+Puro (pGLV3) lentiviral shuttle vector
  • the preparation method of the recombinant vector containing the Tud RNA comprises the following steps: (1) Designing a DNA sequence ⁇
  • Tud-148a-152-185 antisense strand [0020] Tud-148a-152-185 antisense strand:
  • the 5' end of the sense strand template is added with GATCC, which is complementary to the sticky end formed by BamHI digestion; the AATTC is added to the 5' end of the antisense strand template, which is complementary to the sticky end formed by EcoRI digestion;
  • the Tud RNA of the present invention which inhibits the expression of human miR-148a, miR-152 and miR-185 is effective for inhibiting the expression of human miR-148a, miR-152 and miR-185.
  • the Tud RNA, which inhibits the expression of human miR-148a, miR-152 and mi R-185 is constructed on a lentiviral vector, and is effective not only for dividing and non-dividing cells, but also for in vivo and in vitro studies.
  • FIG. 1 is a structural diagram of a pGLV3 vector
  • FIG. 2 is a miRNA expression level of each group of cells, wherein, a.
  • miR-148a Expression of miR-148a, b. expression of miR-152, c. expression of miR-185.
  • TuD RNA oligonucleosides targeting miR-148a, miR-152 and miR-185 were designed based on the sequence information of miR-148a, miR-152 and miR-185 provided in the TuD RNA design sequence and miRBase.
  • Tud-148a-152-185 antisense strand [0035] Tud-148a-152-185 antisense strand:
  • the 5' end of the sense strand template is added with GATCC, which is complementary to the sticky end formed by BamHI digestion; the AATTC is added to the 5' end of the antisense strand template, which is complementary to the sticky end formed by EcoRI digestion.
  • the pGLV3 vector (Gimma Gene Co., Ltd.) was digested with BamH I and EcoR I, and the vector was linearized.
  • the conditions of the digestion were as follows: pGLV3 vector (10 ⁇ , BamH I) Dicer (5 L, Fermentas), EcoR I endonuclease (5 L, Fermentas), FastDigest buffer (8 (VL, TOYOBO) were mixed, placed at 37 ° C for 1 hour, using a gel back kit ( Axygen) recovered the linear vector fragment and diluted its concentration to 50 ng ⁇ L.
  • the plasmid was purified and verified by sequencing to prepare a recombinant plasmid pGLV3-Tud-148a-152-185.
  • the recombinant vector pGLV3-Tud-148a-152-185 was co-transfected into packaging cells pGag/Pol, pRev, pVSV-G to 293T cells, and the culture supernatant was collected to obtain virus particles containing the desired Tud RNA. can be use on Transfect the target cells.
  • 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-148a-152-185 cell line.
  • TuD-148a-152-185 cells were inoculated into 6-well plates (about 300,000 per well), and the cells were cultured for about 24 hours to a degree of fusion of 80%.
  • the miRNAs of these cells were extracted using the miRcute miRNA extraction and isolation kit, followed by S-Poly(T) hsa-miR-148a 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 levels of miR-148a, miR-152 and miR-185 were detected by real-time PCR.
  • the experiment was repeated 3 times, and 3 parallel samples were set per well, with snord 44 as the internal reference. .
  • Fig. 2 it was found that the expression level of miR-148a with TuD-148a-152-185 cells was 60% lower than that of 16HBE cells, and the expression level of mi R-152 was 62% lower than that of 16HBE cells, miR-185 The expression level is 59% lower than that of 16HBE cells.
  • the difference was statistically significant (/? ⁇ 0.01), indicating that the TuD-148a-152-185 cell line was successfully constructed.
  • the Tud RNA of the present invention which inhibits the expression of human miR-148a, miR-152 and miR-185 is effective for inhibiting the expression of human miR-148a, miR-152 and miR-185.
  • the Tud RNA, which inhibits the expression of human miR-148a, miR-152 and mi R-185 is constructed on a lentiviral vector, and is effective not only for dividing and non-dividing cells, but also for in vivo and in vitro studies.

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

Abstract

L'invention concerne un ARN TuD et son utilisation. L'ARN TuD peut inhiber efficacement l'expression des miR-148a, miR-152 et miR-185 humains. La séquence d'ADN codant pour l'ARN TuD est telle que représentée ci-dessous : 5'-ggcgctaggatcatcaactccccgaccgaaatctaggagaccagcaaagtcacgtactatctgtcttgaacccaaagtcacgtgatgatcttcttgaaacacaagtattctggtcacagaatacaactccccgaccgaaatctaggagaccagcaaagtcacgtactatctgtcttgaacccaaagtcacgtgatgatcttcttgaaacacaagatgatcctagcgccaccttttt-3'. L'utilisation de l'ARN TuD consiste à construire l'ARN TuD sur un vecteur lentiviral pour obtenir un vecteur recombiné; et à appliquer le vecteur recombiné aux cellules humaines pour inhiber l'expression des miR-148a, miR-152 et miR-185 humains.
PCT/CN2017/089927 2017-06-26 2017-06-26 Arn tud inhibant efficacement l'expression des mir-148a, mir-152 et mir-185 humains et son utilisation Ceased WO2019000147A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/089927 WO2019000147A1 (fr) 2017-06-26 2017-06-26 Arn tud inhibant efficacement l'expression des mir-148a, mir-152 et mir-185 humains et son utilisation

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PCT/CN2017/089927 WO2019000147A1 (fr) 2017-06-26 2017-06-26 Arn tud inhibant efficacement l'expression des mir-148a, mir-152 et mir-185 humains et son utilisation

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010138263A2 (fr) * 2009-05-28 2010-12-02 University Of Massachusetts Nouveaux virus adéno-associés (aav) et leurs utilisations
CN102851291A (zh) * 2006-04-03 2013-01-02 桑塔里斯制药公司 包含抗微小rna 反义寡核苷酸的药物组合物
CN104357451A (zh) * 2014-12-02 2015-02-18 广州市番禺区中心医院 针对dd3基因的小干扰rna及其表达载体构建与应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102851291A (zh) * 2006-04-03 2013-01-02 桑塔里斯制药公司 包含抗微小rna 反义寡核苷酸的药物组合物
WO2010138263A2 (fr) * 2009-05-28 2010-12-02 University Of Massachusetts Nouveaux virus adéno-associés (aav) et leurs utilisations
CN104357451A (zh) * 2014-12-02 2015-02-18 广州市番禺区中心医院 针对dd3基因的小干扰rna及其表达载体构建与应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SU, QUANQIU ET AL.: "The Research Progress of miR-185 in Tumor", JOURNAL OF MODERN MEDICINE & HEALTH, 15 February 2015 (2015-02-15), pages 380 - 383, ISSN: 1009-5519 *

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