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WO2018170750A1 - Arn tud pour supprimer les expressions de miarn-29a, de miarn-148a, et de miarn-152, et application de celui-ci - Google Patents

Arn tud pour supprimer les expressions de miarn-29a, de miarn-148a, et de miarn-152, et application de celui-ci Download PDF

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Publication number
WO2018170750A1
WO2018170750A1 PCT/CN2017/077591 CN2017077591W WO2018170750A1 WO 2018170750 A1 WO2018170750 A1 WO 2018170750A1 CN 2017077591 W CN2017077591 W CN 2017077591W WO 2018170750 A1 WO2018170750 A1 WO 2018170750A1
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WIPO (PCT)
Prior art keywords
mirna
mir
tud
rna
tud rna
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Ceased
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PCT/CN2017/077591
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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/CN2017/077591 priority Critical patent/WO2018170750A1/fr
Publication of WO2018170750A1 publication Critical patent/WO2018170750A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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  • the present invention relates to a Tud RNA for inhibiting expression of small RNA-29a, 148a and 152 and its use, and belongs to the field of genetic engineering technology.
  • 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-29a is a small RNA closely related to cell proliferation. It is involved in many diseases and can act as a tumor suppressor gene in a variety of tumors. It has the ability to grow and invade cells such as human gastric cancer and bladder cancer. Correlation, its expression level is an important reference for evaluating the benign and malignant glioma, and it is also associated with diseases such as atherosclerosis liver fibrosis, and has important potential application value for the treatment of various tumors; miR-148a is in recent years A more studied micr 0 RNA.
  • miR-148a is closely related to exogenous substance metabolism, apoptosis, occurrence, development and epigenetics of various cancers; miR-152 is a multifunctional miRNA, and the study found that miR-152 Related to 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 is A tumor suppressing microRNA associated with many diseases such as pre-eclampsia, trophoblastic tumor, bladder cancer, gastrointestinal cancer, and ovarian cancer.
  • miR-29a, miR-148a and miR-152 peers can work synergistically with other drugs to provide new epigenetic ideas for the treatment of cancer.
  • MiRNA functional studies often require miRNA silencing techniques, including anti-miR, antago miR, miRNA Sponge et al., these technologies are all transient transfection techniques, unable to maintain long-term stable silencing of the target miRNA, and the silencing effect is far from optimal.
  • 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 present invention provides a Tud RNA which inhibits the expression of small RNA-29a, 148a and 152, and has a nucleotide sequence ⁇ IJ as shown in SEQ ID NO: 1, mainly inhibiting human miR-29a, miR-148a. And application in miR-152 expression preparations.
  • the present invention also provides a method for preparing a shRNA interference carrier, and the steps are as follows:
  • the interference fragment obtained in the step (1) was ligated to a plasmid vector linearized by BamHI and Hindlll to obtain a Tud RNA vector.
  • the interference fragment obtained in the step (1) was ligated to the p-Ge nesil 1.0 plasmid vector linearized by BamHI and Hindlll to obtain the Tud RNA vector p-Genesil-Tud-29a-148a-152.
  • a method of preparing an RNA vector for use in inhibiting expression of human miR-29a, miR-148a and miR-152 expression preparations is provided.
  • Tud RNA sequences were designed by comparative analysis of human miR-29a, miR-148a and miR-152 sequences.
  • the homologous interference miR-29a, miR-148a and miR-152 TuD RNA sequences designed by the present invention have a stem-loop structure and are not easily degraded, and the double-stranded Tud RNA is relatively common with the currently used single-stranded miRNA sponge.
  • the binding efficiency is higher, and the same target for three targets can better achieve the interference of three miRNAs and improve the efficiency of miRNA function research.
  • FIG. 1 miRNA expression levels of each group of cells, wherein, a. miR-29a expression, b.
  • pGenesill.O vector was purchased from Biovector Plasmid Vector Culture Cell Genetic Collection Center; Reverse Transcription Kit
  • SYBR fluorescence real-time quantitative PCR kits were purchased from Takara Reagent Co., Ltd.; endotoxin plasmid extraction kits were purchased from Tiangen Biochemical.
  • TuD RNA oligonucleosides targeting miR-29a, miR-148a and miR-152 were designed based on the sequence information of miR-29a, miR-148a and miR-152 provided in the TuD RNA design sequence and miRBase.
  • the Tud-29a-148a-152 sequence and p-Genesill.0 were separately treated with BamHI and Hindlll enzymes, respectively, and electrophoresed and recovered, and then ligated with T4 DNA ligase at 4 ° C overnight, and then transformed into competent E. coli ToplO, Single colonies were cultured and sent to Shanghai Biotech for sequencing.
  • the correctly sequenced pG eneS il-T U d-29a-14 8a-152 vector was constructed.
  • the p-Genesil-Tud-29a-148a-152 vector was extracted using an endotoxin plasmid extraction kit.
  • 16HBE cells were seeded in 6-well plates at 1000000 cells per well, and the cell density was approximately 60% after 18 h.
  • the p-Genesil-Tud-29a-148a-152 vector was transduced with 16HBE cells using jetPrime transfection reagent. After 4 h of reaction, the medium was changed to fresh DMEM complete medium and culture was continued for 24 h.
  • 16HBE cells and 16HBE cells transduced with p-Genesil-Tud-29a-148a-152 vector were used to extract miRNAs from these cells using the miRcute miRNA extraction and isolation kit, followed by S-Poly(T) hsa-miR- 29a qPCR-assay primer set, S-Poly(T) hsa-miR-148a qPCR-assay primer set and S-Poly(T) hsa-miR-152 qPCR-assay primer set kit (both purchased from Shenzhen Anran Bio) The miRNA is reverse transcribed and tailed to obtain the corresponding cDNA. Take 2 kinds of cells of cDNA 2
  • the homologous interference miR-29a, miR-148a and miR-152 TuD RNA sequences designed by the present invention have a stem-loop structure and are not easily degraded, and the double-stranded Tud RNA is more efficient than the currently used single-stranded miRNA sponge. High, and the same target for three targets, can better achieve the interference of three miRNAs, improve the efficiency of miRNA function research.

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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un ARN leurre résistant (ARN Tud) pour supprimer les expressions de miARN-29a, de miARN-148a et de miARN-152. Une séquence nucléotidique de l'ARN Tud est représentée par la SEQ ID NO. 1. La présente invention concerne également un vecteur d'interférence d'ARN Tud pour supprimer les expressions de miARN-29a, de miARN-148a et de miARN-152. Une méthode de préparation du vecteur d'interférence d'ARN Tud comprend : la synthèse, selon les séquences des miR-29a, miR-148a, et miR-152a humains comparées et analysées, d'un fragment ayant des sites de coupure des enzymes de restriction BamHI et HindIII, connectant le fragment sur un vecteur plasmidique soumis à une linéarisation par BamHI et HindIII, et une filtration pour obtenir un vecteur d'interférence efficace.
PCT/CN2017/077591 2017-03-21 2017-03-21 Arn tud pour supprimer les expressions de miarn-29a, de miarn-148a, et de miarn-152, et application de celui-ci Ceased WO2018170750A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/077591 WO2018170750A1 (fr) 2017-03-21 2017-03-21 Arn tud pour supprimer les expressions de miarn-29a, de miarn-148a, et de miarn-152, et application de celui-ci

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/077591 WO2018170750A1 (fr) 2017-03-21 2017-03-21 Arn tud pour supprimer les expressions de miarn-29a, de miarn-148a, et de miarn-152, et application de celui-ci

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WO2018170750A1 true WO2018170750A1 (fr) 2018-09-27

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Citations (2)

* 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
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

Patent Citations (2)

* 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
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

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LINWENSI ZHU ET AL.: "The Structure and Clinical Roles of MicroRNA in Colorectal Cancer", GASTROENTEROLOGY RESEARCH AND PRACTICE, vol. 2016, 31 December 2016 (2016-12-31), pages 1 - 6, XP055540703, ISSN: 1687-630X *
XIE, XING ET AL.: "Construction of a Human Bronchial Epithelial Hsa-miR-148a-3p Knockdown Cell Line", CARCINOGENESIS, TERATOGENESIS & MUTAGENESIS, vol. 26, no. 3, 14 May 2014 (2014-05-14), pages 204 - 208, 212, XP055447857, ISSN: 1004-616X, DOI: doi:10.3969/j.issn.1004-616x.2014.03.010 *

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