+

WO2018170752A1 - Arn tud pour le knock-down de miarn-29a, miarn-148a, et miarn-424, et application associée - Google Patents

Arn tud pour le knock-down de miarn-29a, miarn-148a, et miarn-424, et application associée Download PDF

Info

Publication number
WO2018170752A1
WO2018170752A1 PCT/CN2017/077593 CN2017077593W WO2018170752A1 WO 2018170752 A1 WO2018170752 A1 WO 2018170752A1 CN 2017077593 W CN2017077593 W CN 2017077593W WO 2018170752 A1 WO2018170752 A1 WO 2018170752A1
Authority
WO
WIPO (PCT)
Prior art keywords
mirna
mir
tud
tud rna
bamhi
Prior art date
Application number
PCT/CN2017/077593
Other languages
English (en)
Chinese (zh)
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/CN2017/077593 priority Critical patent/WO2018170752A1/fr
Publication of WO2018170752A1 publication Critical patent/WO2018170752A1/fr

Links

Definitions

  • the present invention relates to a Tud RN ⁇ application for knockdown of miRNA-29a, miRNA-148a and miRNA-424, 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. miRNAs are widely distributed in plants, animals, and multicellular organisms, and are important. In the study of human miRNAs, it was found that the expression of miRNAs in normal tissues was significantly different. Some miRNAs were lowly expressed in tumor tissues and highly expressed in tumor tissues, indicating that miRNAs are involved in tumorigenesis. Has played a crucial role in the '
  • the target gene which is involved in the regulation of target gene regulation 4 , affects the biological effects and development of tumor cells, plays a role similar to oncogenes, suppresses cancer, or promotes and inhibits tumor invasion and metastasis.
  • miR-424 is a multifunctional miRr associated with cervical cancer, pancreatic cancer and other cell invasion and metastasis; with inflammatory factors such as IL-6, TNF-oc; since the miR-424 promoter region has a CpG island, it Methylation-induced gene silencing also controls the expression of miR-29a, miR-148a, and miR-424, and synergizes with other drugs.
  • miR miRNA
  • Tough Decoy RNA is a novel miRNA-inhibiting method for the adsorption of target miRNAs into double-stranded RNA to inhibit miRNAs. Since the bow I is double-stranded and has a secondary structure of a stem loop, it is resistant to intracellular nuclease degradation and stably and efficiently inhibits miRNA.
  • the present invention provides a Tud RN nucleotide sequence which knocks down miRNA-29a, miRNA-148a and miRNA-424 as shown in SEQ ID NO: 1, mainly in inhibiting human miR-29a, miR-148a and n Application in the expression product.
  • 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 the BamHI and Hindlll linearized vector to obtain a Tud RNA vector.
  • the interference fragment obtained in the step (1) was ligated to the linearized nesil 1.0 plasmid vector via BamHI and Hindlll to obtain the Tud RNA vector p-Genesil-Tud-29a-148a-424.
  • the preparation method of the RNA vector is used to inhibit the expression of human miR-29a, 11 11 -148 & and 1 ⁇ 1 -424 expression preparations.
  • the effective interference fragment constructed and screened is Tud-29a-148a-424.
  • the homology designed by the present invention interferes with the miR-29a, miR-148a and miR-424 TuD RNA sequence structures, which are not easily degraded, and the double-stranded Tud RNA is more efficient than the currently used single-stranded miRNA spons. And the same target for three targets, can better achieve the efficiency of the dry high-miRNA function of the three miRNAs.
  • FIG. 1 miRNA expression levels of cells in each group, wherein a. miR-29a expression, b. miR-148a expression, c. miR-424 expression.
  • the pGenesill.O vector was purchased from the Biovector Plasmid Vector Culture Cell Collection; Reverse Transcription -
  • SYBR fluorescence real-time quantitative PCR kits were purchased from Takara Reagent Co., Ltd.; endotoxin plasmid ⁇ was purchased from Tiangen Biochemical.
  • TuD RNA oligonuclear ⁇ ijTud targeting miR-29a, miR-148a and miR-424 was designed based on the tuD RNA design sequence and miR-29a, miR-148a and miR-4 column information provided in miRBase.
  • -29a-148a-424, whose sequence ⁇ ij is shown in SEQ ID NO 1, was commissioned by Shanghai Biotech to be synthesized by gene.
  • the Tud-29a-148a-424 sequence and p-Genesill.0 were separately treated with BamHI and Hindlll enzymes, and the fractions were recovered, ligated with T4 DNA ligase at 4 ° C overnight, and then transformed into competent E. coli ToplO colonies and cultured. Sended to Shanghai Shenggong for sequencing. The correctly sequenced p-Genesil- ⁇ 8a-424 vector was constructed. Extraction of p-Genesil-Tud-29a-148a-424 using the endotoxin plasmid extraction kit :
  • HeLa cells were seeded in 6-well plates at 1000000 cells per well. After 18 h, the cell density was approximately transduced into Hela cells by jetPrime transfection reagent, p-Genesil-Tud-29a-148a-424 vector, ⁇ After that, the medium was changed to fresh DMEM complete medium, and then cultured for 24 h.
  • the homology designed by the present invention interferes with the miR-29a, miR-148a and miR-424 TuD RNA sequences, and is not easily degraded.
  • the double-stranded Tud RNA is more efficient than the commonly used single-stranded miRNA spons, and the same ⁇
  • the efficiency of dry miRNA high miRNA function studies of three miRNAs can be well achieved.

Landscapes

  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un ARN Tud pour le knock-down de miARN-29a, miARN-148a et miARN-424. Une séquence nucléotidique de l'ARN Tud est représentée par SEQ ID NO : 1. L'invention concerne également un vecteur d'interférence ARN Tud pour supprimer les expressions de miARN-29a, miARN-148a et miARN-424, et son procédé de préparation. Le procédé de préparation comprend les étapes suivantes : synthèse, selon les séquences de miR-29a, miR-148a et miR-424 humaines comparées et analysées, d'un fragment ayant des sites de coupure par enzymes de restriction BamHI et HindIII, liaison du fragment sur un vecteur plasmidique soumis à une linéarisation de BamHI et HindIII, et filtration pour obtenir le vecteur d'interférence.
PCT/CN2017/077593 2017-03-21 2017-03-21 Arn tud pour le knock-down de miarn-29a, miarn-148a, et miarn-424, et application associée WO2018170752A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/077593 WO2018170752A1 (fr) 2017-03-21 2017-03-21 Arn tud pour le knock-down de miarn-29a, miarn-148a, et miarn-424, et application associée

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/077593 WO2018170752A1 (fr) 2017-03-21 2017-03-21 Arn tud pour le knock-down de miarn-29a, miarn-148a, et miarn-424, et application associée

Publications (1)

Publication Number Publication Date
WO2018170752A1 true WO2018170752A1 (fr) 2018-09-27

Family

ID=63584870

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/077593 WO2018170752A1 (fr) 2017-03-21 2017-03-21 Arn tud pour le knock-down de miarn-29a, miarn-148a, et miarn-424, et application associée

Country Status (1)

Country Link
WO (1) WO2018170752A1 (fr)

Citations (4)

* 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
CN103589730A (zh) * 2013-11-13 2014-02-19 东北农业大学 一种抑制IRS1基因表达的shRNA及应用
CN103623425A (zh) * 2012-08-27 2014-03-12 苏州圣诺生物医药技术有限公司 应用双靶标拮抗寡核酸抑制新血管增生疾病的药物
WO2016091747A1 (fr) * 2014-12-09 2016-06-16 Pierfrancesco Tassone Inhibiteurs du groupe mir-17-92 destinés à une activité dans le myélome multiple et autres tumeurs malignes

Patent Citations (4)

* 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
CN103623425A (zh) * 2012-08-27 2014-03-12 苏州圣诺生物医药技术有限公司 应用双靶标拮抗寡核酸抑制新血管增生疾病的药物
CN103589730A (zh) * 2013-11-13 2014-02-19 东北农业大学 一种抑制IRS1基因表达的shRNA及应用
WO2016091747A1 (fr) * 2014-12-09 2016-06-16 Pierfrancesco Tassone Inhibiteurs du groupe mir-17-92 destinés à une activité dans le myélome multiple et autres tumeurs malignes

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
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 e43, XP002656483, ISSN: 0305-1048 *
XIE, XING ET AL.: "Construction of a Human Bronchial Epithelial hsa-miR-148a-3p Knockdown Cell Line", CARCINOGENESIS, TERATOGENESIS & MUTAGENESIS, 14 May 2014 (2014-05-14), pages 204 - 208, ISSN: 1004-616X *
ZHAO, MING: "MicroRNA-137 and MicroRNA-29a Expression and the Clinical Significance in Non-Small Cell Lung Cancer", MEDICINE & PUBLIC HEALTH, CHINA MASTER'S THESES FULL-TEXT DATABASE, 15 December 2014 (2014-12-15), ISSN: 1674-0246 *

Similar Documents

Publication Publication Date Title
Ji et al. The miR-17-92 microRNA cluster is regulated by multiple mechanisms in B-cell malignancies
EP3222723B1 (fr) Développement de médicaments et vaccins universels contre le cancer
WO2021263124A2 (fr) Éléments génétiques commandant la traduction d'arn circulaire et procédés d'utilisation
JP7432521B2 (ja) 新規小分子活性化ｒｎａ
Sun et al. Construction of lentivirus-based inhibitor of hsa-microRNA-338-3p with specific secondary structure
WO2018170752A1 (fr) Arn tud pour le knock-down de miarn-29a, miarn-148a, et miarn-424, et application associée
KR101286154B1 (ko) 안티센스 올리고뉴클레오타이드를 포함하는 줄기세포로부터 연골세포 분화 촉진용 및 항암 약제학적 조성물
WO2018165929A1 (fr) Vecteur d'expression inhibant le micro-arn double brin, son procédé de mise au point et son application
CN111411155B (zh) lncRNA IGFL2-AS1作为结肠癌诊断标志物的应用
CN104293782B (zh) 调控FABP6基因的两个microRNA及其应用
US9540644B2 (en) Small interference RNA for inhibiting intracellular expression of ribosomal protein S3
Priyadarshini et al. Downregulation of ribosomal RNA (rRNA) genes in human head and neck squamous cell carcinoma (HNSCC) cells correlates with rDNA promoter hypermethylation
CN108192895B (zh) 靶向NOB1基因的siRNA分子及其应用
WO2018170750A1 (fr) Arn tud pour supprimer les expressions de miarn-29a, de miarn-148a, et de miarn-152, et application de celui-ci
CN101434629B (zh) 抑制人Akt2基因表达的siRNA序列及其融合表达载体
WO2018170751A1 (fr) Arn tud destiné à antagoniser l'expression d'un micro-arn, et son application
WO2018170753A1 (fr) Arn tud pour l'inactivation de miarn-29a, de miarn-152, et de miarn-185, et application de celui-ci
CN102229928A (zh) 人rbbp6基因的小干扰rna及其应用
CN1927877A (zh) 抑制人Ezrin基因表达的siRNA及其表达载体
KR20120116766A (ko) Ａｄｅｎｉｎｅ ｎｕｃｌｅｏｔｉｄｅ ｔｒａｎｓｌｏｃａｔｏｒ ２ 발현 또는 활성 억제제를 이용한 간암 치료 방법
WO2017219166A1 (fr) Vecteur lentiviral pour l'inhibition simultanée de l'expression de deux miarn, et application associée
Taylor Regulation of Diabetic Cardiomyopathy Through Mitochondrial Import of Long Non-Coding RNAs
WO2018170623A1 (fr) Méthode de réduction des expressions de miarn-152 et de miarn-424
WO2017214953A1 (fr) Construction et application d'un vecteur lentiviral pour l'inhibition spécifique de l'expression de l'arnmi-424 humain
CN119082108A (zh) 一种基于仿效微小rna杂合体的小核酸药物制备方法

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: 17902300

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: 17902300

Country of ref document: EP

Kind code of ref document: A1

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载