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WO2018170652A1 - Arn tud pour le knockdown multiple de trois miarn et application associée - Google Patents

Arn tud pour le knockdown multiple de trois miarn et application associée Download PDF

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Publication number
WO2018170652A1
WO2018170652A1 PCT/CN2017/077207 CN2017077207W WO2018170652A1 WO 2018170652 A1 WO2018170652 A1 WO 2018170652A1 CN 2017077207 W CN2017077207 W CN 2017077207W WO 2018170652 A1 WO2018170652 A1 WO 2018170652A1
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Prior art keywords
tud
mir
rna
vector
mirnas
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PCT/CN2017/077207
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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/CN2017/077207 priority Critical patent/WO2018170652A1/fr
Publication of WO2018170652A1 publication Critical patent/WO2018170652A1/fr
Anticipated expiration legal-status Critical
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  • the present invention relates to the field of gene editing and epigenetics, and specifically relates to a Tud RNA that knocks down three mi RNAs and its application.
  • 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. Related, its expression level is an important reference for evaluating the benign and malignant glioma, and it is also associated with diseases such as atherosclerotic liver fibrosis, and has important potential application value for the treatment of various tumors; miR-140 and various diseases The occurrence and development are closely related, 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 TGFBR1 and other gene expression.
  • miR-140 is highly expressed in articular cartilage and plays a vital role in the pathogenesis of osteoarthritis. Under various mechanisms, miR-140 plays an oncogene role in some tumors, and plays a tumor suppressor role in other tumors, and is involved in a variety of tumor chemotherapy resistance; miR-185 is a 22 nt miRNA , located in human chromosome 22ql l.21, plays an important role as a tumor suppressor gene in the occurrence and invasion of tumors such as colon cancer, gastric cancer, esophageal cancer, lung cancer, liver cancer, etc.
  • MiRNA functional studies often require miRNA silencing techniques, including anti-miR, antago miR, miRNA
  • 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.
  • Lentiviral vector is currently used to construct a stable cell line. Compared with expression vectors such as retrovirus and adenovirus, it can simultaneously infect dividing cells and non-dividing cells, and has high transfection efficiency. And high stability, can make the experimental target gene long-term stable expression
  • the primary object of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a Tud RNA that multiple knockdown of three mi RNAs.
  • Another object of the present invention is to provide the use of the Tud RNA of the multiple knockdown of three miRNAs.
  • a further object of the present invention is to provide a recombinant vector containing the TudRNA.
  • the object of the present invention is achieved by the following technical scheme: a Tud RNA with multiple knockdown of three miRNAs, the DNA sequence ij ij is shown as SEQ ID NO 1;
  • the RNA is applied to inhibit the expression of miRNA-29a, miR-140 and miR-185, and the Tud RNA is preferably constructed on a lentiviral vector, and the recombinant plasmid consisting of the Tud RNA and the lentiviral vector is applied to the T24 cell.
  • the purpose of inhibiting the expression of miRNA-29a, miR-140 and miR-185 is achieved;
  • the lentiviral vector is preferably a CS-RfA-EG lentiviral vector
  • the Tud RNA is constructed on a CS-RfA-EG lentiviral vector, and comprises the following steps:
  • the TuD RNA sequence designed to inhibit human miR-29a, miR-140 and miR-185 has a stem-loop structure and is 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 can better achieve the interference of the three miRNAs for the three targets, and improve the efficiency of the miRNA function research.
  • FIG. 1 is a schematic diagram showing the structure of a CS-RfA-EG lentiviral vector
  • FIG. 2 is an example of T24 cells transfected with T24 cells transfected with C S-RfA-EG-Tud-29a-140-185 lentivirus.
  • miRNA expression level wherein, a. Expression of miR-29a, b. expression of miR-140, c. expression of miR-185.
  • the temperature was lowered to room temperature at ° C/s, and 2 times of frozen absolute ethanol (added 0.1 times pH 5.6 of 3 mol/L NaAc) was precipitated to obtain a double-stranded DNA fragment;
  • pENTR/U6 vector 20 ng T4 DNA ligase (purchased from NEB Corporation) ⁇
  • ddH20 is supplemented to 20 ⁇ 1;
  • reaction was carried out at 25 ° C for 6 hours, and the reaction was carried out at 37 ° C for 10 minutes to remove excess LR.
  • Clonase II enzyme Then, the ⁇ competent Escherichia coli Stbl3 (purchased from Quanjin Company) was cultured at 30 ° C for 22 to 24 hours in LB solid medium containing 10 (Vg/ml Ampicillin), and the clone that grew was a positive clone. Positive clones were picked and cultured in LB liquid medium containing 10 (Vg/ml Ampicillin, cultured at 30 ° C for 20 hours, and the plasmid was purified and sent for sequencing. The correct sequencing result was obtained by correct sequencing.
  • This recombinant plasmid was defined as: CS-RfA-EG-Tud-29a-140-185, a Tud RNA lentiviral plasmid that inhibits the expression of human mi RNA-29a, miR-140 and miR-185. Extraction with endotoxin-free plasmid The recombinant plasmid was extracted from the box (purchased from Tiangen Biochemical).
  • CS-RfA-EG-Tud-29a-140-185, pCMV-VSV-G-RSV-Rev and pCAG-HIVgp plasmid were introduced into human embryonic kidney 293T cells in a ratio of 5:2:2 with Lipofectamine 2000. The supernatant of the cell culture was collected, and filtered with a 0.45 ⁇ filter to obtain a virus containing the CS-RfA-EG-Tud-29a-140-185 plasmid. (6) virus transfection of T24 cells
  • T24 cells were infected with a virus containing the CS-RfA-EG-Tud-29a-140-185 plasmid.
  • the infection steps are as follows: 50,000 T24 cells and 100,000 TU virus solution were suspended in DMEM medium, and prepared according to the amount of ⁇ mixture/well. Each ⁇ mixture also contained 10% FBS and 8 g/ml.
  • the amine was mixed and placed in a 96-well plate. The reaction was carried out at 37 ° C for 24 h.
  • the culture medium was replaced with fresh medium (DMEM + 10% FBS), and the culture was continued for 24 h and subcultured.
  • the normal T24 cells were uncontrolled, and the miRNAs of normal T24 cells and T24 cells transfected with CS-Rf A-EG-Tud-29a-140-185 lentivirus were extracted with miRcute miRNA extraction and isolation kit, respectively, and reverse transcription and After tailing, the corresponding cDNA was obtained. Take 2 kinds of cells of cDNA 2
  • the expression levels of miR-29a, miR-140 and miR-185 were detected by real-time PCR, and the experiment was repeated 3 times with 3 parallel samples per well, with snord 44 as an internal reference.
  • Fig. 3 it can be seen that the expression level of miR-29a with TuD-29a-140-185 cells is 58 ⁇ 3 ⁇ 4 lower than that of T24 cells, and the expression level of miR-140 is 54% lower than that of T24 cells, miR-185 The expression level is 67% lower than that of T24 cells. The difference was statistically significant (/? ⁇ 0.01), indicating that the TuD-29a-140-185 cell line was successfully constructed.
  • the TuD RNA sequence designed to inhibit human miR-29a, miR-140 and miR-185 of the present invention has a stem-loop structure and is 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 can better achieve the interference of the three miRNAs for the three targets, and improve the efficiency of the miRNA function research.

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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne un ARN Tud pour inhiber les expressions des miARN-29a, miR-140 et miR-185 humains. La séquence d'ADN de l'ARN Tud est représentée par SEQ ID NO : 1. L'invention concerne également une application de l'ARN Tud pour inhiber les expressions des miARN-29a, miR-140 et miR-185 humains. L'application comprend : d'abord la conception de la séquence d'ADN de l'ARN Tud qui inhibe les des miARN-29a, miR-140 et miR-185 humains ; puis le clonage de la séquence d'ADN sur un vecteur pENTR/U6 de façon à obtenir un vecteur recombinant pENTR/U6-Tud-29a-140-185 ; et le transfert de la séquence d'ADN sur le vecteur recombinant pENTR/U6-Tud-29a-140-185 à un vecteur lentiviral CS-RfA-EG au moyen d'une technologie de passerelle de façon à obtenir un plasmide recombinant CS-RfA-EG-Tud-29a-140-185. Le plasmide recombinant peut inhiber les expressions des miARN-29a, miR-140 et miR-185 humains.
PCT/CN2017/077207 2017-03-19 2017-03-19 Arn tud pour le knockdown multiple de trois miarn et application associée Ceased WO2018170652A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101838648A (zh) * 2010-01-20 2010-09-22 暨南大学 抑制小鼠TRAF6基因表达的shRNA及其应用
WO2010138263A2 (fr) * 2009-05-28 2010-12-02 University Of Massachusetts Nouveaux virus adéno-associés (aav) et leurs utilisations
CN102218144A (zh) * 2010-04-13 2011-10-19 江苏命码生物科技有限公司 一种调节生物体内微小核糖核酸含量的方法及其用途
CN103987858A (zh) * 2011-11-22 2014-08-13 英特芒尼公司 诊断和治疗特发性肺纤维化的方法

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
CN101838648A (zh) * 2010-01-20 2010-09-22 暨南大学 抑制小鼠TRAF6基因表达的shRNA及其应用
CN102218144A (zh) * 2010-04-13 2011-10-19 江苏命码生物科技有限公司 一种调节生物体内微小核糖核酸含量的方法及其用途
CN103987858A (zh) * 2011-11-22 2014-08-13 英特芒尼公司 诊断和治疗特发性肺纤维化的方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHU, LINWENSI 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 - 7, XP055538564, ISSN: 1687-630X *

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