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WO2018170753A1 - Arn tud pour l'inactivation de miarn-29a, de miarn-152, et de miarn-185, et application de celui-ci - Google Patents

Arn tud pour l'inactivation de miarn-29a, de miarn-152, et de miarn-185, et application de celui-ci Download PDF

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Publication number
WO2018170753A1
WO2018170753A1 PCT/CN2017/077594 CN2017077594W WO2018170753A1 WO 2018170753 A1 WO2018170753 A1 WO 2018170753A1 CN 2017077594 W CN2017077594 W CN 2017077594W WO 2018170753 A1 WO2018170753 A1 WO 2018170753A1
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WIPO (PCT)
Prior art keywords
mirna
mir
tud
rna
tud rna
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PCT/CN2017/077594
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English (en)
Chinese (zh)
Inventor
毛吉炎
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深圳市博奥康生物科技有限公司
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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/CN2017/077594 priority Critical patent/WO2018170753A1/fr
Publication of WO2018170753A1 publication Critical patent/WO2018170753A1/fr

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  • the present invention relates to a Tud RNA which knocks down miRNA-29a, miRNA-152 and miRNA-185 and an application thereof, 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.
  • miR-152 is a kind of Multi-functional miRNA, the study found that miR-152 is related to methylation, such as the methyltransferase DNMT1 content and enzyme activity, miR-152 can be methylated by endometrial cancer DNA into a silent gene, and its It is involved in the development of various cancers. 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 in human chromosome 22ql l.21, plays an important role as a tumor suppressor gene in the occurrence and invasion of colon cancer, gastric cancer, esophageal cancer, lung cancer, liver cancer and other tumors. Further it is one methylation-related inhibition of miRNA, may affect the level of the whole genome methylation by direct targeted expression of DNMT1, further regulation of the methylation status of certain modification genes, gene expression. By controlling the expression of miR-2 9a, miR-152 and miR-185, the synergy with other drugs can provide new epigenetic ideas for the treatment of cancer.
  • 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 can inhibit miRNAs in a long-term, stable and efficient manner.
  • the present invention provides a knockdown of miRNA-29a, miRNA-152 and miRNA-185 of Tud RNA, the nucleotide sequence of which is shown in SEQ ID NO: 1, mainly in inhibiting human miR-29a, miR-152 And miR-185 expression products are used.
  • 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-152-185.
  • RNA vector preparation in inhibiting human miR-29a, miR-152 and miR-185 expression products
  • Tud RNA sequences were designed by comparative analysis of human miR-29a, miR-152 and miR-185 sequences.
  • human miR-29a, 1 ⁇ 11-152 and 1 ⁇ 11-185 sequences single-stranded Tud RNA fragments with BamHI and Hindlll cleavage sites were synthesized, renatured into double strands, ligated to The p-Genesill.O vector linearized by BamHI and Hindlll. The effective interference fragment constructed and screened is Tud-29a-152-185.
  • the same design of the present invention interferes with miR-29a, !
  • the ⁇ 11-152 and 1 ⁇ 11-185 TuD RNA sequences have a stem-loop structure and are not easily degraded.
  • the double-stranded Tud RNA has higher binding efficiency than the currently used single-stranded miRNA sponge, and its Target, can better achieve the interference of three miRNAs, improve the efficiency of mi RNA 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 designed for miR-29a, miR-152 and miR-185 was designed.
  • RNA oligonucleotide sequence ⁇ lJTud-29a-152-185 whose sequence ⁇ ij is shown in SEQ ID NO 1, was commissioned by Shanghai Biotech to synthesize by gene synthesis.
  • the Tud-29a-152-185 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 p-Genesil-Tud-29a-152-185 vector was constructed.
  • the p-Genesil-Tud-29a-152-185 vector was extracted using an endotoxin plasmid extraction kit.
  • T24 cells were seeded in 6-well plates at 1000000 cells per well, and the cell density was about 60% after 18 h.
  • the p-Genesil-Tud-29a-152-185 vector was transduced into T24 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.
  • the miRNA extraction and isolation kit extracts the miRNAs of these cells, and then uses S-Poly(T) hsa-miR-29a qPCR-assay primer set, S-Poly(T) hsa-miR-152 qPCR-assay primer set and S-Poly (T) hsa-miR-185 qPCR-assay primer set kit (both purchased from Shenzhen Anran Bio) Reverse transcription and tailing of miRNA to obtain the corresponding cDNA. Take 2 kinds of cells of cDNA 2
  • the expression levels of miR-29a, miR-152 and miR-185 were detected by real-time PCR, and the experiment was repeated 3 times, and 3 parallel samples were set per well, and snord 44 was used as an internal reference.
  • the results are shown in Figure 1. It can be seen that the expression level of miR-29a in TuD-29a-152-185 cells is 53 ⁇ 3 ⁇ 4 lower than that in T24 cells, and the expression level of miR-152 is 69% lower than that in T24 cells, miR-185 The expression level is 62% lower than that of T24 cells. The difference is statistical Significance ( ⁇ 0.01), indicating that the TuD-29a-152-185 cell line was successfully constructed.
  • the present invention is designed to interfere with the miR-29a, 1 ⁇ &-152 and 1 ⁇ !-185 TuD RNA sequences with a stem-loop structure, which 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 at the same time, the interference of the three miRNAs can be well achieved for the three targets, and the efficiency of the miRNA function research is improved.

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Abstract

L'invention concerne un ARN Tud pour l'inactivation de miARN-29a, de miARN-152, et de miARN-424. Une séquence nucléotidique codant pour l'ARN Tud est représentée par la SEQ ID NO. 1. L'invention concerne également une méthode de préparation d'un vecteur d'interférence contenant la séquence nucléotidique codant pour l'ARN Tud.
PCT/CN2017/077594 2017-03-21 2017-03-21 Arn tud pour l'inactivation de miarn-29a, de miarn-152, et de miarn-185, et application de celui-ci WO2018170753A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/077594 WO2018170753A1 (fr) 2017-03-21 2017-03-21 Arn tud pour l'inactivation de miarn-29a, de miarn-152, et de miarn-185, et application de celui-ci

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/077594 WO2018170753A1 (fr) 2017-03-21 2017-03-21 Arn tud pour l'inactivation de miarn-29a, de miarn-152, et de miarn-185, et application de celui-ci

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WO2018170753A1 true WO2018170753A1 (fr) 2018-09-27

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Citations (1)

* 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

Patent Citations (1)

* 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

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE Genbank [O] 11 December 2014 (2014-12-11), YU , M.: "Homo sapiens microRNA 185 (MIR185), microRNA", XP055540744, Database accession no. NR_029706 *
DATABASE Genbank [O] 21 May 2015 (2015-05-21), SERAFIN, A.: "Homo sapiens microRNA 29a (MIR29A), microRNA", XP055540730, Database accession no. NR_029503 *
DATABASE Genbank [O] 21 May 2015 (2015-05-21), YAO, Y.: "Homo sapiens microRNA 152 (MIR152), microRNA", XP055540739, Database accession no. NR_029687 *
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 - 6, XP055538564, ISSN: 1687-630X *

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