WO2019222986A1 - Arn non codant à chaîne longue et son utilisation - Google Patents

Arn non codant à chaîne longue et son utilisation Download PDF

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Publication number
WO2019222986A1
WO2019222986A1 PCT/CN2018/088308 CN2018088308W WO2019222986A1 WO 2019222986 A1 WO2019222986 A1 WO 2019222986A1 CN 2018088308 W CN2018088308 W CN 2018088308W WO 2019222986 A1 WO2019222986 A1 WO 2019222986A1
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WO
WIPO (PCT)
Prior art keywords
long
coding rna
chain non
lnc00705
klf6
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PCT/CN2018/088308
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English (en)
Chinese (zh)
Inventor
钱政江
杨海洋
李燕娇
韦命余
李伟丽
李翔
Original Assignee
中国科学院深圳先进技术研究院
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Priority to PCT/CN2018/088308 priority Critical patent/WO2019222986A1/fr
Publication of WO2019222986A1 publication Critical patent/WO2019222986A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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

Definitions

  • the present application relates to the field of biomedicine, and in particular to a long-chain non-coding RNA and application thereof.
  • LncRNA Long non-coding RNA
  • LncRNAs usually have an mRNA-like structure, which has a polyA tail and a promoter structure after splicing, and has dynamic expression and different splicing methods during differentiation. Most LncRNAs have distinct spatiotemporal expression specificity during tissue differentiation and development, and have characteristic expression patterns in tumors and other diseases. Studies have shown that LncRNA plays an important role in many life activities such as dose compensation effect, epigenetic regulation, cell cycle regulation and cell differentiation regulation, and has become a hotspot in genetics research.
  • LncRNAs A large number of mechanisms have been reported on the role of LncRNAs, including: 1) binding to a specific protein, changing the cellular localization of that protein, 2) forming a nucleic acid protein complex with a protein as a structural component, and 3) a transcript of a gene encoding a protein Form complementary double-strands, generate endogenous siRNA under the action of Dicer enzyme, 4) form complementary double-strands with transcripts encoding protein genes, interfere with the splicing of mRNAs and form different splicing forms, 5) with small-molecule RNA (Such as miRNA, piRNA) specific binding, regulating the activity and function of small molecule RNA.
  • small-molecule RNA Small-molecule RNA (Such as miRNA, piRNA) specific binding, regulating the activity and function of small molecule RNA.
  • KLF6 Kruppel-like factor 6 gene is a tumor suppressor gene. Its inactivation or abnormal expression is involved in the development of many tumors.
  • the KLF6 gene is approximately 7 kb in length and contains four exons, 218, 574, 124, and 525 bp, respectively. Mutations often occur in exons 2 and 3, and the mRNA is 1383 bp long.
  • the KLF6 expression product is a 283 amino acid-containing protein with three consecutive C2H2 zinc finger structures shared by the three KLF6 families at its C-terminus. It can bind to GC-rich or CACCC-rich sequences in various gene promoter regions and interact with DNA. effect.
  • KLF6 gene is related to processes such as growth and development, cell differentiation, growth-related cell signal transduction, cell proliferation, apoptosis, angiogenesis, and injury response. Its deletion, mutation, and inactivation are related to a variety of tumors. Occurrence and development are closely related. Therefore, the KLF6 gene has great potential as a new drug target for the treatment of related diseases. Numerous studies have shown that LncRNA can regulate the expression of small molecule RNAs such as miRNA and play an important regulatory function in the occurrence of various diseases.
  • the long-chain non-coding RNA is LncRNA00705, which is located downstream of the KLF6 gene, and uses LncRNA00705 as miR-181a and KLF6 abnormal
  • LncRNA00705 is located downstream of the KLF6 gene
  • the regulatory factors of the diseases caused by the diseases are widely used in the preparation of drugs for related diseases and have broad application prospects and market value.
  • the present application provides a long-chain non-coding RNA.
  • the gene of the long-chain non-coding RNA is Lnc00705. Its nucleotide sequence is shown in SEQ ID No. 1. The nucleotide sequence of its transcript is as follows. SEQ ID No.2.
  • SEQ ID No.1 is as follows:
  • SEQ ID No. 2 is as follows:
  • the inventor found a LncRNA named Knc6 downstream from the KLF6 gene, and further analysis found that the transcript of Lnc00705 has four miR-181a Binding sites; analysis of the binding effect of Lnc00705 to miR-181a using the luciferase reporter system; designing siRNA sequences for Lnc00705, and using gastric mucosal cell line GES-1 cells to analyze miR-181a and KLF6 expression and cell proliferation after inhibiting Lnc00705 And apoptosis; a long-chain non-coding RNA that specifically targets miR-181a and then regulates KLF6 expression was identified.
  • long-chain non-coding RNA gene is located downstream of the KLF6 gene.
  • transcript of the long-chain non-coding RNA gene has four binding sites that bind to miR-181a.
  • the four binding sites correspond to positions 1-7, 1696-1703, 2344-2351, and 2529-2536 of the Lnc00705 gene, respectively.
  • the identification method of this application is as follows:
  • Lnc00705 According to the UCSC online database (http://genome.ucsc.edu/), an LncRNA named Lnc00705 was found downstream of the KLF6 gene, and further analysis revealed that the transcript of Lnc00705 has four binding sites of miR-181a;
  • the present application provides the use of the long-chain non-coding RNA according to the first aspect as a miR-181a and / or KLF6 gene regulator.
  • the present application provides a recombinant vector comprising the long-chain non-coding RNA according to the first aspect.
  • the present application provides a host cell comprising the long-chain non-coding RNA according to the first aspect
  • the host cell includes any one or a combination of at least two of GES-1, MGC-803, SGC-7901, or BGC823, and is preferably a GES-1 cell.
  • the present application provides an siRNA.
  • the siRNA is designed by Lnc00705, and its nucleotide sequence is shown in SEQ ID No. 3.
  • SEQ ID No. 3 is as follows: CUGACCAGCAUAGAUGUUA.
  • the present application provides a use of the recombinant vector according to the third aspect, the host cell according to the fourth aspect, or the siRNA according to the fifth aspect as a miR-181a regulator.
  • the present application provides a use of the recombinant vector according to the third aspect, the host cell according to the fourth aspect, or the siRNA according to the fifth aspect as a KLF6 gene regulator.
  • the present application provides a recombinant vector according to the third aspect, a host cell according to the fourth aspect, or an siRNA according to the fifth aspect, for preparing a medicament for treating a prostate disease, a nervous system disease, and a malignant tumor. And / or use of reagents.
  • the malignant tumor includes any one or a combination of at least two of gastric cancer, prostate cancer, lung cancer or liver cancer.
  • Lnc00705 is located downstream of the KLF6 gene, and the transcription product has four binding sites of miR-181a; this application found that Lnc00705 can inhibit the expression of miR-181a, and then affect the expression of KLF6; the long-chain non-coding RNA provided by this application It can be used as a new regulator to treat diseases caused by the abnormal expression of miR-181a and KLF6. Therefore, the long-chain non-coding RNA of the present application can be used as a new regulator to prepare for the treatment of inflammatory diseases, Drugs for diseases of the prostate, nervous system and various malignancies.
  • FIG. 1 is an analysis result of an online database of the present application, wherein FIG. 1 (A) is a position of Lnc00705 in a KLF6 gene, and FIG. 1 (B) is a schematic diagram of four binding sites of miR-181a in a transcript of Lnc00705;
  • FIG. 2 is an analysis result chart of a luciferase report of the present application
  • Figure 3 is the expression map of miR-181a and KLF6 after inhibition of Lnc00705 in this application.
  • Figure 3 (A) is the expression map of miR-181a
  • Figure 3 (B) is the expression map of KLF6 transcription level
  • Figure 3 (C) KLF6 protein level expression map
  • FIG. 4 is a diagram of the effect of inhibiting the expression of Lnc00705 on the proliferation and apoptosis of GES-1 cells, where FIG. 4 (A) is a line chart of cell proliferation, and FIG. 4 (B) is a flow chart of apoptosis, FIG. 4 (C) is a histogram of apoptosis.
  • miR-181a has a targeted regulatory effect on KLF6; therefore, analysis of long-chain non-coding RNA transcripts revealed that one of the long-chain non-coding RNAs, the transcription product of Lnc00705, has four binding sites for miR-181a Point, see Figure 1 (B);
  • the nucleotide sequence of Lnc00705 is shown in SEQ ID No. 1, and the nucleotide sequence of its transcript is shown in SEQ ID No. 2.
  • SEQ ID No.1 is as follows:
  • SEQ ID No. 2 is as follows:
  • FIG. 1 (B) It can be known from FIG. 1 (B) that the four binding sites correspond to positions 1-7, 1696-1703, 2344-2351, and 2529-2536 of the Lnc00705 gene, respectively.
  • HEK293A cells purchased from ATCC, Manassas, VA
  • DMEN medium containing 10% fetal bovine serum.
  • the cells were transfected by the calcium phosphate method and co-transfected with KLF6 gene 3 ' -UTR dual luciferase reporter vector, Lnc00705 overexpression vector, miR-181a binding site mutated vector, miR-181a analogue and corresponding control.
  • luciferase activity was reduced after transfection of miR-181a, and luciferase activity was restored after co-transfection of miR-181a and Lnc00705;
  • the mutation partially restored luciferase activity, and Lnc00705 was able to inhibit the targeted regulation of KLF6 by miR-181a.
  • the gastric mucosal cell line GES-1 was used for routine culture. When the cell density reached 70%, Lnc00705 siRNA was transfected with Lipofectamine 2000 (Invitrogen). After 6 hours of transfection, the medium was changed and the culture was continued for 48 hours for experiments;
  • the nucleotide sequence of Lnc00705 siRNA is shown in SEQ ID No. 3:
  • the present application provides a long-chain non-coding RNA and its application.
  • the long-chain non-coding RNA is LncRNA00705, which is located downstream of the KLF6 gene.
  • LncRNA00705 is used as a regulator of diseases caused by miR-181a and KLF6 abnormalities. , Used to prepare drugs for related diseases, has broad application prospects and market value.

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  • Health & Medical Sciences (AREA)
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Abstract

L'invention concerne un ARN non codant à chaîne longue et son utilisation, l'ARN non codant à chaîne longue étant le Lnc00705, situé en aval du gène KLF6, et dont le produit de transcription peut cibler spécifiquement le miR-181a, régulant ainsi l'expression du KLF6.
PCT/CN2018/088308 2018-05-25 2018-05-25 Arn non codant à chaîne longue et son utilisation WO2019222986A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104971363A (zh) * 2015-05-22 2015-10-14 浙江大学 靶向抑制hotair的小rna在制备抗前列腺癌药物中的应用
CN104975023A (zh) * 2015-04-02 2015-10-14 中南大学 人宫颈癌转移相关的新长链非编码rna序列、分离方法及其用途
CN105233304A (zh) * 2015-10-30 2016-01-13 中南大学 长链非编码rna基因loc553103在制备鼻咽癌细胞抑制剂上的应用
EP3067422A2 (fr) * 2015-03-13 2016-09-14 Sabanci Üniversitesi Inhibiteurs ct-1

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3067422A2 (fr) * 2015-03-13 2016-09-14 Sabanci Üniversitesi Inhibiteurs ct-1
CN104975023A (zh) * 2015-04-02 2015-10-14 中南大学 人宫颈癌转移相关的新长链非编码rna序列、分离方法及其用途
CN104971363A (zh) * 2015-05-22 2015-10-14 浙江大学 靶向抑制hotair的小rna在制备抗前列腺癌药物中的应用
CN105233304A (zh) * 2015-10-30 2016-01-13 中南大学 长链非编码rna基因loc553103在制备鼻咽癌细胞抑制剂上的应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE GenBank 19 August 2004 (2004-08-19), POUSTKA, A, XP002583562, Database accession no. CR749391 *

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