WO2018170621A1 - Vecteur d'expression réduisant un micro-r-148a et un micro-r-424 et son application - Google Patents
Vecteur d'expression réduisant un micro-r-148a et un micro-r-424 et son application Download PDFInfo
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- WO2018170621A1 WO2018170621A1 PCT/CN2017/077171 CN2017077171W WO2018170621A1 WO 2018170621 A1 WO2018170621 A1 WO 2018170621A1 CN 2017077171 W CN2017077171 W CN 2017077171W WO 2018170621 A1 WO2018170621 A1 WO 2018170621A1
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- WIPO (PCT)
- Prior art keywords
- mir
- sequence
- vector
- tud
- mirna
- Prior art date
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- 108091027034 miR-148a stem-loop Proteins 0.000 title claims abstract description 24
- 108091030938 miR-424 stem-loop Proteins 0.000 title claims abstract description 24
- 239000013598 vector Substances 0.000 title claims abstract description 19
- 230000014509 gene expression Effects 0.000 title claims abstract description 17
- 239000002679 microRNA Substances 0.000 claims abstract description 36
- 239000013604 expression vector Substances 0.000 claims abstract description 11
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 8
- 230000008685 targeting Effects 0.000 claims abstract description 5
- 108091070501 miRNA Proteins 0.000 claims description 35
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 21
- 230000009977 dual effect Effects 0.000 claims description 11
- 239000013612 plasmid Substances 0.000 claims description 11
- 108020004414 DNA Proteins 0.000 claims description 7
- 102000053602 DNA Human genes 0.000 claims description 6
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- 108020004682 Single-Stranded DNA Proteins 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 6
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- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 4
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- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 238000011160 research Methods 0.000 claims description 4
- 238000003786 synthesis reaction Methods 0.000 claims description 4
- 241000894006 Bacteria Species 0.000 claims description 3
- 108010054576 Deoxyribonuclease EcoRI Proteins 0.000 claims description 3
- 241000588724 Escherichia coli Species 0.000 claims description 3
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 claims description 3
- 229960000723 ampicillin Drugs 0.000 claims description 3
- 230000000295 complement effect Effects 0.000 claims description 3
- 238000005516 engineering process Methods 0.000 claims description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 2
- 102000012410 DNA Ligases Human genes 0.000 claims description 2
- 108010061982 DNA Ligases Proteins 0.000 claims description 2
- 238000012163 sequencing technique Methods 0.000 claims description 2
- 230000001225 therapeutic effect Effects 0.000 claims description 2
- 230000035807 sensation Effects 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 6
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- 230000005764 inhibitory process Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 21
- 108090000623 proteins and genes Proteins 0.000 description 6
- 108020004999 messenger RNA Proteins 0.000 description 4
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- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
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- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 108091029523 CpG island Proteins 0.000 description 1
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- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
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- 108091030146 MiRBase Proteins 0.000 description 1
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- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- 102000044209 Tumor Suppressor Genes Human genes 0.000 description 1
- 108700025716 Tumor Suppressor Genes Proteins 0.000 description 1
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- 210000000424 bronchial epithelial cell Anatomy 0.000 description 1
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- 230000001973 epigenetic effect Effects 0.000 description 1
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- 229950010131 puromycin Drugs 0.000 description 1
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- 238000012216 screening Methods 0.000 description 1
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- 210000004881 tumor cell Anatomy 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to the field of molecular biology, and more particularly to a vector for reducing the expression of miR-148a and miR-424 and uses thereof.
- RNA is an important substance in living organisms and plays various functions in life activities. RNA can be classified into messenger RNA (mRNA) and non-coding RNA depending on whether the protein is encoded or not.
- mRNA messenger RNA
- non-coding RNA depending on whether the protein is encoded or not.
- RNA non-coding RNA, ncRNA.
- Small RNA small RNA
- RNA is an important class of ncRNAs. miRNAs are endogenous small RNAs in organisms that are typically 20-24 nt in length. The miRNA is part of the pri-miRNA (primary RNA) and is initially expressed in the nucleus by RNA polymerase ⁇ . The mature miRNA acts as a guiding molecule, binds to the target gene mRNA according to the principle of base pairing, and directs the silencing complex (RISC) to degrade mRNA or hinder its translation, thereby exerting a negative regulation effect on the expression of the target gene.
- RISC silencing complex
- miR-148a is a microRNA that has been studied more in recent years. It has been reported that miR-148a is closely related to exogenous substance metabolism, apoptosis, occurrence, development and epigenetics of various cancers, so it is important to study the function of miR-148a; miR-424 is in recent years A miRNA discovered, which acts on a target gene in a variety of tumors and participates in a signal pathway of target gene regulation, thereby affecting the biological effects and development of tumor cells, exerting effects similar to oncogenes, tumor suppressor genes, or promoting Inhibit the invasion and metastasis of tumors.
- miR-424 is a multifunctional miRNA, which is associated with cell invasion and metastasis of cervical cancer, pancreatic cancer, etc.; it is associated with the expression of inflammatory factors such as IL-6 and TNF-ot; since the miR-424 promoter region has CpG islands It is also associated with methylation-induced gene silencing.
- 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.
- the technical problem to be solved by the present invention is to provide a dual miRNA suppression expression vector which is simple in structure, low in cost and simple in operation.
- a Tud RNA targeting a dual miRNA the nucleotide sequence of which is shown in SEQ ID NO: 1 in the Sequence Listing.
- a dual miRNA-inhibiting expression vector comprising the sequence of the invention SEQ ID NO: 1.
- the double miRNA-inhibiting expression vector can inhibit the expression of miR-148a and miR-424 after transformation of Hela cells.
- a method for constructing a dual miRNA suppression expression vector according to the present invention comprises the following steps:
- the synthesized sequence is two complementary single-stranded DNAs.
- the two single-stranded DNAs were dissolved in ddH20, mixed in an equimolar ratio, treated at 95 ° C for 5 min, and then allowed to cool to room temperature at room temperature.
- the digested vector was recovered using the MinElute Reaction Cleanup Kit, and the TuD obtained in the previous step was further treated with T4 DNA ligase.
- RNA sequence was ligated into the vector pLKO.l-puro to form the recombinant vector pLKO-TuD-148a-424, and finally the ligation product was transformed into competent E. coli Stbl3 and plated onto a plate containing ampicillin LB medium. Incubate at 37 °C for 14 h. Single colonies were picked and sequenced. The correct bacteria were amplified and cultured and extracted with a small amount of endotoxin-free extraction kit. The extracted plasmid is the homologous interference pLKO- required for the invention. Plasmid for TuD-148a-424.
- the homologous interference miR-148a and miR-424 TuD RNA sequences designed by the present invention have a stem-loop structure and are not easily degraded, and the double-stranded Tud RNA has higher binding efficiency than the currently used single-stranded miRNA sponge. And the same target for two targets, can better achieve the interference of two miRNAs, improve the efficiency of miRNA function research.
- Figure 1 shows the miRNA expression levels of 16HBE cells and TuD-148a-424 cells, wherein a.
- the lentiviral plasmid pLKO.l-puro vector used in the present invention was purchased from Addgene; the human bronchial epithelial cells (16HBE cell strain) used in the present invention were purchased from ATCC, USA.
- the TuD RNA oligonucleotide sequence targeting miR-148a and miR-424 was designed, and its sequence is SEQ ID.
- the synthesized sequence is two complementary single-stranded DNAs.
- the two single-stranded DNAs were dissolved in ddH20, mixed at an equimolar ratio, treated at 95 ° C for 5 min, and allowed to cool to room temperature by allowing them to stand at room temperature.
- RNA sequence was ligated into the vector pLKO.l-puro to form the recombinant vector pLKO-Tud-148a-424, and finally the ligation product was transformed into competent E. coli Stbl3 and plated onto a plate containing ampicillin LB medium. Incubate at 37 °C for 14 h. Single colonies were picked and sequenced. The correct sequencing bacteria were expanded and extracted with a non-endotoxin plasmid miniprep kit. The extracted plasmid was the plasmid for the homologous interference pLKO-TuD-148a-424 required by the present invention.
- 16HBE cells were seeded in 6-well plates, 1000000 cells per well, and the cell density was about 60% after 18 hours.
- the plasmid pLKO-TuD-148a-424 was transduced into 16HBE cells with Lipfectamine 2000, and culture was continued for 48 h. Thereafter, the cells were cultured for 3 days in DMEM medium containing 1.0 g/ml puromycin, and the cell line obtained by the screening was named TuD-148a-424 cell line.
- the homologous interference miR-148a and miR-424 TuD RNA sequences designed by the present invention have a stem-loop structure and are not easily degraded, and the double-stranded Tud RNA has higher binding efficiency than the currently used single-stranded miRNA sponge. And the same target for two targets, can better achieve the interference of two miRNAs, improve the efficiency of miRNA function research.
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
L'invention concerne un vecteur d'expression réduisant un micro-R-148a et un micro-R-424 et son application. Le procédé de mise au point d'un vecteur d'expression inhibant le micro-R-148a et le micro-R-424 consiste à lier une cible avec l'ARN Tud de micro-R-424 et de micro-R148a sur un vecteur de clonage pLKO.1-puro afin d'obtenir un vecteur d'expression inhibant le micro-ARN double brin pLKO-Tud-140-424. Le vecteur d'expression inhibe l'action de has-micro-R-148a et de has-micro-R-424.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2017/077171 WO2018170621A1 (fr) | 2017-03-18 | 2017-03-18 | Vecteur d'expression réduisant un micro-r-148a et un micro-r-424 et son application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2017/077171 WO2018170621A1 (fr) | 2017-03-18 | 2017-03-18 | Vecteur d'expression réduisant un micro-r-148a et un micro-r-424 et son application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2018170621A1 true WO2018170621A1 (fr) | 2018-09-27 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2017/077171 WO2018170621A1 (fr) | 2017-03-18 | 2017-03-18 | Vecteur d'expression réduisant un micro-r-148a et un micro-r-424 et son application |
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| Country | Link |
|---|---|
| WO (1) | WO2018170621A1 (fr) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
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- 2017-03-18 WO PCT/CN2017/077171 patent/WO2018170621A1/fr active Application Filing
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
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