CN116726181B - Use of agent for inhibiting NAT9 gene expression - Google Patents
Use of agent for inhibiting NAT9 gene expression Download PDFInfo
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- CN116726181B CN116726181B CN202310995413.7A CN202310995413A CN116726181B CN 116726181 B CN116726181 B CN 116726181B CN 202310995413 A CN202310995413 A CN 202310995413A CN 116726181 B CN116726181 B CN 116726181B
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- 230000014509 gene expression Effects 0.000 title claims abstract description 16
- 230000002401 inhibitory effect Effects 0.000 title abstract description 10
- 101150090948 nat9 gene Proteins 0.000 title abstract description 3
- 201000008968 osteosarcoma Diseases 0.000 claims abstract description 31
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 18
- 206010027476 Metastases Diseases 0.000 claims abstract description 11
- 230000009401 metastasis Effects 0.000 claims abstract description 11
- 239000003814 drug Substances 0.000 claims abstract description 8
- 239000003795 chemical substances by application Substances 0.000 claims description 11
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- 230000005764 inhibitory process Effects 0.000 claims description 4
- 238000012226 gene silencing method Methods 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 230000030279 gene silencing Effects 0.000 claims description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 claims 1
- 229940079593 drug Drugs 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 27
- 230000006907 apoptotic process Effects 0.000 description 12
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 5
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- 102000004121 Annexin A5 Human genes 0.000 description 2
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- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 2
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- 101000602350 Homo sapiens N-acetyltransferase 9 Proteins 0.000 description 1
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- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- 101710202061 N-acetyltransferase Proteins 0.000 description 1
- 102100037155 N-acetyltransferase 9 Human genes 0.000 description 1
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- IWUCXVSUMQZMFG-AFCXAGJDSA-N Ribavirin Chemical compound N1=C(C(=O)N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 IWUCXVSUMQZMFG-AFCXAGJDSA-N 0.000 description 1
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- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
Abstract
The invention provides an application of a reagent for inhibiting NAT9 gene expression, belonging to the technical field of medicines. The invention can inhibitNAT9The gene expression reagent is used as a medicament for inhibiting osteosarcoma and inhibiting metastasis of osteosarcoma, and provides a new choice for clinically treating osteosarcoma and metastasis thereof.
Description
Technical Field
The invention belongs to the field of medical engineering, and in particular relates to an inhibition methodNAT9Use of a reagent for gene expression.
Background
Osteosarcoma is a common primary bone malignancy, which occurs in children and adolescents, and the current standard treatment is a combination of neoadjuvant chemotherapy-surgery-adjuvant chemotherapy based on a regimen comprising doxorubicin, cisplatin, and large doses of methotrexate (MAP regimen). Although perioperative chemotherapy greatly improves the 5-year survival rate and the limb protection rate of patients with osteosarcoma, the 5-year survival rate of patients with limited period is 60% -70%, the survival benefit brought by chemotherapy does not obtain further breakthrough in more than 40 years. 10% -15% of osteosarcoma patients are accompanied with metastasis at the initial diagnosis, the most common metastasis site is lung, the survival rate of the patients with metastatic or recurrent osteosarcoma is only 20% in 5 years, the prognosis is extremely poor, and improvement is needed in the current state of treatment.
NAT9UniProt accession number Q9BTE0, N-acetyltransferase, is a member of the NAT family of genes. Not seeNAT9Reports on osteosarcoma.
Disclosure of Invention
The invention aims to provide inhibitionNAT9The application of gene expression reagent in inhibiting osteosarcoma and its metastasis.
The invention provides a method for inhibitingNAT9Use of an agent for gene expression in the manufacture of a medicament for the prevention and/or treatment of osteosarcoma.
The invention also provides inhibition ofNAT9Use of an agent that expresses a gene in the preparation of a medicament for reducing osteosarcoma metastasis.
Wherein the agent is a knockoutNAT9And (3) a reagent of a gene. Preferably, the reagent comprises a nucleotide sequence shown in SEQ ID NO. 1-2.
Wherein the reagent is a reagent for causingNAT9Agents for gene silencing.
Wherein the reagent isNAT9Inhibitors of genes.
The invention also provides a medicament for inhibiting osteosarcoma or osteosarcoma metastasis, which comprises inhibitingNAT9A reagent for gene expression and pharmaceutically acceptable auxiliary materials.
Wherein the agent is a knockoutNAT9Gene reagent and gene expression systemNAT9Agents or agents for gene silencingNAT9Inhibitors of genes.
Wherein the reagent comprises a nucleotide sequence shown in SEQ ID NO. 1-2.
The invention has the beneficial effects that the invention is knocked downNAT9Can remarkably inhibit proliferation of osteosarcoma cells, remarkably promote apoptosis of osteosarcoma cells, inhibit migration of osteosarcoma cells, and inhibit proliferation of osteosarcoma cellsNAT9The gene expression reagent is used as a medicament for inhibiting osteosarcoma and inhibiting metastasis of osteosarcoma, and provides a new choice for clinically treating osteosarcoma and metastasis thereof.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
FIG. 1NAT9And (5) a graph of gene expression proportion results.
Fig. 2 knockdownNAT9Inhibition of 143b cell proliferation patterns; FIG. A shows the growth index of 143b cells after the dynamic detector detects knockdown of NAT 9; panel B is a graph of cell viability statistics.
Fig. 3 knockdownNAT9A graph of apoptosis promotion 143 b; a is a knockdown NAT9 flow cytometry analysis apoptosis graph, and B is an apoptosis proportion statistical result graph.
Fig. 4 knockdownNAT9Inhibition 143b cell migration profile; a is a stained, perforated cell map, and B is a statistical map of the number of migrating cells.
Detailed Description
The raw materials and equipment used in the invention are all known products and are obtained by purchasing commercial products.
143b cells: osteosarcoma cells purchased from the biological technology Co.Ltd
Lipofectamine ™ 3000 transfection reagent: eukaryotic transfection reagents were purchased from thermosusher under the designation L3000001.
Annexin V-APC/PI apoptosis detection kit: apoptosis was detected. Propidium Iodide (PI) is a nucleic acid dye that does not penetrate intact cell membranes, but cells in the middle and late stages of apoptosis and dead cells due to increased cell membrane permeability, PI can penetrate cell membranes and stain the nucleus with red. By matching Annexin V to PI, cells at different stages of apoptosis can be distinguished. The product number KGA1030, the Yu Kaiji organism was purchased.
Example 1,NAT9Effects of knockout on osteosarcoma
1. Experimental method
(one)NAT9Knock-out
143b Cell line was inoculated in an e-plate96 well plate (DMEM medium) and placed in an RTCA Cell dynamic detector, and sh-NAT9 and transfection reagent were added when the CI (Cell Index, indicator of Cell growth) was 1, to add universal negative control NC-treated cells as controls.
sh-NAT9:
sense CCACGAGTGGATGAAATCA (SEQ ID NO.1)
antisense GGTGCTCACCTACTTTAGT(SEQ ID NO.2)。
shNC group: is a negative transfection control group.
(II) inspection method
Apoptosis: 143b cell knockdownNAT9 After 48h, the cell suspension was collected. The cell suspension was centrifuged for 5 min at 250 g, the supernatant was aspirated, washed once with PBS, centrifuged for 5 min at 250 g, and the collected cell pellet was used for staining. After resuspension of the cells with 500. Mu.L of PBS buffer (Punuocele, cat. PB 180327), 5. Mu.L of Annexin V was added and gently blown up, followed by addition of 5. Mu.L of PIHomogenizing; incubate at room temperature for 10 min under light protection, and perform detection analysis using a flow analyser (model: cytoflex manufacturer: beckman) instrument.
Cell migration: serum-free DMEM 200. Mu.L and 2X 10 were added to the Transwell chamber 4 143b cells, 600. Mu.L of 10% serum DMEM was added to the lower chamber at 5% CO 2 The cell incubator was incubated for 24 hours, the cells passing through the membrane pores were stained with 1% crystal violet in a 4% paraformaldehyde fixed cell, photographed under a normal microscope, the number of perforated cells calculated, and the cell mobility calculated.
2. Experimental results
NAT9The result of the gene expression ratio is shown in FIG. 1, the lower the gene expression ratio is, the higher the knocking down efficiency is,NAT9the knockdown efficiency was 64%.
As can be seen from fig. 2, knockdownNAT9Significantly inhibited 143b cell proliferation. As can be seen from fig. 3, knockdownNAT9Significantly promoting 143b apoptosis.
As can be seen from fig. 4, knockdownNAT9Significantly inhibiting 143b cell migration.
The invention knocks down in osteosarcoma cellsNAT9Can significantly inhibit 143b cell proliferation (fig. 2), significantly promote 143b apoptosis (fig. 3), and significantly inhibit 143b cell migration (fig. 4).
In conclusion, the invention is realized by knocking downNAT9Can remarkably inhibit proliferation of osteosarcoma cells, remarkably promote apoptosis of osteosarcoma cells, and remarkably inhibit migration of osteosarcoma cells, which indicates inhibitionNAT9The gene expression reagent can effectively inhibit osteosarcoma and can also inhibit metastasis of osteosarcoma, and has important theoretical and practical significance for clinical osteosarcoma prevention and treatment.
Claims (5)
1. Inhibition ofNAT9Use of an agent for gene expression in the manufacture of a medicament for the treatment of osteosarcoma.
2. Inhibition ofNAT9Use of an agent for gene expression in the manufacture of a medicament for reducing osteosarcoma metastasis.
3. According to claim 1 or 2The application is characterized in that: the reagent is knockdownNAT9And (3) a reagent of a gene.
4. Use according to claim 3, characterized in that: the reagent comprises nucleotide sequences shown in SEQ ID NO. 1-2.
5. Use according to claim 1 or 2, characterized in that: the reagent is to makeNAT9Agents for gene silencing.
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CN116726181B true CN116726181B (en) | 2023-10-20 |
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Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2003020930A1 (en) * | 2001-08-29 | 2003-03-13 | The Board Of Trustees Of The University Of Illinois | IDENTIFICATION AND USE OF MAMMALIAN p21 INHIBITORS |
WO2008076857A2 (en) * | 2006-12-14 | 2008-06-26 | Wisconsin Alumni Research Foundation | Method and compositions for inhibiting mage protein interaction with kap-1 |
CN106794125A (en) * | 2014-04-03 | 2017-05-31 | 剑桥企业有限公司 | For treating or preventing lamin sick, aging and cancer NAT10 conditioning agents |
CN110951873A (en) * | 2019-12-03 | 2020-04-03 | 中山大学 | Bone and sarcoma marker, application thereof and kit |
CN111424082A (en) * | 2019-01-09 | 2020-07-17 | 上海中医药大学附属龙华医院 | Application of lncRNA-SNHG6 gene in preparation of medicine for treating osteosarcoma |
WO2020257401A1 (en) * | 2019-06-21 | 2020-12-24 | The Children's Medical Center Corporation | Methods and compositions for the treatment of cancer |
WO2021232094A1 (en) * | 2020-05-19 | 2021-11-25 | Hudson Institute of Medical Research | Hedgehog signaling -dependent cancer treatment |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
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GB0915877D0 (en) * | 2009-09-10 | 2009-10-14 | E Therapeutics Plc | Cancer cell apoptosis |
JP2014519496A (en) * | 2011-05-11 | 2014-08-14 | ナノヴァレント ファーマシューティカルズ,インコーポレイテッド | Enhanced osteosarcoma growth inhibition by cytotoxic polymerized liposome nanoparticles targeting ALCAM cell surface receptors |
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- 2023-08-09 CN CN202310995413.7A patent/CN116726181B/en active Active
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Publication number | Priority date | Publication date | Assignee | Title |
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WO2003020930A1 (en) * | 2001-08-29 | 2003-03-13 | The Board Of Trustees Of The University Of Illinois | IDENTIFICATION AND USE OF MAMMALIAN p21 INHIBITORS |
WO2008076857A2 (en) * | 2006-12-14 | 2008-06-26 | Wisconsin Alumni Research Foundation | Method and compositions for inhibiting mage protein interaction with kap-1 |
CN106794125A (en) * | 2014-04-03 | 2017-05-31 | 剑桥企业有限公司 | For treating or preventing lamin sick, aging and cancer NAT10 conditioning agents |
CN111424082A (en) * | 2019-01-09 | 2020-07-17 | 上海中医药大学附属龙华医院 | Application of lncRNA-SNHG6 gene in preparation of medicine for treating osteosarcoma |
WO2020257401A1 (en) * | 2019-06-21 | 2020-12-24 | The Children's Medical Center Corporation | Methods and compositions for the treatment of cancer |
CN114269920A (en) * | 2019-06-21 | 2022-04-01 | 儿童医疗中心有限公司 | Methods and compositions for treating cancer |
CN110951873A (en) * | 2019-12-03 | 2020-04-03 | 中山大学 | Bone and sarcoma marker, application thereof and kit |
WO2021232094A1 (en) * | 2020-05-19 | 2021-11-25 | Hudson Institute of Medical Research | Hedgehog signaling -dependent cancer treatment |
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Identification of a molecular component of the mitochondrial acetyltransferase programme: a novel role for GCN5L1;Iain SCOTT 等;Biochem. J.;第443卷(第3期);第657页左栏、Supplementary Figure S1 * |
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