CN111411112B - Targeting inhibitor of MAPK6P2 gene and application thereof - Google Patents
Targeting inhibitor of MAPK6P2 gene and application thereof Download PDFInfo
- Publication number
- CN111411112B CN111411112B CN202010424256.0A CN202010424256A CN111411112B CN 111411112 B CN111411112 B CN 111411112B CN 202010424256 A CN202010424256 A CN 202010424256A CN 111411112 B CN111411112 B CN 111411112B
- Authority
- CN
- China
- Prior art keywords
- mapk6p2
- gene
- inhibitor
- cells
- targeted
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 60
- 239000003112 inhibitor Substances 0.000 title claims abstract description 48
- 230000008685 targeting Effects 0.000 title abstract description 8
- 241000282414 Homo sapiens Species 0.000 claims abstract description 9
- 239000003814 drug Substances 0.000 claims abstract description 7
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 6
- 238000002360 preparation method Methods 0.000 claims abstract description 6
- 208000005017 glioblastoma Diseases 0.000 claims abstract 6
- 201000010915 Glioblastoma multiforme Diseases 0.000 claims abstract 2
- 108091027967 Small hairpin RNA Proteins 0.000 claims description 14
- 230000014509 gene expression Effects 0.000 claims description 14
- 239000004055 small Interfering RNA Substances 0.000 claims description 14
- 230000000692 anti-sense effect Effects 0.000 claims description 8
- 108091081021 Sense strand Proteins 0.000 claims description 7
- 239000002552 dosage form Substances 0.000 claims description 2
- 239000007972 injectable composition Substances 0.000 claims description 2
- 238000005516 engineering process Methods 0.000 abstract description 6
- 230000009368 gene silencing by RNA Effects 0.000 abstract description 6
- 230000009545 invasion Effects 0.000 abstract description 6
- 230000005012 migration Effects 0.000 abstract description 6
- 238000013508 migration Methods 0.000 abstract description 6
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 abstract description 5
- 229940079593 drug Drugs 0.000 abstract description 5
- 230000035755 proliferation Effects 0.000 abstract description 5
- 238000001415 gene therapy Methods 0.000 abstract description 4
- 229940126585 therapeutic drug Drugs 0.000 abstract description 3
- 238000011281 clinical therapy Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 45
- 206010018338 Glioma Diseases 0.000 description 31
- 208000032612 Glial tumor Diseases 0.000 description 30
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 239000000243 solution Substances 0.000 description 11
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 8
- 238000000034 method Methods 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 238000004113 cell culture Methods 0.000 description 6
- 230000012292 cell migration Effects 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 238000011282 treatment Methods 0.000 description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 5
- 108091008109 Pseudogenes Proteins 0.000 description 5
- 102000057361 Pseudogenes Human genes 0.000 description 5
- 230000033228 biological regulation Effects 0.000 description 5
- 238000007664 blowing Methods 0.000 description 5
- 239000006285 cell suspension Substances 0.000 description 5
- 108010087230 Sincalide Proteins 0.000 description 4
- 229960000583 acetic acid Drugs 0.000 description 4
- 238000010609 cell counting kit-8 assay Methods 0.000 description 4
- 239000012362 glacial acetic acid Substances 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 238000013518 transcription Methods 0.000 description 4
- 230000035897 transcription Effects 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- 239000012124 Opti-MEM Substances 0.000 description 3
- 230000004791 biological behavior Effects 0.000 description 3
- 201000007455 central nervous system cancer Diseases 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000030279 gene silencing Effects 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 238000007808 Cell invasion assay Methods 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 108010019160 Pancreatin Proteins 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 210000001130 astrocyte Anatomy 0.000 description 2
- 230000004709 cell invasion Effects 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 238000003570 cell viability assay Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000003828 downregulation Effects 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 229940055695 pancreatin Drugs 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 230000007480 spreading Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- BRZYSWJRSDMWLG-DJWUNRQOSA-N (2r,3r,4r,5r)-2-[(1s,2s,3r,4s,6r)-4,6-diamino-3-[(2s,3r,4r,5s,6r)-3-amino-4,5-dihydroxy-6-[(1r)-1-hydroxyethyl]oxan-2-yl]oxy-2-hydroxycyclohexyl]oxy-5-methyl-4-(methylamino)oxane-3,5-diol Chemical compound O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H]([C@@H](C)O)O2)N)[C@@H](N)C[C@H]1N BRZYSWJRSDMWLG-DJWUNRQOSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 101000950710 Homo sapiens Mitogen-activated protein kinase 6 Proteins 0.000 description 1
- 102100037801 Mitogen-activated protein kinase 6 Human genes 0.000 description 1
- 108700005084 Multigene Family Proteins 0.000 description 1
- 206010067482 No adverse event Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108010057163 Ribonuclease III Proteins 0.000 description 1
- 102000003661 Ribonuclease III Human genes 0.000 description 1
- 108020004459 Small interfering RNA Proteins 0.000 description 1
- BPEGJWRSRHCHSN-UHFFFAOYSA-N Temozolomide Chemical compound O=C1N(C)N=NC2=C(C(N)=O)N=CN21 BPEGJWRSRHCHSN-UHFFFAOYSA-N 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000003527 anti-angiogenesis Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 201000007983 brain glioma Diseases 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 208000025997 central nervous system neoplasm Diseases 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 239000006481 glucose medium Substances 0.000 description 1
- 238000012333 histopathological diagnosis Methods 0.000 description 1
- 210000003917 human chromosome Anatomy 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- 238000010232 migration assay Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000011242 molecular targeted therapy Methods 0.000 description 1
- 238000009126 molecular therapy Methods 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 230000009437 off-target effect Effects 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000002271 resection Methods 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 239000008925 shengjing Substances 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 229960004964 temozolomide Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
Images
Classifications
-
- 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
-
- 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
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
-
- 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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Veterinary Medicine (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- General Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Epidemiology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention belongs to the technical field of biological medicines, and particularly relates to a targeted inhibitor of MAPK6P2 gene and application thereof. A targeted inhibitor of the MAPK6P2 gene, which has the targeting sequence as follows: 5'-GCGAGTGAAGTTAACCATTAC-3' (SEQ ID No. 1). The MAPK6P2 gene targeted inhibitor is applied to the preparation of medicaments for treating human glioblastoma multiforme. The invention develops a targeting inhibitor of MAPK6P2 gene by using RNA interference technology, the inhibitor can be specifically combined with MAPK6P2 gene to silence MAPK6P2 gene, thereby inhibiting the proliferation, migration and invasion capacity of glioblastoma cells and achieving the purpose of treating glioblastoma. The MAPK6P2 gene targeted inhibitor plays an important role in the field of glioblastoma gene therapy, and provides a new targeted therapeutic drug for clinical therapy of glioblastoma.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a targeted inhibitor of MAPK6P2 gene and application thereof.
Background
Glioma is the most common primary central nervous system tumor, the annual incidence rate is 3-8 people/10 ten thousand people, and the composition accounts for 45.2% of all malignant central nervous system tumors and 80% of all primary malignant central nervous system tumors. According to the current treatment standard, firstly, the operation resection with the maximum safety range is carried out, then, the operation radiotherapy is carried out, and meanwhile, chemotherapy such as temozolomide and the like are supplemented, and the treatment of anti-angiogenesis medicines is carried out. However, patients with low differentiation and high grade in histopathological diagnosis in glioma have poorer prognosis, and the median survival time of patients with stage IV glioma is only 12-18 months. In recent years, in research on the pathogenesis of glioma, the regulation and control effect of pseudogenes on the biological behavior of glioma cells at the level of transcription, posttranscription and the like is highly emphasized by more researchers. Therefore, the role and mechanism of pseudogene regulation of glioma cell biological behavior are deeply researched, and a new target can be provided for glioma molecular therapy.
Pseudogenes are also called pseudogenes, DNA sequences which are similar to normal genes but lose gene function. It has been thought that it is a non-functional residue formed during the evolution of gene families, and is not generally transcribed, so it has no clear physiological significance and is often present in multigene families of eukaryotes. Recent studies indicate that pseudogenes have functions similar to non-coding RNA, play important molecular regulation and control roles at the level of transcription, posttranscription and the like, participate in the regulation of the occurrence and development of various malignant tumors, and play an important regulation role in tumors of various systems such as the respiratory system, the digestive system, the nervous system and the like of human beings.
The MAPK6P2 gene maps to 21q21.1, and its parent MAPK6 maps to human chromosome 15q 21.2. The coding nucleotide sequence is 4036bp in length. No study report on the relevance of MAPK6P2 in glioma has been retrieved. The principle of RNA interference technology is that Dicer enzyme is utilized to cut RNA molecules to form an RNA silencing complex, and the process of target RNA molecule combination and RNA molecule degradation is targeted, so that the RNA interference technology can play a therapeutic role in medical application. The shRNA is an artificial RNA consisting of two short reverse complementary sequences with a stem-loop interval, and is used as a common RNA interference tool, and the shRNA is combined with target gene mRNA to play a role. Because siRNA has off-target effect or influences normal gene expression, the prior shRNA technology is safer and more reliable.
At present, the role and the related mechanism of MAPK6P2 in the development of tumors are not reported, and the application of MAPK6P2 in glioma gene therapy is not clear at present. Therefore, the development of a MAPK6P 2-related drug is a problem to be solved.
Disclosure of Invention
In view of the problems in the prior art, the invention aims to provide a targeted inhibitor of MAPK6P2 gene and application thereof. Experiments of the invention prove that the MAPK6P2 gene is highly expressed in glioma tissues, and the proliferation, migration and invasion capacities of glioma cells are inhibited by inhibiting the expression of the MAPK6P2 gene. The invention develops a targeting inhibitor of MAPK6P2 gene by RNA interference technology, the inhibitor can be specifically combined with MAPK6P2 gene to silence MAPK6P2 gene, thereby inhibiting the proliferation, migration and invasion capacity of glioma cells and achieving the purpose of treating glioma. The targeted inhibitor of the MAPK6P2 gene plays an important role in the fields of glioma gene therapy and molecular targeted therapy, and provides a new targeted therapeutic drug for clinical treatment of glioma.
In order to achieve the above object, the present invention adopts the following technical solutions.
A targeted inhibitor of the MAPK6P2 gene, which has the targeting sequence as follows: 5'-GCGAGTGAAGTTAACCATTAC-3' (SEQ ID No. 1).
Further, the targeted inhibitor of the MAPK6P2 gene can inhibit shRNA sequence expressed by the MAPK6P2 gene, the shRNA template sequence comprises a sense strand and an antisense strand, and the sense strand and the antisense strand are respectively:
sense strand:
5’-CACCGCGAGTGAAGTTAACCATTACTTCAAGAGAGTAATGGTTAACTTCACTCGCTTTTTTG-3’ (SEQ ID No.2)。
antisense strand:
5’-GATCCAAAAAAGCGAGTGAAGTTAACCATTACTCTCTTGAAGTAATGGTTAACTTCACTCGC-3’ (SEQ ID No.3)。
further, a transcription product for transcribing the shRNA is obtained, and the sequence is as follows:
5’-GCGAGTGAAGTTAACCATTACTTCAAGAGAGTAATGGTTAACTTCACTCGCTT-3’ (SEQ ID No.4)。
preferably, the inhibitor is in the form of an injectable formulation.
Preferably, the inhibitor is in any pharmaceutically therapeutically acceptable dosage form.
Preferably, the inhibitor is in any pharmaceutically therapeutically acceptable dose.
The MAPK6P2 gene targeted inhibitor is applied to the preparation of the drugs for treating human brain glioma.
Compared with the prior art, the invention has the following beneficial effects.
1) The MAPK6P2 gene targeted inhibitor provided by the invention has strong specificity, high silencing efficiency and low off-target rate, and inhibits the expression of the MAPK6P2 gene.
2) The invention relates to a method for inhibiting the expression of human MAPK6P2 gene by an inhibitor of MAPK6P2 gene, thereby inhibiting the proliferation, migration and invasion capabilities of glioma cells and providing a brand-new treatment method for glioma gene therapy.
3) The inhibitor of the MAPK6P2 gene provided by the invention can obviously reduce the drug resistance problem of the traditional therapeutic drugs by targeted therapy.
4) The inhibitor of the MAPK6P2 gene provided by the invention is applied and researched on the in vitro cytology level, and experiments prove that the targeted treatment effect of the inhibitor of the MAPK6P2 gene is exact and no adverse reaction occurs.
Drawings
Fig. 1 is a bar graph of the significant increase in expression levels of the MAPK6P2 gene in glioma tissues and cells using realtome PCR.
Figure 2 is a bar graph of the significant down-regulation of MAPK6P2 expression levels in glioma cells following the application of MAPK6P2 gene inhibitor using realtome PCR.
FIG. 3 is a statistical chart of the ability of CCK-8 cell viability assay to inhibit glioma cell proliferation using MAPK6P2 gene inhibitors.
FIG. 4 is a photograph and a statistical chart of the ability of a Transwell cell migration assay to detect inhibition of glioma cell migration using an inhibitor of the MAPK6P2 gene.
FIG. 5 is a photograph and a statistical chart of the ability of a Transwell cell invasion assay to detect inhibition of glioma cell invasion by the application of MAPK6P2 gene inhibitor.
Detailed Description
The technical solutions and effects of the present invention will be described in detail below with reference to specific embodiments and accompanying drawings. The following examples are only preferred embodiments of the present invention and are not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art without departing from the spirit and the principle of the present invention, and any modifications, equivalents, improvements, etc. made within the scope of the present invention are intended to be covered by the present invention.
Example design of shRNA and preparation of interference vector and application of MAPK6P2 gene inhibitor.
Firstly, cell culture.
Human glioma cell lines U87, U251, U373 and Human Astrocytes (HA) were purchased from the Shanghai institute bioscience cell resource center. The cells of U87, U251, U373, and HA were cultured in a 100mm cell culture dish in DMEM high-glucose medium (astrocyte medium AM) containing 10% fetal bovine serum, and the medium was changed every 2 days, and the cells grew to a monolayer in about 2-3 days.
Secondly, real-time quantitative PCR is used for detecting the expression of MAPK6P 2.
1. Total RNA was extracted from the cells by Trizol method.
Washing the collected cells with cold PBS, adding 1ml Trizol reagent, blowing for several times, observing the cells under a mirror to form oil drops (fully cracking), transferring into a 1.5ml EP tube, and standing for 5 minutes to fully crack the cells; adding 0.2ml of chloroform into the sample, manually and violently shaking the sample, and standing the sample for 3 minutes at room temperature; centrifuging at 12000g at 4 deg.C for 15 min, adding the upper water phase into a new EP tube, adding 0.5ml isopropanol, turning upside down, mixing, and standing at room temperature for 10 min; centrifuging at 12000g at 4 deg.C for 15 min, discarding supernatant, and adding 1ml 75% ethanol; after centrifuging at 7500g for 5 minutes at 4 ℃ and drying for 15 minutes, 40. mu.l of DEPC water is added, and the sample can be frozen in a refrigerator at-80 ℃.
2. The expression of MAPK6P2 is detected by a one-step dye method qRT-PCR.
The CT value was determined and the relative expression level of MAPK6P2 was calculated using B-ACTIN as the internal reference and 2^ (Δ Ct). Statistical plots of significantly increased expression levels of the MAPK6P2 gene in glioma tissues and cells were examined, as shown in figure 1.
Thirdly, preparation and application of MAPK6P2 gene inhibitor.
Designing an interference sequence of the MAPK6P2 gene, and selecting an inhibitor targeting sequence which targets the human MAPK6P2 gene and specifically inhibits the expression of the MAPK6P2 gene as follows: 5'-GCGAGTGAAGTTAACCATTAC-3' are provided.
The GCGAGTGAAGTTAACCATTAC sequence is input in the homologous sequence alignment analysis nucleotide blast of NCBI for alignment analysis, and the result shows that the sequence has no high homology with other human mRNA genes and can be used as a specific sequence for specifically interfering MAPK6P2 gene. The shRNA sequence of the targeted inhibitor was further designed to include the sense and antisense strands as follows.
The sense strand sequence of the shRNA is as follows:
5’-CACCGCGAGTGAAGTTAACCATTACTTCAAGAGAGTAATGGTTAACTTCACTCGCTTTTTTG-3’。
the sequence of the shRNA antisense strand is as follows:
5’-GATCCAAAAAAGCGAGTGAAGTTAACCATTACTCTCTTGAAGTAATGGTTAACTTCACTCGC-3’。
the sequence of the transcription product of the shRNA is as follows:
5’-GCGAGTGAAGTTAACCATTACTTCAAGAGAGTAATGGTTAACTTCACTCGCTT-3’。
sequence information corresponding plasmids were designed and synthesized as MAPK6P2 gene inhibitors.
Transfection with MAPK6P2 gene inhibitor: plasmid U6/GFP/Neo of sh-NC and sh-MAPK6P2 enables expression of MAPK6P2 to be silent, and an empty plasmid without MAPK6P2 sequence or shRNA is used as an experimental negative control; culturing glioma cells by using a 24-hole culture plate, and performing transfection when the cell growth reaches about 80%; plasmid, Opti-MEM, required for the preparation of transfections®I and LTX and Plus reagent (Life Technologies) transfection reagents. Tube A: dissolving a hole according to 1 mu g plasmid DNA in 50 mu l Opti-MEM I +1 mu l p3000 for 5min, placing a tube B: the wells were dissolved in 50 μ l Opti-MEM according to 1 μ l LTX and Plus®In the step I; evenly mixing A, B two tubes, and standing for 5 min; sucking out the culture solution, adding 100 mul of transfection mixed solution into each hole, and adding 400 mul of EBM-2 culture solution; after 48 h, the selection was carried out in a medium containing the antibiotic G418 at a concentration of 0.4 mg/ml, and the concentration of G418 was increased continuously, so that a glioma cell line capable of stably silencing MAPK6P2 was obtained after about 4 weeks. In subsequent experiments, the groups were divided into 3 groups, each of which was: blank Control (Control) without any treatment; negative control group (sh-NC) transfected with MAPK6P2 silent empty plasmid; experimental group transfected with MAPK6P2 silencing plasmid (sh-MAPK6P 2). Statistical plots of significant down-regulation of MAPK6P2 expression levels in glioma cells following MAPK6P2 gene inhibitor, as shown in figure 2.
Example 2 the use of MAPK6P2 gene inhibitors significantly inhibited the malignant biological behavior of gliomas.
Firstly, a CCK-8 cell viability method is used for detecting the influence of the MAPK6P2 gene inhibitor on the proliferation capacity of glioma cells.
U251 cells were trypsinized and single cell suspensions were prepared separately in normal medium. Cells were counted and seeded at a concentration of 2000 cells/well in 96-well cell culture plates. Each group is provided with 5 multiple holes, and each hole accounts for 200 mu l. After 24h, 10 mul CCK-8 was added. After 2 hours, the light absorption value of each well at a wavelength of 450nm was measured by a microplate reader. A statistical chart of inhibition of U251 cell proliferation by the CCK-8 cell viability assay using MAPK6P2 gene inhibitors is shown in FIG. 3.
Secondly, a Transwell cell migration experiment detects the influence of the MAPK6P2 gene inhibitor on the migration capacity of glioma cells.
Detecting the migration capacity of the cells: adding 600 μ l of culture solution containing serum to each well of a 24-well plate, and then placing a Transwell chamber; digesting different groups of U251 cells by pancreatin, slightly blowing the cells apart, centrifuging the cells, adding a serum-free culture solution to resuspend the cells, adding 100 mu l of cell suspension into each small chamber, uniformly paving the cells in an upper chamber, wherein the cell suspension is about 10 mu l5And (4) cells. PlacingCulturing in a cell culture box at 37 ℃ for 24 h; after 24h, the chamber was removed, the upper chamber was wiped down gently with a cotton swab, the chamber was washed with PBS and dried. Then preparing a stationary liquid according to the ratio of methanol to glacial acetic acid = 3: 1, namely blowing and uniformly mixing 750 mul of methanol and 250 mul of glacial acetic acid; immersing the chamber into a fixing solution to fix the cells for 30 min; washing the small chamber by using PBS and drying, spreading 200 mul of Sa dye of Jimu at the bottom of the inverted small chamber, and dyeing for at least 30 min; the chamber was washed twice with PBS, placed under an inverted microscope, observed under a 400 Xmicroscope, and 5 fields were randomly selected for cell count to demonstrate cell migration ability. Transwell cell migration experiments detected a significant reduction in the number of U251 cells migrated after the application of MAPK6P2 gene inhibitor, as shown in figure 4.
And thirdly, detecting the influence of the MAPK6P2 gene inhibitor on the invasion capacity of glioma cells by a Transwell cell invasion experiment.
And (3) detecting the invasion capacity of the cells: adding 600 mu l of culture solution containing serum into each well of a 24-well plate, then placing a Transwell chamber, adding 50 mu l of Matrigel collagen solution into the chamber, and culturing for 30 mins in a cell culture box at 37 ℃; digesting different groups of U251 cells by pancreatin, slightly blowing the cells apart, centrifuging the cells, adding a serum-free culture solution to resuspend the cells, adding 100 mu l of cell suspension into each small chamber, uniformly paving the cells in an upper chamber, wherein the cell suspension is about 10 mu l5And (4) cells. Culturing in a cell culture box at 37 deg.C for 24 hr; after 24h the chamber was removed, washed with PBS and dried. Then preparing a stationary liquid according to the ratio of methanol to glacial acetic acid = 3: 1, namely blowing and uniformly mixing 750 mul of methanol and 250 mul of glacial acetic acid; immersing the chamber into a fixing solution to fix the cells for 30 min; washing the small chamber by using PBS and drying, spreading 200 mul of Sa dye of Jimu at the bottom of the inverted small chamber, and dyeing for at least 30 min; the chamber was washed twice with PBS, placed under an inverted microscope, observed under a 400 Xmicroscope, and 5 fields were randomly selected for cell count to demonstrate the ability to invade cells. Transwell cell invasion assay detection the number of U251 cell migration inhibition was significantly reduced after application of MAPK6P2 gene inhibitor, as shown in fig. 5.
The statistical method comprises the following steps: all the above experimentsAll were performed in triplicate individually and the data are presented as mean ± sd. The statistical software SPSS 19.0 is used for analyzing the data of the plurality of groups by using a one-way analysis of variance (ANOVA) method, whether the difference exists between the groups is determined, and when the difference exists, the difference is determinedPValue of<0.05 was considered statistically significant. IC50 was calculated using Graphpad software.
Sequence listing
<110> Shengjing Hospital affiliated to Chinese medical university
<120> targeting inhibitor of MAPK6P2 gene and application thereof
<130> 4
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213> Artificial sequence
<400> 1
gcgagtgaag ttaaccatta c 21
<210> 2
<211> 62
<212> DNA
<213> Artificial sequence
<400> 2
caccgcgagt gaagttaacc attacttcaa gagagtaatg gttaacttca ctcgcttttt 60
tg 62
<210> 3
<211> 62
<212> DNA
<213> Artificial sequence
<400> 3
gatccaaaaa agcgagtgaa gttaaccatt actctcttga agtaatggtt aacttcactc 60
gc 62
<210> 4
<211> 51
<212> DNA
<213> Artificial sequence
<400> 4
gcgagtgaag ttaaccatta cttcaagaga gtaatggtta acttcactcg ctt 53
Claims (4)
1. The application of a targeted inhibitor of MAPK6P2 gene in the preparation of a medicament for treating human glioblastoma multiforme is characterized in that the targeted inhibitor of the MAPK6P2 gene is a shRNA sequence capable of inhibiting the expression of the MAPK6P2 gene, the shRNA template consists of a sense strand and an antisense strand, and the sense strand and the antisense strand are respectively:
sense strand:
5’-CACCGCGAGTGAAGTTAACCATTACTTCAAGAGAGTAATGGTTAACTTCACTCGCTTTTTTG-3’；
antisense strand:
5’-GATCCAAAAAAGCGAGTGAAGTTAACCATTACTCTCTTGAAGTAATGGTTAACTTCACTCGC-3’。
2. the use of claim 1, wherein the inhibitor is in any pharmaceutically acceptable dosage form.
3. The use of claim 2, wherein the inhibitor is in the form of an injectable formulation.
4. The use of claim 1, wherein the inhibitor is in any pharmacotherapeutically acceptable dose.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010424256.0A CN111411112B (en) | 2020-05-19 | 2020-05-19 | Targeting inhibitor of MAPK6P2 gene and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010424256.0A CN111411112B (en) | 2020-05-19 | 2020-05-19 | Targeting inhibitor of MAPK6P2 gene and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111411112A CN111411112A (en) | 2020-07-14 |
| CN111411112B true CN111411112B (en) | 2022-02-22 |
Family
ID=71490495
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010424256.0A Active CN111411112B (en) | 2020-05-19 | 2020-05-19 | Targeting inhibitor of MAPK6P2 gene and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111411112B (en) |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005021793A1 (en) * | 2003-08-29 | 2005-03-10 | Pantarhei Bioscience B.V. | Prenatal diagnosis of down syndrome by detection of fetal rna markers in maternal blood |
-
2020
- 2020-05-19 CN CN202010424256.0A patent/CN111411112B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN111411112A (en) | 2020-07-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhou et al. | Reduction of miR-21 induces glioma cell apoptosis via activating caspase 9 and 3 | |
| CA2812650C (en) | Use of mirnas for the diagnosis, prophylaxis, treatment and follow-up of diseases involving macroautophagy abnormalities | |
| CN111411112B (en) | Targeting inhibitor of MAPK6P2 gene and application thereof | |
| EP2872634B1 (en) | Micrornas sensitize cancers to therapy | |
| CN102533755A (en) | Human miR-328 antisense nucleic acid and application thereof | |
| CN102382824A (en) | Human miR-145 antisense nucleic acid and application thereof | |
| CN111020036B (en) | Application of human circ-STXBP5L and related product | |
| CN105873617A (en) | Treatment of diseases associated with PGC 1-alpha by modulation of microRNAs miR-130a and miR-130b | |
| CN108251528A (en) | Applications of the LINC01814 in gastric cancer diagnosis and treatment | |
| CN118147140A (en) | A targeted inhibitor of RRN3P2 gene and its use | |
| CN111534516B (en) | Target inhibitor of SNORD83A gene and application thereof | |
| CN111419866A (en) | Application of miR-29a-3p in the preparation of drugs for peripheral nerve injury | |
| CN111635946B (en) | A molecular biomarker for diagnosis and treatment of glioma and its application | |
| EP2654801B1 (en) | Targeting glioma stem cells by sequence-specific functional inhibition of pro-survival oncomir-138 | |
| CN111607595B (en) | Targeting inhibitor of DBF4P1 gene and application thereof | |
| CN100410373C (en) | siRNA duplexes inhibiting bcl-2 gene expression | |
| CN111763670B (en) | Targeting inhibitor of hsa_circ_0008821 gene and application thereof | |
| CN100395335C (en) | siRNA duplexes inhibiting bcl-2 gene expression | |
| CN116334226B (en) | Application of lncRNA SNHG26 in the preparation of tumor prevention and treatment drugs | |
| CN102382825B (en) | Human miR-1826 antisense nucleic acid and its application | |
| CN103667430A (en) | Application and relevant medicament of nucleotide octamer binding protein expression gene | |
| CN102140462B (en) | Human miR-1260 antisense nucleic acid and application thereof | |
| CN102643806B (en) | The antisense oligonucleotide of people miR-1913 and application thereof | |
| CN109536498B (en) | A kind of PABPC5 gene inhibitor and use thereof | |
| CN100395334C (en) | siRNA duplexes inhibiting bcl-2 gene expression |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |