CN114934051B - LncRNA TAB2-AS and application thereof in porcine ovarian granulosa cells - Google Patents
LncRNA TAB2-AS and application thereof in porcine ovarian granulosa cells Download PDFInfo
- Publication number
- CN114934051B CN114934051B CN202210582494.3A CN202210582494A CN114934051B CN 114934051 B CN114934051 B CN 114934051B CN 202210582494 A CN202210582494 A CN 202210582494A CN 114934051 B CN114934051 B CN 114934051B
- Authority
- CN
- China
- Prior art keywords
- lncrna
- tab2
- cells
- granulosa cells
- porcine
- 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
- 108091046869 Telomeric non-coding RNA Proteins 0.000 title claims abstract description 104
- 210000002503 granulosa cell Anatomy 0.000 title claims abstract description 70
- 230000002611 ovarian Effects 0.000 title claims abstract description 47
- 210000004027 cell Anatomy 0.000 claims abstract description 58
- 230000006907 apoptotic process Effects 0.000 claims abstract description 27
- 210000001672 ovary Anatomy 0.000 claims abstract description 27
- 239000013598 vector Substances 0.000 claims abstract description 26
- 239000000074 antisense oligonucleotide Substances 0.000 claims abstract description 13
- 238000012230 antisense oligonucleotides Methods 0.000 claims abstract description 13
- 230000035755 proliferation Effects 0.000 claims abstract description 13
- 108091034117 Oligonucleotide Proteins 0.000 claims abstract description 6
- 238000011161 development Methods 0.000 claims abstract description 4
- 108020005198 Long Noncoding RNA Proteins 0.000 claims abstract 11
- 108020004414 DNA Proteins 0.000 claims description 28
- 230000014509 gene expression Effects 0.000 claims description 22
- 239000003814 drug Substances 0.000 claims description 21
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 17
- 102000053602 DNA Human genes 0.000 claims description 16
- 229940079593 drug Drugs 0.000 claims description 13
- 230000004663 cell proliferation Effects 0.000 claims description 12
- 239000012620 biological material Substances 0.000 claims description 10
- 108091027963 non-coding RNA Proteins 0.000 claims description 9
- 102000042567 non-coding RNA Human genes 0.000 claims description 9
- 230000002441 reversible effect Effects 0.000 claims description 9
- 108020000948 Antisense Oligonucleotides Proteins 0.000 claims description 7
- 230000001737 promoting effect Effects 0.000 claims description 7
- 239000002299 complementary DNA Substances 0.000 claims description 6
- 238000012408 PCR amplification Methods 0.000 claims description 4
- 230000002401 inhibitory effect Effects 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 239000012634 fragment Substances 0.000 claims description 2
- 238000000338 in vitro Methods 0.000 claims description 2
- 108091008146 restriction endonucleases Proteins 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 1
- 230000002018 overexpression Effects 0.000 abstract description 20
- 230000000694 effects Effects 0.000 abstract description 16
- 238000000034 method Methods 0.000 abstract description 15
- 238000002474 experimental method Methods 0.000 abstract description 14
- XAUDJQYHKZQPEU-KVQBGUIXSA-N 5-aza-2'-deoxycytidine Chemical compound O=C1N=C(N)N=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 XAUDJQYHKZQPEU-KVQBGUIXSA-N 0.000 abstract description 11
- 108010048367 enhanced green fluorescent protein Proteins 0.000 abstract description 9
- 230000007067 DNA methylation Effects 0.000 abstract description 6
- 238000000926 separation method Methods 0.000 abstract description 6
- 238000013518 transcription Methods 0.000 abstract description 6
- 230000035897 transcription Effects 0.000 abstract description 6
- 102000037865 fusion proteins Human genes 0.000 abstract description 4
- 108020001507 fusion proteins Proteins 0.000 abstract description 4
- 230000004960 subcellular localization Effects 0.000 abstract description 4
- 230000007246 mechanism Effects 0.000 abstract description 2
- 230000002194 synthesizing effect Effects 0.000 abstract description 2
- 229940126190 DNA methyltransferase inhibitor Drugs 0.000 abstract 1
- 239000003968 dna methyltransferase inhibitor Substances 0.000 abstract 1
- 210000002394 ovarian follicle Anatomy 0.000 abstract 1
- 238000011160 research Methods 0.000 abstract 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 21
- 239000002953 phosphate buffered saline Substances 0.000 description 21
- 238000011529 RT qPCR Methods 0.000 description 17
- 238000005406 washing Methods 0.000 description 14
- 239000006228 supernatant Substances 0.000 description 12
- 241000282898 Sus scrofa Species 0.000 description 9
- 238000001514 detection method Methods 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 238000001890 transfection Methods 0.000 description 8
- 238000012258 culturing Methods 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 5
- 108010040476 FITC-annexin A5 Proteins 0.000 description 5
- 108700026244 Open Reading Frames Proteins 0.000 description 5
- 239000013604 expression vector Substances 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 238000012406 Annexin V-FITC/PI double staining Methods 0.000 description 4
- 210000000805 cytoplasm Anatomy 0.000 description 4
- 238000004043 dyeing Methods 0.000 description 4
- 230000003325 follicular Effects 0.000 description 4
- 230000008217 follicular development Effects 0.000 description 4
- 230000002452 interceptive effect Effects 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 238000001179 sorption measurement Methods 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 210000000349 chromosome Anatomy 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 230000016087 ovulation Effects 0.000 description 3
- 230000036961 partial effect Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 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
- 108700024394 Exon Proteins 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 229940123379 Methyltransferase inhibitor Drugs 0.000 description 2
- 102000043141 Nuclear RNA Human genes 0.000 description 2
- 108020003217 Nuclear RNA Proteins 0.000 description 2
- 108091081024 Start codon Proteins 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000001516 cell proliferation assay Methods 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 238000013401 experimental design Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 238000012165 high-throughput sequencing Methods 0.000 description 2
- 239000003697 methyltransferase inhibitor Substances 0.000 description 2
- QPUKANZXOGADOB-UHFFFAOYSA-N n-dodecyl-n-methylnitrous amide Chemical compound CCCCCCCCCCCCN(C)N=O QPUKANZXOGADOB-UHFFFAOYSA-N 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 230000000149 penetrating effect Effects 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000001850 reproductive effect Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 108090000672 Annexin A5 Proteins 0.000 description 1
- 108020005544 Antisense RNA Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 101000674728 Homo sapiens TGF-beta-activated kinase 1 and MAP3K7-binding protein 2 Proteins 0.000 description 1
- 108060004795 Methyltransferase Proteins 0.000 description 1
- 102000016397 Methyltransferase Human genes 0.000 description 1
- 108091092724 Noncoding DNA Proteins 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 230000004570 RNA-binding Effects 0.000 description 1
- 102100021227 TGF-beta-activated kinase 1 and MAP3K7-binding protein 2 Human genes 0.000 description 1
- 101150011375 Tab2 gene Proteins 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 238000003782 apoptosis assay Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000007444 cell Immobilization Methods 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000003184 complementary RNA Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 108091092330 cytoplasmic RNA Proteins 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 238000012921 fluorescence analysis Methods 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 210000001733 follicular fluid Anatomy 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 239000003163 gonadal steroid hormone Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 230000010311 mammalian development Effects 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 210000000287 oocyte Anatomy 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000004681 ovum Anatomy 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000008823 permeabilization Effects 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 230000008288 physiological mechanism Effects 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000012342 propidium iodide staining Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
-
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
-
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
-
- 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
- C12N2510/00—Genetically modified cells
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Reproductive Health (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Cell Biology (AREA)
- Gynecology & Obstetrics (AREA)
- Pregnancy & Childbirth (AREA)
- Endocrinology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses LncRNA TAB2-AS and application thereof in porcine ovarian granulosa cells. The invention takes lncRNA TAB2-AS AS a research object, treats cells through a DNA methyltransferase inhibitor 5-Aza-CdR, and detects the influence of DNA methylation on transcription; detecting subcellular localization and encoding capacity of EGFP fusion protein vectors of the lncRNA through a nucleoplasm separation experiment; the influence of the lncRNA on the function of the porcine ovarian granulosa cells is detected by constructing an overexpression vector of the lncRNA and synthesizing an antisense oligonucleotide of the lncRNA. The invention verifies that DNA methylation can inhibit transcription of lncRNA TAB 2-AS; illustrating its effect on porcine ovarian granulosa cells: it can promote proliferation of pig ovary granulosa cells, and inhibit apoptosis of pig ovary granulosa cells. The method has good application value for researching the development and locking mechanism of ovarian follicles.
Description
Technical Field
The invention belongs to the technical fields of cell engineering and genetic engineering, and particularly relates to lncRNA TAB2-AS and application thereof in pig ovary granular cells.
Background
The ovary is the most important reproductive organ of female animals, and the main function of the ovary is to produce and discharge mature ovum and secretion sex hormone, and the growth and development condition of the ovary determines the reproductive capacity and the utilization period of the female animals. Follicles are the basic functional units of the ovaries, located in the cortex of the ovaries, and consist of oocytes and surrounding granulosa cells and membranous cells. During mammalian development, most follicular occlusion degenerates, with only a few mature and ovulating. Among them, the basic physiological mechanism of follicular occlusion is granulosa apoptosis, and the key to follicular maturation and ovulation is granulosa proliferation and differentiation.
Long non-coding RNAs (lncRNA) are a class of RNAs that are more than 200 nucleotides in length, have a certain function but have no coding ability; plays a role in regulating gene expression and participates in various biological regulation processes such as physiology, pathology and the like. At present, studies have shown that lncRNA can play an important role in the processes of follicular development, ovulation and the like by influencing proliferation, differentiation, apoptosis and the like of ovarian granulosa cells. Thus, studies of lncRNA have helped to further understand the molecular mechanisms of follicular development and ovulation.
Disclosure of Invention
To solve the problems, the primary objective of the present invention is to provide a long-chain non-coding RNA.
Another object of the invention is to provide the use of the long non-coding RNA described above in porcine ovarian granulosa cells.
In order to achieve the above object, the present invention adopts the following technical scheme:
a long non-coding RNA, named lncRNA TAB2-AS, has a nucleotide sequence shown in SEQ ID NO: 1.
The long-chain non-coding RNA related biological material is any one or a combination of more of the following biological materials:
1) A DNA molecule encoding the long non-coding RNA;
2) An expression cassette comprising the DNA molecule of 1);
3) A recombinant vector comprising the DNA molecule described in 1), or a recombinant vector comprising the expression cassette described in 2);
4) Antisense oligonucleotides that inhibit expression of said long non-coding RNAs;
5) A recombinant cell comprising the DNA molecule of 1), or a recombinant cell comprising the expression cassette of 2), or a recombinant cell comprising the recombinant vector of 3), or a recombinant cell comprising the antisense oligonucleotide of 4).
Further, the DNA molecule of 1) is prepared by: extracting RNA of the porcine ovary granulosa cells, reversely transcribing the RNA into cDNA, and carrying out PCR amplification by taking the cDNA as a template to obtain the target fragment.
Further, the primers used for PCR amplification are as follows:
lncRNA TAB2-AS Forward：5′-CGCGGATCCACTGCCCCAACAGTAAAGCA-3′；
lncRNA TAB2-AS Reverse：5′-CCGCTCGAGGCCCTCATAAGTGCCGATGT-3′；
wherein the black bolded font part is a protective base, and the underlined is an enzyme cutting site.
Further, the recombinant vector in 3) is prepared by the following method: the DNA molecule was ligated to pcDNA3.1 vector digested with restriction enzymes BamHI and XhoI to give a recombinant vector.
Further, the antisense oligonucleotide in 4) has the sequence:
ASO-lncRNA TAB2-AS-2：5′-AAAGGGCTGTAGGTACTGCT-3′。
the long-chain non-coding RNA or the biological material related to the long-chain non-coding RNA is applied to porcine ovarian granulosa cells, and the long-chain non-coding RNA positively regulates any one or two functional phenotypes of cell proliferation and cell apoptosis in an in vitro environment.
Further, exogenous lncRNA TAB2-AS is added to promote cell proliferation and inhibit apoptosis; inhibit lncRNA TAB2-AS expression, inhibit cell proliferation, and promote apoptosis.
The application of the long-chain non-coding RNA or DNA molecule or the expression cassette or the recombinant vector in the preparation of medicaments is characterized in that the medicaments are any one or a combination of more of the following medicaments:
i, a drug for promoting proliferation of porcine ovary granulosa cells;
II, a drug for inhibiting apoptosis of porcine ovary granular cells;
III, a medicine for promoting follicular development.
LncRNA TAB2-AS (Long non-coding RNA TAB2 antisense RNA, temporarily named) is lncRNA obtained by RNA-SEQ against porcine ovarian granulosa cells, and the nucleotide sequence is shown in SEQ ID NO: 1.
The inventors previously treated porcine ovarian granulosa cells with 5-Aza-CdR and found that DNA methylation could regulate expression of lncRNA TAB2-AS by high throughput sequencing of RNAs, affecting porcine ovarian granulosa cell function, but the specific role was not yet clear.
The results of the validation of this study are as follows:
1. effect of DNA methylation on lncRNA TAB2-AS transcription
Pig ovarian granulosa cells were treated with 2. Mu. MDNA methyltransferase inhibitor 5-Aza-CdR (available from MCE company) and the effect of 5-Aza-CdR on the transcription of lncRNA TAB2-AS was detected by qRT-PCR. qRT-PCR results showed that expression levels of lncRNA TAB2-AS were significantly increased after 5-Aza-CdR treatment (FIG. 1).
2. Partial characterization of lncRNA TAB2-AS
After treatment of ovarian granulosa cells with 5-Aza-CdR, RNAs were sequenced at high throughput and analyzed by bioinformatics to give lncRNA TAB2-AS on chromosome 1 (16533983-16534304 bp) of swine with 2 exons (16533983-16534131 bp, 16534211-16534304 bp) (FIG. 2 a).
Subcellular localization of lncRNA TAB2-AS was examined using a nucleoplasm isolation assay, which showed that: lncRNA TAB2-AS was expressed in both the nucleus and cytoplasm of porcine ovarian granulosa cells, with more cytoplasm (b in fig. 2).
The encoding capacity of lncRNTAB2-AS is detected by constructing an overexpression vector of EGFP fusion protein and transfecting the EGFP fusion protein into porcine ovarian granulosa cells. The experimental results showed that lncRNA TAB2-AS had no coding ability (c in fig. 2).
3. Selection of optimal concentrations of lncRNA TAB2-AS overexpression vectors and antisense oligonucleotides
200, 400, 1000 and 1500ng of lncRNA TAB2-AS over-expression vector (pcDNA3.1-lncRNA TAB 2-AS) were transfected into porcine ovarian granulosa cells, respectively, and the expression level of lncRNA TAB2-AS was detected by qRT-PCR after 24 hours (FIG. 3 a). According to the over-expression efficiency and the sum thereof3000 transfection kit, 1000ng of pcDNA3.1-lncRNA TAB2-AS was selected for subsequent experiments.
2 antisense oligonucleotides of lncRNA TAB2-AS (ASO-lncRNA TAB 2-AS) were synthesized, and their interference efficiency was screened and examined. ASO-lncRNA TAB2-AS was transferred to pig ovarian granulosa cells at 50 and 100nM, respectively, and the expression level of lncRNA TAB2-AS was detected by qRT-PCR after 48 hours (b in FIG. 3). And finally, according to the interference efficiency, selecting 100nMASO-lncRNA TAB2-AS-2 with the best interference effect for subsequent experiments.
ASO-lncRNA TAB2-AS-1：5′-GTCAGTATATAGTGTGACCT-3′；
ASO-lncRNA TAB2-AS-2：5′-AAAGGGCTGTAGGTACTGCT-3′。
4. Effect of LncRNA TAB2-AS on proliferation of porcine ovarian granulosa cells
In order to study the influence of lncRNA TAB2-AS on the proliferation of porcine ovarian granulosa cells, pcDNA3.1-lncRNA TAB2-AS and ASO-lncRNA TAB2-AS are respectively transfected into porcine ovarian granulosa cells, and then the proliferation of the granulosa cells is detected by adopting an EdU method. The EdU results (FIGS. 4 a and b) show that overexpression of lncRNA TAB2-AS significantly promotes granulosa cell proliferation; interference with lncRNA TAB2-AS significantly inhibited granulosa cell proliferation.
5. Effect of LncRNA TAB2-AS on apoptosis of porcine ovarian granulosa cells
In order to study the influence of lncRNA TAB2-AS on apoptosis of porcine ovarian granulosa cells, pcDNA3.1-lncRNA TAB2-AS and ASO-lncRNA TAB2-AS are respectively transfected into porcine ovarian granulosa cells, and then an Annexin V-FITC/PI double-staining method is adopted to detect apoptosis of granulosa cells. Annexin V-FITC/PI double-stain results (FIGS. 5 a and b) show that overexpression of lncRNA TAB2-AS inhibits granulosa apoptosis; interfering lncRNA TAB2-AS significantly promotes granulosa apoptosis. Compared with the prior art, the invention has the following advantages and effects:
the invention confirms the influence of lncRNA TAB2-AS on the function of ovary granular cells for the first time. The invention sets forth the influence of the lncRNA TAB2-AS on the function of the pig ovary granular cells by constructing an lncRNA TAB2-AS over-expression vector and synthesizing an antisense oligonucleotide thereof and adopting experimental methods such AS qRT-PCR, edU, annexin V-FITC/PI double-dyeing, flow cytometry and the like. Has good application value for researching ovarian follicular development, follicular locking mechanism and the like.
Drawings
FIG. 1 is a graph showing the effect of qRT-PCR detection of DNA methylation on lncRNA TAB2-AS transcription;
FIG. 2 is a partial feature map of the detection of lncRNA TAB2-AS in a nuclear mass separation experiment or the like; wherein a is a schematic diagram of the relative position of lncRNA TAB2-AS on a pig chromosome; b is a nuclear mass separation experiment for detecting the distribution condition of the lncRNA TAB2-AS in the porcine ovarian granulosa cells; c is the encoding capability verification result of the lncRNA TAB 2-AS;
FIG. 3 is a graph showing the over-expression/interference efficiency of qRT-PCR detection of lncRNA TAB2-AS over-expression vector (pcDNA3.1-lncRNA) and 2 antisense oligonucleotides (ASO-lncRNA); wherein a is the over-expression efficiency of the pcDNA3.1-lncRNA detected by a qRT-PCR method; b is the interference efficiency of detecting ASO-lncRNA by qRT-PCR method;
FIG. 4 is a graph showing the effect of EdU assay for the detection of lncRNA TAB2-AS on porcine ovarian granulosa cell proliferation; wherein a is the influence of overexpression of lncRNA TAB2-AS on proliferation of porcine ovarian granulosa cells; b is the effect of interfering lncRNA TAB2-AS on porcine ovarian granulosa cell proliferation;
FIG. 5 is a graph showing the effect of Annexin V-FITC/PI double-staining method for detecting lncRNA TAB2-AS on apoptosis of porcine ovarian granulosa cells; wherein a is the influence of overexpression of lncRNA TAB2-AS on apoptosis of porcine ovarian granulosa cells; b is the effect of interfering lncRNA TAB2-AS on apoptosis of porcine ovarian granulosa cells.
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. It should be understood that the embodiments described in this specification are only for explaining the present invention, and are not intended to limit the present invention, and parameters, proportions, etc. of the embodiments may be selected according to the circumstances without materially affecting the results. The examples are, unless otherwise indicated, all the reagents and method steps conventional in the art. The reagents and starting materials used in the present invention are commercially available unless otherwise specified.
Example 1: construction of lncRNA TAB2-AS overexpression vector
According to the position and sequence information of the lncRNA TAB2-AS obtained by high-throughput sequencing of RNAs, searching the front and back sequences of the lncRNA TAB2-AS on an NCBI website, and designing an amplification primer of the lncRNA TAB 2-AS; the restriction sites existing on the lncRNA TAB2-AS sequence are searched by Primer5 software analysis, and the two restriction sites of BamH I and XhoI are not found on the lncRNA TAB2-AS sequence, and the restriction sites exist in the pcDNA3.1 vector, so that BamH I and XhoI restriction site sequences are respectively added on the upstream and downstream of the lncRNA TAB2-AS amplification Primer.
The cDNA of the RNA reverse transcription of the porcine ovary granular cells is used AS a template, the TAB2-AS sequence of the lncRNA is amplified, and pcDNA3.1 is subjected to purification recovery, pcDNA3.1 vector connection, transformation, screening and sequencing identification, and then endotoxin-free plasmids (a small amount of endotoxin-free plasmid extraction kit is purchased from Magen company, guangzhou) are extracted, and the obtained product is named pcDNA3.1-lncRNA.
The lncRNA TAB2-AS amplification primers used in the present invention:
lncRNA TAB2-AS Forward：5′-CGCGGATCCACTGCCCCAACAGTAAAGCA-3′(SEQ ID NO.3)；
lncRNA TAB2-AS Reverse：5′-CCGCTCGAGGCCCTCATAAGTGCCGATGT-3′(SEQ ID NO.4)。
note that: the black bolded font is the protecting base, and the underlined is the cleavage site.
The presence of 1 Open Reading Frame (ORF) in the lncRNA TAB2-AS sequence was found by ORF Finder function prediction of NCBI. Connecting an Enhanced Green Fluorescent Protein (EGFP) sequence to pcDNA3.1 to construct a pcDNA3.1-EGFP recombinant vector; deleting an initiation codon (ATG) in the EGFP sequence, and connecting the EGFP sequence to pcDNA3.1 to construct a pcDNA3.1-EGFP Mut recombinant vector; the ORF of lncRNA TAB2-AS was ligated to EGFP sequence (deletion of the initiation codon ATG) and to pcDNA3.1 to construct pcDNA3.1-ORF-EGFP Mut recombinant vector.
The sequence of lncRNA TAB2-AS used in the present invention (SEQ ID No. 1):
CCAACAGTAAAGCATATAAATTTCTCTCCAAAAAAAGGGCTGTAGGTACTGCTATTGTTGCTGGTTTTGAAACTGTACTTTCCCAACAGTAATCTCTGGTGAAAGTTCTGTGGGACACCGGCAGGTAAAGCAAGCTTAACCTTGTTGCTGTCAGTATATAGTGTGACCTACAGACATAAGTCTTGTAATATTGTTTGCAGTTCACTGTGCAGCATCGTATGTCTTCACAGAACATCGGCACTT。
ORF sequence of lncRNA TAB2-AS (SEQ ID NO. 2) used in the present invention:
ATGTTCTGTGAAGACATACGATGCTGCACAGTGAACTGCAAACAATATTACAAGACTTATGTCTGTAGGTCACACTATATACTGACAGCAACAAGGTTAAGCTTGCTTTACCTGCCGGTGTCCCACAGAACTTTCACCAGAGATTACTGTTGGGAAAGTACAGTTTCAAAACCAGCAACAATAGCAGTACCTACAGCCCTTTTTTTGGAGAGAAATTTATATGCTTTACTGTTG。
example 2: culture of porcine ovary granulosa cells
(1) In the invention, pig ovary granulosa cells are purchased from Guangzhou white Yun Oukong Wangji slaughterhouse, healthy and normal ovaries are selected and stored in PBS containing 1% of double antibody (penicillin, streptomycin, the same applies below), and the ovaries are quickly brought back to a laboratory for subsequent treatment;
(2) Washing ovary with PBS containing 1% diabody for several times, soaking in 75% alcohol for 30min, washing with PBS containing 1% diabody for several times, sealing and transferring to cell room;
(3) The ovary is clamped by using forceps, 1-2mL of follicular fluid is sucked by a 1mL disposable sterile syringe into a 15mL sterile centrifuge tube containing 4mL DMEM high-sugar culture medium, the mixture is centrifuged at 1000rpm for 5min at room temperature, and the supernatant is discarded;
(4) Washing the cells with a proper amount of preheated PBS containing 1% of double antibody for 2 times, centrifuging at 1000rpm for 5min at room temperature, and discarding the supernatant;
(5) 3mL of cell complete medium (DMEM high-sugar medium containing 1% diabody and 10% fetal bovine serum) was added, the resuspended cells were gently swirled, and then the cells were inoculated at 75cm 2 Cell culture flask (12 mL of complete cell culture medium was added in advance) and placed at 37℃in 5% CO 2 Culturing in an incubator;
(6) After 48 hours, the growth condition of the porcine ovarian granulosa cells is observed under an inverted microscope, and a subsequent experiment is carried out according to the growth condition.
Example 3: transfection and drug treatment of porcine ovarian granulosa cells
(1) When the confluence of the cultured granulosa cells in example 2 reached 70-90%, the culture medium was discarded, washed 2 times with pre-warmed PBS containing 1% of double antibody, centrifuged at 1000rpm for 5min at room temperature, and the supernatant was discarded;
(2) Adding 5mL trypsin to digest cells, placing the cells into an incubator for 3-5 min, observing that most cells float under a microscope, and immediately adding an equivalent amount of complete culture medium to terminate digestion;
(3) Washing with 1% diabody-containing PBS for 2 times, centrifuging at 1000rpm for 5min at room temperature, and discarding supernatant;
(4) Resuspension the cells with a proper amount of complete culture medium, uniformly inoculating the cells into each well of a cell culture plate, completely culturing to supplement the volume, gently shaking, and standing at 37deg.C in 5% CO 2 Culturing in an incubator;
(5) Observing the cell state after about 24 hours, and carrying out transfection or drug treatment when the cell confluence reaches about 80%;
(6) Transfection: transfection procedure reference Invitrogen company3000 transfection kit instruction, after transfection, continue to 37 ℃ and 5% CO 2 Culturing in an incubator followed by subsequent experiments according to the experimental design, at least 3 replicates per group.
(7) And (3) drug treatment: DNA methyltransferase 5-Aza-CdR (available from MCE Co.) was prepared as a 1mM working solution and porcine ovarian granulosa cells were treated with 2. Mu.M 5-Aza-CdR and placed at 37℃in 5% CO 2 Culturing in an incubator followed by subsequent experiments according to the experimental design, at least 3 replicates per group.
Example 4: nuclear mass separation experiment
The subcellular localization of the lncRNA TAB2-AS is detected by using a nuclear mass separation experiment, and the specific operation steps are AS follows according to the specification of Cytoplasmic & Nuclear RNA Purification Kit of Norgen company:
(1) Preparation of cell components: discarding the culture medium in the 6-hole cell culture plate, washing the cells with a proper amount of PBS for 2 times, and discarding the PBS; 100. Mu.L of precooled Lysis Buffer J was added to the culture plate, gently shaken for several minutes, then placed on ice to lyse the cells, after completion of the Lysis the mixture was transferred to a 1.5mLRNase-free centrifuge tube, centrifuged at maximum speed for 10min, the transfer supernatant (containing cytoplasmic RNA) was placed in a fresh 1.5mL RNase-free centrifuge tube, and the pellet (containing nuclear RNA) was retained in the original 1.5mL centrifuge tube.
(2) Cytoplasmic (or nuclear) RNA binding to the adsorption column: adding 200 mu L (or 400 mu L) Buffer SK into a centrifuge tube filled with supernatant (or sediment), and mixing by vortex; adding 200 mu L of absolute ethyl alcohol, stirring, transferring the mixed solution to an adsorption column sleeved with a collecting pipe, centrifuging at 3500rpm for 1min or more, and discarding the filtrate.
(3) Washing: respectively adding 400 mu LWAsh Solution A into the adsorption column in the step (2), washing the column, centrifuging for 2min, and discarding the filtrate; this step was repeated 2 times. The column was left free for 2min, the collection tube was discarded and the column was placed in a new 1.7mL elution tube.
(4) Eluting: 50. Mu.L of the filtration Buffer was added to the column of (3), centrifuged at 200rpm for 2min and at 14000rpm for 1min, the adsorption column was discarded, and the eluted RNA was stored at-80℃for use.
Example 5: qRT-PCR
qRT-PCR detection in the present invention was performed using a Maxima SYBR Green qPCR Master Mix (2X) kit (available from Thermo Scientific). The experiment adopts a Ct value comparison method to detect the content of the sample genes, and the specific calculation formula is as follows:
relative expression level of gene=2 { (experimental group target gene Ct value-experimental group internal reference gene Ct value-experimental group target gene Ct value-control group internal reference gene Ct value-experimental group internal reference gene Ct value ]
The invention uses GAPDH as reference gene.
The qRT-PCR primers used in the invention are as follows:
qRT-PCR-lncRNA TAB2-AS Forward：5′-GGGCTGTAGGTACTGCTATTGTT-3′(SEQ ID NO.5)；
qRT-PCR-lncRNA TAB2-AS Reverse：5′-GGTCACACTATATACTGACAGCAAC-3′(SEQ ID NO.6)；
qRT-PCR-GAPDH Forward：5′-GGACTCATGACCACGGTCCAT-3′(SEQ ID NO.7)；
qRT-PCR-GAPDH Reverse：5′-TCAGATCCACAACCGACACGT-3′(SEQ ID NO.8)。
the extraction of total RNA of cells is described in the following steps according to the TRIzol operation instruction of Takara company:
(1) After washing the granulosa cells 2 times with PBS, the granulosa cells were directly washed at 10cm 2 TRIzol was added in a ratio of/mL, and the mixture was allowed to stand on ice for 10min to lyse the cells sufficiently, centrifuged at 12000Xg for 5min at 4℃and the supernatant was transferred to a fresh 1.5mL RNase-free tube;
(2) Adding 0.2mL chloroform (1 mL TRIzol), shaking vigorously for 15-30 s, standing on ice for 5min, centrifuging at 4 ℃ and 12000xg for 15min, and transferring the upper water phase into a new 1.5mL RNase-free tube;
(3) Adding 0.5mL of isopropanol (1 mL of TRIzol), mixing, standing on ice for 10min, centrifuging at 12000Xg for 10min at 4deg.C, and discarding the supernatant;
(4) 1mL of 75% ethanol-DEPC water (per 1mL TRIzol) was added to wash RNA, and the RNA was centrifuged at 12000Xg for 5min at 4℃and the supernatant was discarded;
(5) Vacuum drying for 5-10min, and taking care to avoid excessive drying of RNA precipitate;
(6) 15-20. Mu.L of DEPC water was added to dissolve the RNA pellet.
PrimeScript using TaKaRa Corp TM The RT Master Mix (Perfect Real Time) cDNA reverse transcription kit reverse transcribes total RNA.
Example 6: granulosa cell proliferation assay
The invention uses EdU method to detect Cell proliferation, and refers to Cell-Light of Ruibo company TM The EdU Apollo 567In vitro Kit instruction is carried out by the following specific operation steps:
(1) Inoculating a proper amount of cells into a 48-pore plate, and culturing the cells until the fusion degree is 50-80%;
(2) Preparation of 50. Mu. MEdU medium (complete medium: edU solution=1000:1), 200. Mu.L of 50. Mu. MEdU medium was added to each well, and the mixture was placed at 37℃in 5% CO 2 Incubating for 2h in an incubator, and washing the cells for 2 times by PBS (phosphate buffered saline) for 3-5 min each time;
(3) Cell immobilization: 200 mu L of 80% acetone (diluted by PBS) is added into each hole, the cells are incubated for 15 to 30min at room temperature, and the cells are washed by PBS for 2 times and 3 to 5min each time;
(4) Cell permeabilization: adding 200 mu L of penetrating agent (PBS containing 0.5% TritonX-100) into each well to permeabilize the cells for 10min, and washing the cells with PBS for 2 times for 3-5 min each time;
(5) EdU detection: 200. Mu.L of 1X was added to each wellDyeing reaction liquid (prepared in a dark place and used at present), incubating for 30min at room temperature in a dark place, and washing cells for 2 times with PBS (phosphate buffer solution) for 3-5 min each time;
(6) Cell re-permeabilization: adding 200 mu L of penetrating agent (PBS containing 0.5% TritonX-100) into each well to permeabilize the cells for 10min, and washing the cells with PBS for 2 times for 3-5 min each time;
(7) DNA staining: 200 mu L of DAPI reaction solution is added into each hole, and incubated for 30min at room temperature in a dark place;
(8) Fluorescence microscopy and analysis of the results (at least 3 replicates per group).
Example 7: granulosa cell apoptosis assay
The invention uses an Annexin V-FITC/PI double-dyeing technology to detect apoptosis, and refers to an Annexin V-FITC/PI double-dyeing apoptosis detection kit instruction book of Jiangsu Kaiyi company, and the specific operation steps are as follows:
(1) Inoculating a proper amount of cells into a 6-hole plate, culturing the cells until the fusion degree is 50-80%, and washing the cells for 1-2 times by using PBS solution;
(2) Digesting the cells with a proper amount of trypsin without EDTA, adding an equivalent stop solution (DMEM high-sugar culture medium containing 10% fetal calf serum) to stop digestion after digestion is finished, collecting the cells in a 1.5mL centrifuge tube, centrifuging at 1000rpm for 5min at room temperature, and discarding the supernatant;
(3) Washing the cells with PBS for 2 times, centrifuging at 1000rpm for 5min, and discarding the supernatant; cells were gently resuspended in PBS and counted;
(4) Taking 5-10 ten thousand resuspended cells, centrifuging at 1000rpm for 5min, discarding the supernatant, and adding 500 μL of 1 Xannexin V Buffer light and heavy suspension cells;
(5) Adding 5 mu L of Annexin V-FITC and 5 mu L of propidium iodide staining solution, gently mixing, and incubating for 15min at room temperature in a dark place;
(6) Detection was performed immediately using a flow cytometer (at least 3 replicates per group).
Analysis of results
1. Effect of DNA methylation on lncRNA TAB2-AS transcription
After treating porcine ovarian granulosa cells with 2. Mu. MDNA methyltransferase inhibitor 5-Aza-CdR (available from MCE company), the expression level of lncRNA TAB2-AS was detected by qRT-PCR. qRT-PCR results showed that expression levels of lncRNA TAB2-AS were significantly increased after 5-Aza-CdR treatment (FIG. 1).
2. Partial characterization of lncRNA TAB2-AS
After treatment of ovarian granulosa cells with 5-Aza-CdR, RNAs were sequenced at high throughput and analyzed by bioinformatics to give lncRNA TAB2-AS on chromosome 1 (16533983-16534304 bp) with 2 exons (16533983-16534131 bp, 16534211-16534304 bp) on the antisense strand of the 3' non-coding region of the TAB2 gene (FIG. 2 a).
Subcellular localization of lncRNA TAB2-AS was detected using a nucleoplasm separation assay, which showed that: lncRNA TAB2-AS was expressed in both the nucleus and cytoplasm of porcine ovarian granulosa cells, which were more in the cytoplasm (b in fig. 2).
The over-expression vectors (pcDNA3.1, pcDNA3.1-EGFP Mut, pcDNA3.1-ORF-EGFP Mut) of EGFP fusion proteins are constructed and respectively transfected into pig ovary granular cells, and the encoding capacity of lncRNTAB2-AS is detected. The experimental results showed that lncRNA TAB2-AS had no coding ability (c in fig. 2).
3. Selection of optimal concentrations of lncRNA TAB2-AS overexpression vectors and antisense oligonucleotides
200, 400, 1000 and 1500ng of lncRNA TAB2-AS over-expression vector (pcDNA3.1-lncRNA TAB 2-AS) were transfected into porcine ovarian granulosa cells, respectively, and the expression level of lncRNA TAB2-AS was detected by qRT-PCR after 24 hours (FIG. 3 a). According to the over-expression efficiency and the sum thereof3000 transfection kit, 1000ng of pcDNA3.1-lncRNA TAB2-AS was selected for subsequent experiments.
2 antisense oligonucleotides of lncRNA TAB2-AS (ASO-lncRNA TAB 2-AS) were synthesized, and their interference efficiency was screened and examined. ASO-lncRNA TAB2-AS was transferred to pig ovarian granulosa cells at 50 and 100nM, respectively, and the expression level of lncRNA TAB2-AS was detected by qRT-PCR after 48 hours (b in FIG. 3). And finally, according to the interference efficiency, selecting 100nMASO-lncRNATAB2-AS-2 with the best interference effect for subsequent experiments.
ASO-lncRNA TAB2-AS-1：5′-GTCAGTATATAGTGTGACCT-3′(SEQ ID NO.9)；
ASO-lncRNA TAB2-AS-2：5′-AAAGGGCTGTAGGTACTGCT-3′(SEQ ID NO.10)。
4. Effect of LncRNA TAB2-AS on proliferation of porcine ovarian granulosa cells
In order to study the influence of lncRNA TAB2-AS on the proliferation of porcine ovarian granulosa cells, pcDNA3.1-lncRNA TAB2-AS and ASO-lncRNA TAB2-AS are respectively transfected into porcine ovarian granulosa cells, and then the proliferation of the granulosa cells is detected by adopting an EdU method. EdU results indicate that overexpression of lncRNA TAB2-AS can significantly promote granulosa cell proliferation (FIG. 4 a); interference with lncRNA TAB2-AS significantly inhibited granulosa cell proliferation (b in fig. 4).
5. Effect of LncRNA TAB2-AS on apoptosis of porcine ovarian granulosa cells
In order to study the influence of lncRNA TAB2-AS on apoptosis of porcine ovarian granulosa cells, pcDNA3.1-lncRNA TAB2-AS and ASO-lncRNA TAB2-AS are respectively transfected into porcine ovarian granulosa cells, and then an Annexin V-FITC/PI double-staining method is adopted to detect apoptosis of granulosa cells. Annexin V-FITC/PI double-staining results show that overexpression of lncRNA TAB2-AS inhibits granulosa apoptosis (fig. 5 a); interfering lncRNA TAB2-AS significantly promoted granulosa apoptosis (b in fig. 5).
The embodiments described above are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principles of the present invention should be made in the equivalent manner, and are included in the scope of the present invention.
Sequence listing
<110> agricultural university of south China
<120> LncRNA TAB2-AS and its use in porcine ovarian granulosa cells
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 243
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> sequence of lncRNA TAB2-AS
<400> 1
ccaacagtaa agcatataaa tttctctcca aaaaaagggc tgtaggtact gctattgttg 60
ctggttttga aactgtactt tcccaacagt aatctctggt gaaagttctg tgggacaccg 120
gcaggtaaag caagcttaac cttgttgctg tcagtatata gtgtgaccta cagacataag 180
tcttgtaata ttgtttgcag ttcactgtgc agcatcgtat gtcttcacag aacatcggca 240
ctt 243
<210> 2
<211> 234
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> ORF sequence of lncRNA TAB2-AS
<400> 2
atgttctgtg aagacatacg atgctgcaca gtgaactgca aacaatatta caagacttat 60
gtctgtaggt cacactatat actgacagca acaaggttaa gcttgcttta cctgccggtg 120
tcccacagaa ctttcaccag agattactgt tgggaaagta cagtttcaaa accagcaaca 180
atagcagtac ctacagccct ttttttggag agaaatttat atgctttact gttg 234
<210> 3
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> lncRNA TAB2-AS Forward
<400> 3
cgcggatcca ctgccccaac agtaaagca 29
<210> 4
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> lncRNA TAB2-AS Reverse
<400> 4
ccgctcgagg ccctcataag tgccgatg 28
<210> 5
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> qRT-PCR-lncRNA TAB2-AS Forward
<400> 5
gggctgtagg tactgctatt gtt 23
<210> 6
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> qRT-PCR-lncRNA TAB2-AS Reverse
<400> 6
ggtcacacta tatactgaca gcaac 25
<210> 7
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> qRT-PCR-GAPDH Forward
<400> 7
ggactcatga ccacggtcca t 21
<210> 8
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> qRT-PCR-GAPDH Reverse
<400> 8
tcagatccac aaccgacacg t 21
<210> 9
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> ASO-lncRNA TAB2-AS-1
<400> 9
gtcagtatat agtgtgacct 20
<210> 10
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> ASO-lncRNA TAB2-AS-2
<400> 10
aaagggctgt aggtactgct 20
Claims (9)
1. A long non-coding RNA designated lncRNA TAB2-AS, characterized by: the nucleotide sequence is shown as SEQ ID NO: 1.
2. The long-chain non-coding RNA-associated biological material of claim 1, wherein: is any one of the following biological materials:
1) A DNA molecule encoding the long non-coding RNA;
2) An expression cassette comprising the DNA molecule of 1);
3) A recombinant vector comprising the DNA molecule of 1);
4) Antisense oligonucleotides that inhibit expression of said long non-coding RNAs;
the antisense oligonucleotide has the sequence:
ASO-lncRNA TAB2-AS-2：5′- AAAGGGCTGTAGGTACTGCT -3′；
5) A recombinant cell comprising the DNA molecule of 1), or a recombinant cell comprising the antisense oligonucleotide of 4); the recombinant cells are porcine ovary granulosa cells.
3. The long-chain non-coding RNA-associated biological material of claim 2, wherein:
3) Wherein the recombinant vector is a recombinant vector containing the expression cassette described in 2);
5) Wherein the recombinant cell comprising the DNA molecule of 1) is a recombinant cell comprising the expression cassette of 2).
4. The long-chain non-coding RNA-associated biological material of claim 2, wherein:
5) Wherein the recombinant cell containing the DNA molecule of 1) is a recombinant cell containing the recombinant vector of 3).
5. The biomaterial according to any one of claims 2-4, wherein:
1) The DNA molecule is prepared by the following steps: extracting RNA of porcine ovary granulosa cells, reversely transcribing the RNA into cDNA, and carrying out PCR amplification by taking the cDNA as a template to obtain a target fragment;
the primers used for PCR amplification are as follows:
lncRNA TAB2-AS Forward：5′-CGCGGATCCACTGCCCCAACAGTAAAGCA-3′；
lncRNA TAB2-AS Reverse：5′-CCGCTCGAGGCCCTCATAAGTGCCGATGT-3′。
6. the biomaterial according to any one of claims 2-4, wherein:
3) The recombinant vector is prepared by the following steps: the DNA molecule was ligated to pcDNA3.1 vector digested with restriction enzymes BamHI and XhoI to give a recombinant vector.
7. Use of a long non-coding RNA as defined in claim 1 or a biomaterial as defined in any one of claims 2 to 6 for modulating porcine ovarian granulosa cells, characterized in that: in the in-vitro environment, the device comprises a body,
increasing exogenous lncRNA TAB2-AS, promoting cell proliferation, and inhibiting apoptosis; or inhibit lncRNA TAB2-AS expression, inhibit cell proliferation, and promote apoptosis.
8. The use of the long non-coding RNA of claim 1 in the manufacture of a medicament, wherein: the medicine is any one or a combination of the following medicines:
i, a drug for promoting proliferation of porcine ovary granulosa cells;
II, a drug for inhibiting apoptosis of porcine ovary granular cells;
III, a drug for promoting development of pig follicles.
9. Use of a DNA molecule or an expression cassette or a recombinant vector according to any one of claims 2 to 6 for the preparation of a medicament, characterized in that: the medicine is any one or a combination of the following medicines:
i, a drug for promoting proliferation of porcine ovary granulosa cells;
II, a drug for inhibiting apoptosis of porcine ovary granular cells;
III, a drug for promoting development of pig follicles.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210582494.3A CN114934051B (en) | 2022-05-26 | 2022-05-26 | LncRNA TAB2-AS and application thereof in porcine ovarian granulosa cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210582494.3A CN114934051B (en) | 2022-05-26 | 2022-05-26 | LncRNA TAB2-AS and application thereof in porcine ovarian granulosa cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114934051A CN114934051A (en) | 2022-08-23 |
| CN114934051B true CN114934051B (en) | 2023-11-28 |
Family
ID=82865316
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210582494.3A Active CN114934051B (en) | 2022-05-26 | 2022-05-26 | LncRNA TAB2-AS and application thereof in porcine ovarian granulosa cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114934051B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115992135A (en) * | 2022-08-30 | 2023-04-21 | 华南农业大学 | LncRNA IFA and application thereof in porcine ovarian granulosa cells |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111019949A (en) * | 2020-01-02 | 2020-04-17 | 长江大学 | Long-chain RNA Lnc-2193 and application thereof |
| AU2020101084A4 (en) * | 2019-12-05 | 2020-07-23 | Institute Of Animal Science And Veterinary Medicine, Hubei Academy Of Agricultural Sciences | A long non-coding RNA porcine Lnc-000649 and its application |
-
2022
- 2022-05-26 CN CN202210582494.3A patent/CN114934051B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2020101084A4 (en) * | 2019-12-05 | 2020-07-23 | Institute Of Animal Science And Veterinary Medicine, Hubei Academy Of Agricultural Sciences | A long non-coding RNA porcine Lnc-000649 and its application |
| CN111019949A (en) * | 2020-01-02 | 2020-04-17 | 长江大学 | Long-chain RNA Lnc-2193 and application thereof |
Non-Patent Citations (5)
| Title |
|---|
| lncRNA在卵母细胞成熟过程中作用的研究进展;焦亚飞;刘树林;叶升;石德顺;黄奔;;中国畜牧杂志(01);第17-21页 * |
| Long non-coding RNA TCONS_00814106 regulates porcine granulosa cell proliferation and apoptosis by sponging miR-1343;Huiyan Hu等;Molecular and Cellular Endocrinology;第1-9页 * |
| Systematic Analysis of Long Non-Coding RNAs and mRNAs in the Ovaries of Duroc Pigs During Different Follicular Stages Using RNA Sequencing;Yi Liu等;Int. J. Mol. Sci.;第19卷;第1-14页 * |
| TGF-b1 controls porcine granulosa cell states: A miRNA-mRNA network view;Qiqi Li等;Theriogenology;第50-60页 * |
| 非编码RNA在卵泡发育和卵巢疾病中的研究进展;王静;包日强;董麒铭;张春平;;生殖与避孕(02);第135-141页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114934051A (en) | 2022-08-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109750035B (en) | sgRNA for targeting and guiding Cas9 protein to efficiently cleave TCR and B2M gene locus | |
| CN110373416B (en) | Application of RBP1 gene in sow ovarian granulosa cells | |
| CN110467663B (en) | Application of RSPO3 gene in sow ovarian granulosa cells | |
| CN114940992B (en) | Application of lncRNA TAB2-AS regulation TAB2 in pig ovary granular cells | |
| CN114934051B (en) | LncRNA TAB2-AS and application thereof in porcine ovarian granulosa cells | |
| CN109136376B (en) | Application of bladder cancer related cyclic RNA, siRNA and application thereof | |
| CN111718995B (en) | Biomarker for nasopharyngeal carcinoma metastasis diagnosis and/or prognosis evaluation | |
| CN111748559B (en) | Application of CTNNB1 gene in porcine ovary granular cells | |
| CN107557472B (en) | Glioma diagnosis marker circ9:135881633|135883078 and application | |
| CN110317878B (en) | Long-chain non-coding RNA for diagnosis and treatment monitoring of bladder cancer and application thereof | |
| CN111676222A (en) | shRNA for inhibiting Mettl3 gene expression, recombinant adeno-associated virus thereof and application thereof | |
| CN114836424B (en) | lncRNA IFFD and application thereof in porcine ovarian granulosa cells | |
| CN113577259B (en) | Application of composition in preparation of medicines for inhibiting or treating tumors | |
| CN114941010B (en) | Application of TAB2 in pig ovary granular cells | |
| CN111154858B (en) | Long non-coding RNA and application thereof in diagnosis/treatment of preeclampsia | |
| CN111411155B (en) | Application of lncRNA IGFL2-AS1 AS colon cancer diagnosis marker | |
| CN107937523B (en) | Lung cancer diagnosis marker microRNA-3607-3p and application thereof in medicines and diagnosis kit | |
| CN107881237B (en) | Lung cancer diagnosis marker microRNA-4317 and application thereof in medicines and diagnosis kit | |
| CN102965372A (en) | SiRNA interfering GDF9 gene expression and application thereof | |
| CN112626220A (en) | Biomarker and application thereof | |
| CN112143807A (en) | Biomarker and application thereof | |
| CN107184983B (en) | Diagnosis and treatment target for lung adenocarcinoma | |
| LU501763B1 (en) | SMALL INTERFERING RNAs (siRNAs) FOR INTERFERING WITH ZINC FINGER PROTEIN 24 (ZNF24) GENE EXPRESSION AND USE THEREOF IN INHIBITING CELL PROLIFERATION AND MIGRATION | |
| CN111349704B (en) | Diagnostic product and therapeutic composition for liver cancer | |
| CN114875029B (en) | Method for regulating and controlling lncRNA IFFD transcriptional expression in porcine ovarian granulosa cells |
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 |