WO2020181663A1 - 一种长链非编码rna及其应用 - Google Patents

一种长链非编码rna及其应用 Download PDF

Info

Publication number
WO2020181663A1
WO2020181663A1 PCT/CN2019/088310 CN2019088310W WO2020181663A1 WO 2020181663 A1 WO2020181663 A1 WO 2020181663A1 CN 2019088310 W CN2019088310 W CN 2019088310W WO 2020181663 A1 WO2020181663 A1 WO 2020181663A1
Authority
WO
WIPO (PCT)
Prior art keywords
mir
lncrna
loc680254
mirna
cell
Prior art date
Application number
PCT/CN2019/088310
Other languages
English (en)
French (fr)
Inventor
于彬
姚淳
顾晓松
杨宇民
刘炎
吴建成
Original Assignee
南通大学
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by 南通大学 filed Critical 南通大学
Priority to AU2019433418A priority Critical patent/AU2019433418B2/en
Publication of WO2020181663A1 publication Critical patent/WO2020181663A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/08—Drugs for disorders of the urinary system of the prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the invention belongs to the professional field of biomedicine, and relates to a long-chain non-coding RNA and its application.
  • LncRNA Long non-coding RNA
  • LncRNA expression has temporal and spatial specificity, and can regulate gene expression at various levels such as chromatin remodeling, transcription regulation, and post-transcriptional processing.
  • LncRNA is closely related to a variety of diseases. At present, most studies on LncRNA mainly focus on the relationship with cancer and its role as tumor-specific markers, while the research on LncRNA in other physiological activities or diseases is very limited.
  • the present invention utilizes RNA-seq to analyze different LncRNAs in samples at different time points after SD rat sciatic nerve injury to obtain a kind of LncRNA LOC680254 related to Schwann cell proliferation and cell cycle regulation, which has the potential to become a new target for peripheral nerve injury repair.
  • RNA LncRNA LOC680254 A long-chain non-coding RNA LncRNA LOC680254, the cDNA sequence of which is shown in SEQ ID No:1.
  • Another object of the present invention is to provide the aforementioned long-chain non-coding RNA and miR-30d-3p (SEQ ID No: 2), miR-671 (SEQ ID No: 3), miR-3594-3p (SEQ ID No: 4) And miR-3473 (SEQ ID No: 5) as molecular intervention targets in the regulation of cell proliferation, apoptosis and cell cycle.
  • the long non-coding RNA of the present invention can be used as a miRNA molecular sponge to specifically bind miR-30d-3p, miR-671, miR-3594-3p or miR-3473, thereby antagonizing the function of miRNA; miR-30d-3p, miR
  • the inhibited function of -671 can promote the expression of Psrc1 gene; the inhibited function of miRNA-3594-3p and miRNA-3473 can promote the expression of Ska1 gene, thereby regulating cell proliferation, apoptosis and cell cycle.
  • Another object of the present invention is to provide the above-mentioned long-chain non-coding RNA and miR-30d-3p, miR-671, miR-3594-3p and miR-3473 as molecular intervention targets for preparing and treating diseases related to cell proliferation or apoptosis Application in medicine.
  • the above-mentioned diseases include: diseases related to excessive cell proliferation, including tumors, liver fibrosis, pulmonary fibrosis, renal fibrosis, prostatic hypertrophy, essential thrombocythemia, familial polycythemia, rheumatoid arthritis, psoriasis Disease, glomerular interstitial disease, atherosclerosis, etc.
  • Diseases related to cell proliferation defects include nerve cell regeneration, diabetic nephropathy, neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis), aplastic anemia and the like.
  • Diseases related to insufficient apoptosis include tumors and autoimmune diseases caused by T lymphocytes against autoantigens that cannot be effectively eliminated.
  • Cardiovascular diseases such as myocardial ischemia-reperfusion injury, heart failure, neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, etc.), viruses Infection (such as AIDS).
  • cardiovascular diseases such as myocardial ischemia-reperfusion injury, heart failure, neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, etc.), viruses Infection (such as AIDS).
  • long-chain non-coding RNA can be used as a molecular intervention target in preparing and regulating the proliferation of glial cells (Schwann cells) to repair peripheral nerve damage and repair drugs.
  • the present invention uses RNA-seq to analyze different LncRNAs in samples at different time points after SD rat sciatic nerve injury to obtain 30 differentially expressed LncRNAs, wherein the LncRNA LOC680254 of the present invention continues to be up-regulated after nerve injury.
  • qRT-PCR proved that the expression changes of LncRNA and LOC680254 were consistent with the results of RNA-seq.
  • RACE reaction obtains the full-length sequence of LncRNA LOC680254, located at 13q22.
  • Subcellular localization analysis LncRNA LOC680254 is mainly expressed in the cytoplasm of Schwann cells.
  • LncRNA LOC680254 shows that in vitro interference with the expression of LncRNA LOC680254 significantly inhibits Schwann cell proliferation, promotes Schwann cell apoptosis, and blocks the development of Schwann cell cycle.
  • Gene chip analysis and ceRNA prediction combined with Luciferase detection suggest that LncRNA LOC680254 combines with 4 miRNAs to regulate the expression of target genes Psrc1 and Ska1 related to cell proliferation, apoptosis, and cell cycle, among which miR-30d-3p, miR-671 targets To Psrc1 gene; miRNA-3594-3p, miRNA-3473 target Ska1 gene.
  • the research of the present invention suggests that LncRNA LOC680254 may affect the proliferation and cell cycle of Schwann cells by regulating gene expression related to cell cycle, and has the potential to become a new target for peripheral nerve injury repair.
  • Figure 1 shows the expression trend and distribution of LncRNA LOC680254 of the present invention.
  • Figure 1a qRT-PCR detects changes in the expression of LOC680254 in the sciatic nerve tissue after sciatic nerve injury (with GAPDH internal control). Compared with 0d, the expression of LncRNA and LOC680254 in the sciatic nerve after injury continued to increase (**p ⁇ 0.01).
  • Figure 1b qRT-PCR detects the relative expression level of LncRNA and LOC680254 in Schwann cell cytoplasm and nucleus. U6 is used as the internal control in the nucleus and ⁇ -actin is used as the internal control in the cytoplasm. The distribution of LncRNA and LOC680254 in the cytoplasm or nucleus is presented as a percentage of total RNA) .
  • Figure 2 shows the effect of the interfering LncRNA LOC680254 of the present invention on the proliferation, apoptosis and cell cycle of Schwann cells.
  • Figure 2a Edu cell proliferation experiment detects the effect of interfering with LncRNA LOC680254 on Schwann cell proliferation.
  • the lower right bar graph shows the proliferation rate of Schwann cells after siRNA interferes with LncRNA LOC680254.
  • LOC680254 knockout significantly inhibits the proliferation of Schwann cells.
  • Figure 2b Annexin V-TITC/PI staining, flow cytometry to detect the effect of interfering LncRNA LOC680254 on Schwann cell apoptosis.
  • the lower right histogram is a flow cytometric measurement of Schwann cell apoptosis rate statistics in different experimental groups.
  • Figure 2c Transfected siRNA And control 48h later, Tunel detected Schwann cell apoptosis.
  • the lower right histogram is a statistical graph of Schwann cell apoptosis rate of different experimental groups detected by Tunel.
  • Figure 2d Flow cytometric detection of the influence of interference LncRNA LOC680254 on Schwann cell cycle. The figure shows the average percentage of cells in each phase of the cell cycle (*p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001)).
  • Figure 3 shows that the LncRNA LOC680254 of the present invention affects the expression of genes Psrc1 and Ska1 by regulating miR-30d-3p, miR-671, miRNA-3594-3p, and miRNA-3473.
  • Figure 3a qRT-PCR detects the expression of genes Psrc1 and Ska1 after interference with LncRNA LOC680254 in Schwann cells.
  • Figure 3b Luciferase reporter assays detects LncRNA LOC680254 and miR-30d-3p, miR-671 and miRNA-3594-3p, miRNA- The binding of 3473.
  • Figure 3c shows that the LncRNA LOC680254 of the present invention affects the expression of genes Psrc1 and Ska1 by regulating miR-30d-3p, miR-671, miRNA-3594-3p, and miRNA-3473.
  • Luciferase reporter assays to detect the binding of miR-30d-3p, miR-671, miRNA-3594-3p, miRNA-3473 to the 3'UTR of Psrc1 and Ska1, respectively.
  • Figure 3d qRT-PCR detection Psrc1 expression in Schwann cells transfected with miR-30d-3p and miR-671.
  • Figure 3e qRT-PCR detects the expression of Ska1 in Schwann cells transfected with miRNA-3594-3p and miRNA-3473 (**p ⁇ 0.01, ***p ⁇ 0.001)).
  • FIG 4 shows that the LncRNA LOC680254 of the present invention regulates Schwann cell proliferation through Psrc1 and Ska1.
  • FIG 4a Edu cell proliferation assay detects the effect of overexpression of LncRNA and LOC680254 in Schwann cells on the proliferation of Schwann cells.
  • Figure 4b Edu cell proliferation assay detects the effect of interference Psrc1 and Ska1 in Schwann cells on overexpression of LncRNA and LOC680254 on the regulation of Schwann cell proliferation ( *p ⁇ 0.05, **p ⁇ 0.01)).
  • Tm>70°C, base number is about 28nt
  • design 5'-GSP primer for LOC 680254 Take SD rat sciatic nerve tissue, use RNeasy Mini Kit (Qiagen) to extract high-quality RNA, strictly follow the instruction system of SMARTer TM RACE (Clotech) kit, respectively use 5'-CDS primer to complete the synthesis of First-strand cDNA , Get the corresponding 5'-cDNA.
  • the primer LOC680254-5-GSP sequence of 5'-GSP was designed for LncRNA LOC680254 as shown in SEQ ID No: 8, and then 5'-RACE was performed using landing PCR to obtain the 5'end of LOC680254. After sequencing, the result was spliced with the original known sequence to obtain the full-length cDNA sequence of LOC680254 and compared with the rat sequence of the UCSC ( http://genome.ucsc.edu/ ) database, which showed that LncRNA LOC680254 is located in 13q22.
  • RNA in cytoplasm and nucleus were extracted from Schwann cells.
  • the 100 ⁇ l RNA solution obtained above was purified according to the RNA cleanup (Qigen) operation in the RNeasy Mini Kit.
  • the two RNAs were eluted with the same volume of RNase-free water (30 ⁇ l) and named as cytoplasm-RNA and nuclear-RNA respectively. ,Measure the concentration.
  • the reverse transcription kit (Takara RR047A), reverse transcription of equal volumes of cytoplasm-RNA and nucleus-RNA solutions into cDNA, using PrimeScript RT-PCR Kit (Takara) for qRT-PCR.
  • PCR reaction program Stage 1: 95°C 2min, Stage 2 (Cycle: 40): 95°C 15s, 60°C 30s; Stage 3: 95°C 15s, 60°C 1min, 95°C 15s.
  • qRT-PCR detects the expression of LncRNA LOC680254, U6, and ⁇ -actin. Compared with U6, which is mainly expressed in the nucleus and ⁇ -actin, which is mainly expressed in the cytoplasm, the results are shown in Figure 1b. The results indicate that LncRNA LOC680254 is mainly expressed in Cytoplasmic expression
  • the cells are purified. First digest the cells with trypsin and transfer them to a 5ml centrifuge tube, centrifuge (1200rpm, 5min) to remove the supernatant; suspend the cells in a complete medium containing anti-thy1.1 (1:1000), incubate on ice for 2h; 1200rpm for 5min Centrifuge to remove the supernatant, add complement (250 ⁇ l Rabbit complement+750 ⁇ l DMEM), incubate at 37°C for 0.5h; wash with 3ml DMEM three times; plant it in a petri dish coated with PLL; change the medium for 8-12h (containing HRG and Forskolin), after the cells are fused, the purity is more than 95%, and it is used for subsequent experiments.
  • complement 250 ⁇ l Rabbit complement+750 ⁇ l DMEM
  • siRNA final concentration: 100nM
  • miRNA minic purchased from Guangzhou Ruibo Biotechnology Co., Ltd., final concentration: 20nM
  • the siRNA 254-si-1 and 254-si-2 sequences for LncRNA LOC680254 are shown in SEQ ID No: 9 5'CCAGCUGUCCAAGAUCAGA dTdT 3'and SEQ ID No: 10 5'UGUGGUUCCUUCAUGACAA dTdT 3'.
  • EdU labeling Dilute EdU solution (reagent A) with cell culture medium at a ratio of 1000:1 to prepare an appropriate amount of 50 ⁇ M EdU medium; add 100 ⁇ l 50 ⁇ M EdU medium to each well of a 96-well plate in a 37°C incubator, incubate for 24 hours, discard Medium: Wash cells with PBS 1 to 2 times, 5 minutes each time.
  • Cell immobilization add 100 ⁇ L of cell fixative (ie 4% paraformaldehyde in PBS) to each well and incubate at room temperature for 30 minutes, discard the fixative; add 50 ⁇ L of 2mg/mL glycine to each well, incubate for 5 minutes on a decolorizing shaker, discard the glycine Solution; add 100 ⁇ l PBS to each well, wash on a decolorizing shaker for 5 minutes, discard PBS; (enhance) add 100 ⁇ l permeant (0.5% TritonX-100 in PBS) to each well and incubate on a decolorizing shaker for 10 minutes; wash once with PBS for 5 minutes .
  • cell fixative ie 4% paraformaldehyde in PBS
  • DNA staining Dilute reagent F with ddH 2 O at a ratio of 100:1, prepare an appropriate amount of 1X Hoechst 33342 reaction solution, and store in the dark; add 100 ⁇ l 1X Hoechst 33342 reaction solution to each well, and incubate for 30 minutes in a decolorizing shaker, protected from light, at room temperature , Discard the staining reaction solution; add 100 ⁇ l PBS to each well to wash 1 to 3 times; add 100 ⁇ l PBS to each well and take pictures with a fluorescence microscope. Two specific siRNAs (254-si-1, 254-si-2) of LncRNA LOC680254 and siRNA Negative Control (NC) were used to transfect the primary cultured Schwann cells.
  • siRNAs (254-si-1, 254-si-2) of LncRNA LOC680254 and siRNA Negative Control (NC) were used to transfect the primary cultured Schwann cells.
  • the Schwann cells were digested with trypsin and transferred to a 5ml centrifuge. Centrifuge at 1000g for 5 minutes, discard the supernatant, collect the cells, resuspend the cells with an appropriate amount of PBS and count. Take 50,000-100,000 resuspended cells, centrifuge at 1000g for 5 minutes, discard the supernatant, and add 195 ⁇ l Annexin V-FITC binding solution to gently resuspend the cells. Add 5 ⁇ l Annexin V-FITC and mix gently. Add 10 ⁇ l PI (propidium iodide) staining solution and mix gently. Incubate for 10-20 minutes at room temperature (20-25°C) in the dark, and then place in an ice bath.
  • PI propidium iodide
  • Tunel reaction mixture take two tubes (tube 1: enzyme concentration Solution, tube 2: labeling solution) for staining 10 samples and 2 negative control groups, each sample uses 50 ⁇ l Tunel mixture, and each control group uses 50 ⁇ l labeling solution. Take 100 ⁇ l of the labeling solution (tube 2) in the control group. Add the total amount (50 ⁇ l) of the enzyme concentrated solution (tube 1) to the remaining 450 ⁇ l labeling solution in tube 2 to prepare 500 ⁇ l Tunel mixture, and mix well to mix the ingredients thoroughly.
  • Labeling Wash the slides with PBS 3 times, 3 minutes each time, carefully suck up the liquid around the sample; add 50 ⁇ l Tunel mixture to the sample; incubate in a humid box at 37°C for 60 minutes in the dark; wash 3 times with PBS; Hoechst stains the nucleus 30min; mount the slide with anti-fluorescence quenching mounting solution, and detect by fluorescent inverted microscope or laser confocal microscope.
  • LncRNA LOC680254 specific siRNA (254-si-1, 254-si-2) interferes with the expression of LncRNA LOC680254 in Schwann cells, 48h after transfection of siRNA, TUNEL staining is performed according to the above method to detect the apoptosis of Schwann cells.
  • TUNEL staining is performed according to the above method to detect the apoptosis of Schwann cells.
  • Figure 2c the results show that compared with the negative control siRNA Negative Control (NC), the interference of LncRNA LOC680254 significantly promotes the apoptosis of Schwann cells.
  • NC negative control siRNA Negative Control
  • Preparation of cell samples digest Schwann cells with trypsin and transfer the cells to a 5ml centrifuge tube. Centrifuge at about 1000g for 5 minutes to pellet the cells. Discard the supernatant, add 1ml of pre-cooled PBS, resuspend the cells and transfer to a 1.5ml centrifuge tube. Centrifuge at 1000g for 5 minutes to pellet the cells, and carefully discard the supernatant. A small amount of PBS can remain to avoid aspirating cells. Flick the bottom of the centrifuge tube to disperse the cells and prevent them from clumping. Cell fixation: Add 1ml of pre-cooled 70% ethanol to the centrifuge tube, pipette gently to mix, and fix at 4°C for 24 hours.
  • PI staining solution Prepare an appropriate amount of PI staining solution according to the number of samples to be tested. Staining: Add 0.5ml PI staining solution to each tube of cell sample, pipette gently with a pipette tip to fully resuspend the cells, and incubate at 37°C for 30 minutes in the dark.
  • the primary cultured Schwann cells were transfected with LncRNA LOC680254 specific siRNA and Negative control (NC), total RNA was extracted, and the expression profile was analyzed with Agilent expression profiling chip, and 548 genes down-regulated by more than 2 times were screened. According to the full-length sequence of LncRNA LOC680254, Tay Y, etc. (Tay Y, Kats L, Salmena L, et al.
  • qRT-PCR detects the expression of genes Psrc1 and Ska1 after interference with LncRNA and LOC680254 in Schwann cells
  • the primary cultured Schwann cells were transfected with the specific siRNA 254-si-2 of LncRNA LOC680254, 48 hours after transfection of siRNA, the cells were harvested, RNA was extracted, reverse transcription, and qRT-PCR was performed to detect the expression of genes Psrc1 and Ska1.
  • the primer sequence of Psrc1 is shown in SEQ ID No: 11 and 12, and the primer sequence of gene Ska1 is shown in SEQ ID No: 13 and 14.
  • the results are shown in Figure 3a, and the results show that when the expression of LncRNA LOC680254 in Schwann cells is interfered, the expression of Psrc1 and Ska1 is also reduced. It shows that LncRNA Loc680254 can regulate the expression of Psrc1 and Ska1.
  • LncRNA LOC680254 was constructed on pmirGLO vector, and pmirGLO-LOC680254-full was co-transfected with 4 miRNAs (miR-30d-3p, miR-671, miRNA-3594-3p, miRNA-3473) and miRNA in 293FT cells mimic (MC), after cell culture for 24 hours, use the dual luciferase reporter gene system to detect the fluorescence values of Firefly and Renilla according to the above method, and calculate the Relative luciferase (Firefly/Renilla), the result is shown in Figure 3b.
  • qRT-PCR detects the influence of miRNAs on target gene expression
  • miRNAs miR-30d-3p, miR-671, miRNA-3594-3p, miRNA-3473 and miRNA mimic (MC) were transfected respectively. 48h after the miRNA was transfected, the cells were harvested, RNA was extracted, reverse transcription was performed, and the expression of the genes Psrc1 and Ska1 was detected by qRT-PCR using the same method as above. The results of qRT-PCR detection are shown in Figures 3d and 3e. The results show that Psrc1 was significantly down-regulated after transfection with miR-30d-3p and miR-671; Ska1 after transfection with miR-3473 and miR-3594-3p The mRNA level is also significantly down-regulated.
  • miR-30d-3p, miR-671, miRNA-3594-3p, and miRNA-3473 can inhibit the expression of Psrc1 and Ska1 by binding to the 3'-UTR of Psrc1 and Ska1, and LncRNA LOC 680254 can competitively bind to miR- 30d-3p, miR-671, miRNA-3594-3p, miRNA-3473, thereby antagonizing miR-30d-3p, miR-671, miRNA-3594-3p, miRNA-3473 to inhibit the expression of Psrc1 and Skal.
  • Example 4 LncRNA LOC 680254 regulates Schwann cell proliferation through Psrc1 and Ska1
  • the Schwann cells expressing LncRNA LOC 680254 were transfected with Psrc1 and Ska1 specific siRNA sequences (SEQ ID No: 15 5'ggaggagauc cuugaugaa dTdT 3'and SEQ ID No: 16 5'gcaucuauga gcucuguga dTdT 3') and Negativecontrol ( NC), after 48h, perform Edu cell proliferation experiment according to the above method. The results are shown in Figures 4a and 4b.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Veterinary Medicine (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Diabetes (AREA)
  • Neurosurgery (AREA)
  • Neurology (AREA)
  • Hematology (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Cardiology (AREA)
  • Urology & Nephrology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • General Engineering & Computer Science (AREA)
  • Virology (AREA)
  • Psychology (AREA)
  • Hospice & Palliative Care (AREA)
  • Epidemiology (AREA)
  • Rheumatology (AREA)
  • AIDS & HIV (AREA)
  • Biochemistry (AREA)
  • Oncology (AREA)
  • Psychiatry (AREA)
  • Communicable Diseases (AREA)

Abstract

提供了一种与细胞增殖和细胞周期调控有关的LncRNA LOC680254，该LncRNA LOC680254通过利用RNA-seq分析SD大鼠坐骨神经损伤后不同时间点样本中差异的LncRNAs获得，可作为miRNA分子海绵特异性结合miR-30d-3p、miR-671、miR-3594-3p或miR-3473，从而拮抗miRNA的功能，促进Psrc1和Ska1基因的表达，从而促进细胞的增殖。

Description

一种长链非编码RNA及其应用 技术领域
本发明属于生物医学专业领域，涉及一种长链非编码RNA及其应用。
技术背景
长链非编码RNA(LncRNA)是一类长度大于200nt，不具有编码功能的RNA。LncRNA表达具有时空特异性，能在染色质重塑、转录调控以及转录后加工等多种水平调节基因的表达。LncRNA与多种疾病密切相关，目前大部分有关LncRNA的研究主要集中于与癌症的关系以及其作为肿瘤特异性标记物的作用，而LncRNA于其他生理活动或者疾病的研究非常有限。
在多细胞生物体内，各类细胞周期存在非常严密的调控系统，使各类细胞能够根据机体的需要进行增殖或处于静止状态。细胞周期除了受到细胞内某些物质的合成、降解或活化的影响，还受细胞内外各信号的种类、强度以及持续时间的控制。细胞周期的调控是细胞中对不同信号进行整合后依靠细胞内的级联翻译完成的，其中任一环节发生异常均可能导致细胞增殖过度或缺陷，并常常伴有细胞分化异常。细胞增殖过度或缺陷以及细胞凋亡过度与不足与多种疾病有关，例如神经退行性疾病、心血管疾病、免疫性疾病等等。因此研究发现与细胞周期调控有关的LncRNA具有很大的临床应用价值。
发明内容
本发明利用RNA-seq分析SD大鼠坐骨神经损伤后不同时间点样本中差异的LncRNAs获得一种与Schwann细胞的增殖和细胞周期调控有关的LncRNA LOC680254，有潜力成为周围神经损伤修复的新靶点。
本发明的具体技术方案如下：
一种长链非编码RNA LncRNA LOC680254，其cDNA序列如SEQ ID No：1所示。
本发明另一目的在于提供上述长链非编码RNA以及miR-30d-3p(SEQ ID No：2)、miR-671(SEQ ID No：3)、miR-3594-3p(SEQ ID No：4)和miR-3473(SEQ ID No：5)作为分子干预靶点在调控细胞增殖、凋亡和细胞周期中的应用。
本发明所述长链非编码RNA能够作为miRNA分子海绵特异性结合miR-30d-3p、miR-671、miR-3594-3p或miR-3473，从而拮抗miRNA的功能；miR-30d-3p、miR-671功能受到抑制后能够促进Psrc1基因的表达；miRNA-3594-3p、miRNA-3473功能受到抑制后能够促进Ska1基因的表达，从而对细胞增殖、凋亡及细胞周期进行调节。
本发明另一目的在于提供上述长链非编码RNA以及miR-30d-3p、miR-671、miR-3594-3p和miR-3473作为分子干预靶点在制备治疗与细胞增殖或凋亡有关疾病的药物中的应用。
上述疾病包括：与细胞增殖过度有关的疾病包括肿瘤、肝纤维化、肺纤维化、肾纤维化、前列腺肥大，原发性血小板增多症、家族性红细胞增多症、类风湿性关节炎、银屑病、肾小球间质性病变、动脉粥样硬化等。
与细胞增殖缺陷有关的疾病包括神经细胞再生、糖尿病肾病、神经退行性疾病(阿尔茨海默病、帕金森病、亨廷顿病、多发性硬化症)、再生障碍性贫血等。
与细胞凋亡不足有关的疾病包括肿瘤以及由于针对自身抗原的T淋巴细胞不能有效予以清除引起的自身免疫疾病等。
与细胞凋亡过度有关的疾病包括心血管疾病如心肌缺血-再灌注损伤、心力衰竭、神经退行性疾病(阿尔茨海默病、帕金森病、亨廷顿病、多发性硬化症等)、病毒感染(如艾滋病)。本发明一项具体的研究应用，长链非编码RNA可以作为分子干预靶点在制备调控神经胶质细胞(Schwann细胞)的增殖进而修复外周神经损伤修复药物中的应用。
本发明利用RNA-seq分析SD大鼠坐骨神经损伤后不同时间点样本中差异的LncRNAs得到30个差异表达的LncRNAs，其中本发明所述LncRNA LOC680254在神经损伤后持续表达上调。qRT-PCR证明LncRNA LOC680254表达变化与RNA-seq结果一致。RACE反应得到LncRNA LOC680254的全长序列，位于13q22。亚细胞定位分析LncRNA LOC680254主要表达于Schwann细胞的细胞质中。
本发明研究表明体外干扰LncRNA LOC680254的表达，显著抑制Schwann细胞增殖、促进Schwann细胞的凋亡，并且阻滞了Schwann细胞周期的发展。基因芯片分析与ceRNA预测结合Luciferase检测提示LncRNA LOC680254与4个miRNAs相结合，调节与细胞增殖、凋亡、细胞周期相关的靶基因Psrc1和Ska1的表达，其中miR-30d-3p、miR-671靶向Psrc1基因；miRNA-3594-3p、miRNA-3473靶向Ska1基因。本发明研究提示LncRNA LOC680254有可能通过调节与细胞周期有关的基因表达影响Schwann细胞的增殖和细胞周期，有潜力成为周围神经损伤修复的新靶点。
附图说明
图1为本发明所述的LncRNA LOC680254的表达趋势及分布。(图1a.qRT-PCR检测坐骨神经损伤后坐骨神经组织中LOC680254的表达变化(以GAPDH内参)。相比于0d，损伤后坐骨神经中的LncRNA LOC680254持续表达上调(**p<0.01)。图1b.qRT-PCR检测LncRNA LOC680254在Schwann细胞细胞质与细胞核中的相对表达水平。U6作为细胞核里内参，β -actin作为细胞质内参。LncRNA LOC680254在细胞质或是细胞核中的分布用其占总RNA的百分比呈现)。
图2为本发明所述的干扰LncRNA LOC680254对Schwann细胞增殖、凋亡及细胞周期的影响。(图2a.Edu细胞增殖实验检测干扰LncRNA LOC680254对Schwann细胞增殖的影响。右下侧柱状图为siRNA干扰LncRNA LOC680254后Schwann细胞的增殖率，LOC680254敲除后显著抑制Schwann细胞的增殖。图2b.Annexin V-TITC/PI染色，流式细胞技术检测干扰LncRNA LOC680254对Schwann细胞凋亡的影响。右下侧柱状图为流式检测不同实验组Schwann细胞凋亡率统计图。图2c.转染siRNA及对照48h后，Tunel检测Schwann细胞的凋亡情况。右下侧柱状图为Tunel检测不同实验组Schwann细胞凋亡率统计图。图2d.通过流式检测干扰LncRNA LOC680254对Schwann细胞周期的影响。图中所示为细胞周期各时期细胞平均百分比(*p<0.05，**p<0.01，***p<0.001))。
图3为本发明所述的LncRNA LOC680254通过调节miR-30d-3p、miR-671及miRNA-3594-3p、miRNA-3473影响基因Psrc1和Ska1的表达。(图3a.qRT-PCR检测Schwann细胞中干扰LncRNA LOC680254后基因Psrc1和Ska1的表达。图3b.Luciferase reporter assays检测LncRNA LOC680254与miR-30d-3p、miR-671及miRNA-3594-3p、miRNA-3473的结合情况。图3c.Luciferase reporter assays检测miR-30d-3p、miR-671及miRNA-3594-3p、miRNA-3473分别Psrc1和Ska1的3’UTR的结合情况。图3d.qRT-PCR检测Schwann细胞中转染miR-30d-3p、miR-671后Psrc1的表达情况。图3e.qRT-PCR检测Schwann细胞中转染miRNA-3594-3p、miRNA-3473后Ska1的表达情况(**p<0.01，***p<0.001))。
图4为本发明所述的LncRNA LOC680254通过Psrc1和Ska1调节Schwann细胞增殖。(图4a.Edu细胞增殖实验检测Schwann细胞中过表达LncRNA LOC680254对Schwann细胞增殖的影响。图4b.Edu细胞增殖实验检测Schwann细胞中干扰Psrc1和Ska1对过表达LncRNA LOC680254调节Schwann细胞增殖的影响(*p<0.05，**p<0.01))。
具体实施方式
以下通过实施例说明本发明的具体步骤，但不受实施例限制。
在本发明中所使用的术语，除非另有说明，一般具有本领域普通技术人员通常理解的含义。
下面结合具体实施例并参照数据进一步详细描述本发明。应理解，这些实施例只是为了举例说明本发明，而非以任何方式限制本发明的范围。
在以下实施例中，未详细描述的各种过程和方法是本领域中公知的常规方法。
下面结合具体实施例对本发明进一步说明。
实施例1考察LncRNA LOC680254的表达及分布特性
1、坐骨神经组织RNA提取及qRT-PCR
取大鼠0d、1d、4d、7d、14d坐骨神经夹伤近端的组织按
Figure PCTCN2019088310-appb-000001
Reagent(Invitrogen)说明书提取组织RNA，逆转录后，采用
Figure PCTCN2019088310-appb-000002
PrimeScript RT-PCR Kit(Takara)进行qRT-PCR，操作按试剂盒说明书进行(以GAPDH作为内参),PCR仪反应程序：Stage 1：95℃2min，Stage 2(Cycle：40)：95℃15s，60℃30s；Stage 3：95℃15s，60℃1min，95℃15s。LncRNA LOC680254引物序列如SEQ ID No：6-7所示。qRT-PCR结果如图1a所示，结果显示与0d相比，损伤的坐骨神经组织中的LncRNA LOC680254表达持续上调。
2、快速扩增cDNA末端(RACE)
根据SMARTer TMRACE(Clotech)说明书要求，Tm>70℃，碱基数在28nt左右，针对LOC 680254设计5’-GSP的引物。取SD大鼠坐骨神经组织，使用RNeasy Mini Kit试剂盒(Qiagen)提取高品质RNA，严格按照SMARTer TMRACE(Clotech)试剂盒的说明书体系，分别进行用5’-CDS primer完成First-strand cDNA的合成，得到相应的5’-cDNA。根据说明书要求，针对LncRNA LOC680254设计5’-GSP的引物LOC680254-5-GSP序列如SEQ ID No：8所示，随后使用降落PCR进行5’-RACE，获取LOC680254的5’末端。测序完成后，将结果和原来已知序列进行拼接，获取LOC680254的cDNA全长序列并与UCSC( http://genome.ucsc.edu/)数据库的大鼠序列进行比对分析，表明LncRNA LOC680254位于13q22。
3、qRT-PCR检测LncRNA的亚细胞定位
按照PARIS TMKit(Ambion公司)，分别从Schwann细胞中提取等份的细胞质与细胞核中的RNA。将上述获取的100μl RNA溶液，按照RNeasy Mini Kit中RNA cleanup(Qigen)操作，纯化RNA，两份RNA用相同体积的RNase-free水洗脱(30μl)，分别命名为细胞质-RNA、细胞核-RNA，测浓度。按照逆转录试剂盒(Takara RR047A)，将等体积细胞质-RNA和细胞核-RNA溶液逆转录为cDNA，采用
Figure PCTCN2019088310-appb-000003
PrimeScript RT-PCR Kit(Takara)进行qRT-PCR。PCR仪反应程序：Stage 1：95℃2min，Stage 2(Cycle：40)：95℃15s，60℃30s；Stage 3：95℃15s，60℃1min，95℃15s。qRT-PCR检测LncRNA LOC680254、U6、β-actin的表达，与主要在细胞核中表达的U6，主要在细胞质中表达的β-actin的相比，结果如图1b所示，结果表明LncRNA LOC680254主要在细胞质中表达
实施例2考察干扰LncRNA LOC680254表达对Schwann细胞增殖、凋亡及细胞周期的影响
1、原代Schwann细胞的培养
取新生1d的SD大鼠的坐骨神经若干只、加入1ml 3mg/ml的胶原酶，剪碎组织、37℃消化30min；室温离心1200rpm，5min，弃除胶原酶，加入1ml胰酶，37℃消化10min；加入3ml完全培养基终止消化，过滤筛网，离心去上清(1200rpm，5min)，再加入3ml完全培养基洗2遍；将细胞种在PLL包被好的培养皿中培养(5％CO 2，37℃)；第二天，换成含有Arac(10μM)的完全培养基，抑制成纤维细胞的快速增殖；第四天，换成含有HRG(50ng/ml)和Forskolin(2μM)的完全培养基刺激Schwann细胞快速生长。待细胞融合后，对细胞进行纯化。先将细胞胰酶消化并转移到5ml离心管中、离心(1200rpm,5min)去除上清；用含有anti-thy1.1(1∶1000)的完全培养基悬浮细胞，冰上孵育2h；1200rpm 5min离心去上清，加入补体(250μl Rabbit complement+750μl DMEM)，37℃，孵育0.5h；3ml DMEM洗三遍；种到PLL包被好的培养皿中；8-12h换液培养(含有HRG和Forskolin)，待细胞融合，纯度达95％以上，用于后续实验。
2、Schwann细胞转染
小分子RNA转染：本实验中，使用Lipofectamine TMRNAiMAX将siRNA(终浓度：100nM)、miRNA minic(购自于广州市锐博生物科技有限公司，终浓度：20nM)转入Schwann细胞，第二天换成完全培养基，根据需要进行后续实验。针对LncRNA LOC680254的siRNA 254-si-1和254-si-2序列如SEQ ID No：9 5’CCAGCUGUCCAAGAUCAGA dTdT 3’和SEQ ID No：10 5’UGUGGUUCCUUCAUGACAA dTdT 3’所示。
质粒DNA转染：先按要求接种细胞，待Schwann细胞密度达到85％以上时，使用Lipofectamine TM3000，按照lip3000：质粒＝2μl：1μg的浓度比，均匀滴加到细胞培养液中，轻轻混匀，4～6h后换成新的完全培养基，第二天再次换成新的完全培养基，根据需要进行后续实验。
3、Edu细胞增殖实验
EdU标记：用细胞培养基按1000：1的比例稀释EdU溶液(试剂A)，配制适量的50μM EdU培养基；96孔板每孔加入100μl 50μM EdU培养基37℃培养箱，孵育24小时，弃培养基；PBS清洗细胞1～2次，每次5分钟。细胞固定化：每孔加入100μL细胞固定液(即含4％多聚甲醛的PBS)室温孵育30分钟，弃固定液；每孔加入50μL 2mg/mL甘氨酸，脱色摇床孵育5分钟后，弃甘氨酸溶液；每孔加入100μl PBS，脱色摇床清洗5分钟，弃PBS；(加强)每孔加入100μl渗透剂(0.5％TritonX-100的PBS)脱色摇床孵育10分钟；PBS清洗1次，5分钟。Apollo染色：每孔加入100μl的1X
Figure PCTCN2019088310-appb-000004
染色反应液，避光、室温、脱色摇床孵育30分钟后，弃染色反应液；加入100μl渗透剂(0.5％TritonX-100的PBS)脱 色摇床清洗2～3次，每次10分钟，弃渗透剂；(加强)每孔每次加入100μl甲醇清洗1～2次，每次5分钟；PBS清洗1次，每次5分钟。DNA染色：用ddH 2O按100：1的比例稀释试剂F，制备适量1X Hoechst33342反应液，避光保存；每孔加入100μl 1X Hoechst 33342反应液，避光、室温、脱色摇床孵育30分钟后，弃染色反应液；每孔每次加入100μl PBS清洗1～3次；每孔加入100μl PBS荧光显微镜拍照。用LncRNA LOC680254的2个特异性的siRNA(254-si-1、254-si-2)与siRNA Negative Control(NC)转染原代培养的Schwann细胞，在转染siRNA 48h后，按上述方法进行EdU标记，Edu实验结果如图2a所示。结果表明：与对照组相比，LncRNA LOC680254的敲除，显著抑制了Schwann细胞的增殖。
4、Annexin V-FITC细胞凋亡检测
用胰酶将Schwann细胞消化下来，转移至5ml离心中。1000g离心5分钟，弃上清，收集细胞，用适量PBS重悬细胞并计数。取5-10万重悬的细胞，1000g离心5分钟，弃上清，加入195μl Annexin V-FITC结合液轻轻重悬细胞。加入5μl Annexin V-FITC，轻轻混匀。加入10μl PI(碘化丙啶)染色液，轻轻混匀。室温(20-25℃)避光孵育10-20分钟，随后置于冰浴中。可以使用铝箔进行避光。孵育过程中可以重悬细胞2-3次以改善染色效果。随即进行流式细胞仪检测，Annexin V-FITC为绿色荧光，碘化丙啶(PI)为红色荧光。通过LncRNA LOC680254特异性的siRNA(254-si-1、254-si-2)干扰Schwann细胞中LncRNA LOC680254的表达，在转染siRNA后48h，按上述方法进行流式细胞技术检测Schwann细胞的凋亡情况，流式结果如图2b所示，结果表明与阴性对照组siRNA Negative Control(NC)相比，干扰LncRNA LOC680254显著促进Schwann细胞的凋亡。
5、TUNEL染色(TMR Red)
在15-25℃下用新鲜的固定液固定风干的Schwann细胞1h；PBS清洗30min；渗透液冰浴(2-8℃)2min；Tunel反应混合物的准备：取两个管(管1：酶浓缩溶液，管2：标记溶液)为10个样本和2个阴性对照组进行染色，每个样本使用50μl Tunel混合液，每个对照组使用50μl标记溶液。取100μl标记溶液(管2)于对照组。将酶浓缩溶液(管1)的总量(50μl)添加到管2中剩余的450μl标记溶液中，以配成500μlTunel混合液，混匀，以使各成分充分混合。标记：用PBS清洗玻片3次，每次3min，小心吸干样品周围的液体；添加50μlTunel混合液到样本中；湿盒中37℃避光孵育60分钟；用PBS清洗3次；Hoechst染核30min；抗荧光淬灭封片液封片，荧光倒置显微镜或是激光共聚焦显微镜检测。通过LncRNA LOC680254特异性的siRNA(254-si-1、254-si-2)干扰Schwann细胞中LncRNA LOC680254的表达，在转染siRNA后48h，按上述方法进行TUNEL染色检测Schwann细胞的凋亡情况如图2c所 示，结果表明，与阴性对照组siRNA Negative Control(NC)相比，干扰LncRNA LOC680254显著促进Schwann细胞的凋亡。
6、流式检测细胞周期
细胞样品的准备：用胰酶消化Schwann细胞，并将细胞转移至5ml离心管中。1000g左右离心5分钟，沉淀细胞。弃上清，加入1ml预冷的PBS，重悬细胞并转移到1.5ml离心管内。1000g离心5min，沉淀细胞，小心弃除上清。可以残留少量PBS，避免吸走细胞。轻弹离心管底，分散细胞，避免细胞聚集成团。细胞固定：向离心管中加入1ml预冷的70％乙醇，用枪头轻轻吹打混匀，4℃固定24小时。1000g离心5分钟，沉淀细胞。弃上清，加入1ml预冷的PBS，重悬细胞。1000g离心5min，沉淀细胞，弃上清，可以残留少量PBS，并轻弹管底以分散细胞，避免细胞成团。PI染色液的配制：根据待检测样品的数量配制适量的PI染色液。染色：每管细胞样品中加入0.5ml PI染色液，用枪头轻轻吹打，充分重悬细胞，37℃避光温浴30分钟。4℃或冰浴避光存放。24小时内完成流式检测。流式检测和分析：用流式细胞仪在激发波长488nm波长处检测红色荧光，同时检测光散射情况。采用适当分析软件进行细胞DNA含量分析和光散射分析。在Schwann细胞中转染LncRNA LOC680254特异性siRNA(254-si-1、254-si-2)以及阴性对照siRNA Negative Control(NC)，48h后按上述方法进行流式检测细胞周期。结果如图2d所示，结果表明，与对照组相比，干扰LncRNA LOC680254，S期的细胞数显著减少。
实施例3考察LncRNA LOC680254通过调节miR-30d-3p、miR-671及miRNA-3594-3p、miRNA-3473影响基因Psrc1和Ska1的表达
1、靶基因筛选
对原代培养的Schwann细胞转染LncRNA LOC680254特异性siRNA与Negative control(NC)，提取总RNA，用Agilent表达谱芯片进行表达谱分析，筛选下调2倍以上的548个基因。依据LncRNA LOC680254的全长序列，用Tay Y等(Tay Y,Kats L,Salmena L,et al.Coding-independent regulation of the tumor suppressor PTEN by competing endogenous mRNAs[J].Cell,2011,147(2):344-357.)建立的生物信息学预测ceRNA的方法，挑选出191个LncRNA LOC680254可能以ceRNA方式调节的靶基因及miRNA。这191个靶基因中下调3倍以上的基因有54个，miRNAs结合位点数不小于2个的基因有82个。两者取交集得到20个基因，其中与细胞增殖和凋亡相关的有12个基因。进一步通过qRT-PCR和双荧光素酶报告检测确认Psrc1和Ska1这两个靶基因。
2、qRT-PCR检测Schwann细胞中干扰LncRNA LOC680254后基因Psrc1和Ska1的表达
用LncRNA LOC680254的特异性siRNA 254-si-2转染原代培养的Schwann细胞，在转染siRNA 48h后，收细胞，提RNA，逆转录，进行qRT-PCR检测基因Psrc1和Ska1的表达，基因Psrc1的引物序列如SEQ ID No：11和12所示，基因Ska1的引物序列如SEQ ID No：13和14所示。结果如图3a所示，结果表明干扰Schwann细胞中LncRNA LOC680254表达时，Psrc1、Ska1的表达也降低。说明LncRNA Loc680254可以调控Psrc1、Ska1的表达。
3、双荧光素酶报告检测
使用
Figure PCTCN2019088310-appb-000005
Reporter Assay System(Promega)试剂盒进行检测。293FT细胞转染后继续培养24h，观察细胞状态，用预冷的PBS洗一次；加入100μl 1×PLB，将培养板摇床室温剧烈摇晃15min；每个孔吹打5次，将裂解液转移EP管中；4℃离心，13000rpm，5min；在96孔板中加入20μl LABII，再加入20μl细胞裂解液，每孔吹打5次，操作均一，放入酶标仪上使用软件Gene5检测Firefly luciferase(Firefly)的荧光强度；再加入20μl Stop&Glo，酶标仪检测Renilla luciferase(Renilla)荧光强度；将两荧光相比(relative luciferase)。实验每次做3个复孔，进行3次以上实验重复，统计分析结果。将LncRNA LOC680254的全长构建到pmirGLO载体上，在293FT细胞中共转染pmirGLO-LOC680254-full与4个miRNAs(miR-30d-3p、miR-671、miRNA-3594-3p、miRNA-3473)及miRNA mimic(MC)，细胞培养24h后，按上述方法，利用双荧光素酶报告基因系统，检测Firefly与Renilla的荧光值，并计算Relative luciferase(Firefly/Renilla)，结果如图3b所示。结果表明：与MC相比，转染了miRNAs的实验组，标准化的荧光素酶活性显著降低，说明LncRNA LOC680254可能与这4个miRNAs都有一定程度的结合。接着又通过luciferase实验，检测上述4个miRNAs与对其应靶基因Psrc1和Ska1的3’-UTR的结合情况，结果如图3c所示。结果表明，与MC相比，转染miRNAs后，荧光素酶活性都出现显著性下调，miR-30d-3p、miR-671可以与Psrc1结合；miR-3594-5p、miR-3473可以与Ska1结合。
4、qRT-PCR检测miRNAs对靶基因表达的影响
在原代培养的Schwann细胞中，分别转染miRNAs(miR-30d-3p、miR-671、miRNA-3594-3p、miRNA-3473)及miRNA mimic(MC)。转染miRNA后48h，收细胞，提RNA，逆转录，同上述方法进行qRT-PCR检测基因Psrc1和Ska1的表达。qRT-PCR检测结果如图3d和3e所示，结果表明，转染了miR-30d-3p，miR-671后Psrc1在mRNA水平显著下调；转染了miR-3473，miR-3594-3p后Ska1在mRNA水平也显著下调。这说明miR-30d-3p、miR-671、miRNA-3594-3p、miRNA-3473可以通过与Psrc1和Ska1的3’-UTR结合，抑制Psrc1和Ska1的表达，LncRNA LOC 680254可以竞争性结合miR-30d-3p、miR-671、miRNA-3594-3p、 miRNA-3473，从而拮抗miR-30d-3p、miR-671、miRNA-3594-3p、miRNA-3473对Psrc1和Ska1表达的抑制。
实施例4 LncRNA LOC 680254通过Psrc1和Ska1调节Schwann细胞增殖
对原代培养的Schwann细胞感染包装有LncRNA LOC 680254全长的慢病毒(LV-254，对照病毒：LV-con)(用Enhanced infection solution(Eni.S)稀释病毒，按照MOI＝20感染)，在Schwann细胞中过表达LncRNA LOC 680254，12h后换回常规培养基，继续培养。再过表达LncRNA LOC 680254的Schwann细胞中转染Psrc1和Ska1特异性siRNA序列(SEQ ID No:15 5’ggaggagauc cuugaugaa dTdT 3’和SEQ ID No:16 5’gcaucuauga gcucuguga dTdT 3’)与Negative control(NC)，48h后按上述方法进行Edu细胞增殖实验。结果如图4a和4b所示。结果表明Schwann细胞中过表达LncRNA LOC 680254后，Schwann细胞增殖明显增加(图4a)，而干扰Psrc1或Ska1的表达，能拮抗LncRNA LOC 680254对Schwann细胞增殖的影响，表明LncRNA LOC 680254通过Psrc1和Ska1调节Schwann细胞增殖(图4b)。

Claims (7)

  1. 一种长链非编码RNA，其特征在于其cDNA序列如SEQ ID No：1所示。
  2. 如权利要求1所述的长链非编码RNA以及miR-30d-3p、miR-671、miR-3594-3p和miR-3473作为分子干预靶点在调控细胞增殖、凋亡和细胞周期中的应用。
  3. 如权利要求2所述的应用，其特征在于所述长链非编码RNA作为miRNA分子海绵特异性结合miR-30d-3p、miR-671、miR-3594-3p或miR-3473，从而抑制miRNA的功能。
  4. 如权利要求3所述的应用，其特征在于所述miR-30d-3p、miR-671功能受到抑制后能够促进Psrc1基因的表达；miRNA-3594-3p、miRNA-3473功能受到抑制后能够促进Ska1基因的表达，从而对细胞增殖、凋亡及细胞周期进行调节。
  5. 如权利要求2所述的应用，其特征在于所述长链非编码RNA以及miR-30d-3p、miR-671、miR-3594-3p和miR-3473作为分子干预靶点在制备治疗与细胞增殖或凋亡有关疾病的药物中的应用。
  6. 如权利要求5所述的应用，其特征在于所述与细胞增殖或凋亡有关疾病包括神经损伤、神经细胞再生、肿瘤、肝纤维化、肺纤维化、肾纤维化、前列腺肥大，原发性血小板增多症、家族性红细胞增多症、类风湿性关节炎、银屑病、肾小球间质性病变、动脉粥样硬化、糖尿病肾病、神经退行性疾病、再生障碍性贫血、由于针对自身抗原的T淋巴细胞不能有效予以清除引起的自身免疫疾病、心肌缺血-再灌注损伤、心力衰竭。
  7. 如权利要求6所述的应用，其特征在于长链非编码RNA作为分子干预靶点在制备调控神经胶质细胞的增殖进而修复外周神经损伤修复药物中的应用。
PCT/CN2019/088310 2019-03-13 2019-05-24 一种长链非编码rna及其应用 WO2020181663A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2019433418A AU2019433418B2 (en) 2019-03-13 2019-05-24 Long-chain non-coding RNA and use thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910187711.7 2019-03-13
CN201910187711.7A CN109897853B (zh) 2019-03-13 2019-03-13 一种长链非编码rna及其应用

Publications (1)

Publication Number Publication Date
WO2020181663A1 true WO2020181663A1 (zh) 2020-09-17

Family

ID=66952163

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/088310 WO2020181663A1 (zh) 2019-03-13 2019-05-24 一种长链非编码rna及其应用

Country Status (3)

Country Link
CN (1) CN109897853B (zh)
AU (1) AU2019433418B2 (zh)
WO (1) WO2020181663A1 (zh)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113930425A (zh) * 2021-09-01 2022-01-14 青岛大学附属医院 调控1型糖尿病cd4+t细胞分化和增殖的生物标记及应用
CN115927321A (zh) * 2022-08-30 2023-04-07 华南农业大学 LncRNA IFA吸附miR-26a在猪卵巢颗粒细胞中的应用
CN116970610A (zh) * 2023-09-21 2023-10-31 浙江大学海南研究院 一种chi-miR-335-5p的新用途及其调控靶基因DKK1表达的方法

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110760583B (zh) * 2019-10-21 2023-03-28 南通大学 一种长链非编码rna bc088259及其应用
CN110819630B (zh) * 2019-10-25 2023-04-07 南通大学 环状RNA circ-01477及其应用
CN111118143B (zh) * 2020-01-16 2020-09-15 西安市红会医院 检测及靶向rp11-754b17.1的试剂及其在关节炎中的应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103316359B (zh) * 2013-07-11 2014-10-15 南京医科大学第二附属医院 长链非编码rna在制备治疗非小细胞肺癌药物中的应用
CN104059887B (zh) * 2014-06-12 2016-08-17 上海交通大学医学院附属瑞金医院 长链非编码RNA Ovol2-AS的应用

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE NUCLEOTIDE 31 July 2016 (2016-07-31), ANONYMOUS: "Rattus norvegicus hypothetical protein LOC680254 (LOC680254), long non-coding RNA", XP055738751, retrieved from NCBI Database accession no. NR_ 027983 *
KE WANG, CHEN MINGWEI, WU WEI: "Analysis of microRNA (miRNA) expression profiles reveals 11 key biomarkers associated with non-small cell lung cancer", WORLD JOURNAL OF SURGICAL ONCOLOGY, vol. 15, no. 175, 19 September 2017 (2017-09-19), XP055738741, ISSN: 1477-7819 *
YAO FANG, CHEN HAI, HU YI, LI QIAN, HU ZHIQIANG, MA TENGFEI, MAO XUHU: "Burkholderia pseudomallei-derived miR-3473 enhances NF-KB via targeting TRAF3 and is associated with different inflammatory responses compared to Burkholderia thiilnndensis in murine macrophages", BMC MICROBIOL., vol. 16, no. 283, 28 November 2016 (2016-11-28), pages 1 - 12, XP055738736, ISSN: 1471-2180 *
YU , YIN ET AL.: "miR-671 promotes prostate cancer cell proliferation by targeting tumor", EUROPEAN JOURNAL OF PHARMACOLOGY, vol. 823, 31 January 2018 (2018-01-31), XP085348197, ISSN: 0014-2999, DOI: 10.1016/j.ejphar.2018.01.016 *
ZHANG, LIANG ET AL.: "Study on Differential Expression of miRNA in Serum of Rats after Renal Injury Induced by Aristolochic Acid I", CHINESE TRADITIONAL AND HERBAL DRUGS, vol. 47, no. 11, 30 June 2016 (2016-06-30), ISSN: 0253-2670 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113930425A (zh) * 2021-09-01 2022-01-14 青岛大学附属医院 调控1型糖尿病cd4+t细胞分化和增殖的生物标记及应用
CN115927321A (zh) * 2022-08-30 2023-04-07 华南农业大学 LncRNA IFA吸附miR-26a在猪卵巢颗粒细胞中的应用
CN116970610A (zh) * 2023-09-21 2023-10-31 浙江大学海南研究院 一种chi-miR-335-5p的新用途及其调控靶基因DKK1表达的方法
CN116970610B (zh) * 2023-09-21 2023-12-29 浙江大学海南研究院 一种chi-miR-335-5p的新用途及其调控靶基因DKK1表达的方法

Also Published As

Publication number Publication date
CN109897853A (zh) 2019-06-18
CN109897853B (zh) 2022-06-14
AU2019433418B2 (en) 2021-08-19
AU2019433418A1 (en) 2021-07-29

Similar Documents

Publication Publication Date Title
WO2020181663A1 (zh) 一种长链非编码rna及其应用
Luo et al. Drosophila tsRNAs preferentially suppress general translation machinery via antisense pairing and participate in cellular starvation response
Ramos et al. The long noncoding RNA Pnky regulates neuronal differentiation of embryonic and postnatal neural stem cells
Wei et al. miR-34s inhibit osteoblast proliferation and differentiation in the mouse by targeting SATB2
Chorghade et al. Poly (A) tail length regulates PABPC1 expression to tune translation in the heart
Dugas et al. Dicer1 and miR-219 Are required for normal oligodendrocyte differentiation and myelination
Judson et al. MicroRNA-based discovery of barriers to dedifferentiation of fibroblasts to pluripotent stem cells
Kim et al. Myc-induced microRNAs integrate Myc-mediated cell proliferation and cell fate
Ikiz et al. The regulatory machinery of neurodegeneration in in vitro models of amyotrophic lateral sclerosis
Wang et al. Long non-coding RNA CYP4B1-PS1-001 inhibits proliferation and fibrosis in diabetic nephropathy by interacting with nucleolin
Wei et al. Long noncoding RNA Lnc-SEMT modulates IGF2 expression by sponging miR-125b to promote sheep muscle development and growth
Shen et al. circINSR promotes proliferation and reduces apoptosis of embryonic myoblasts by sponging miR-34a
Verjans et al. Functional screening identifies microRNAs as multi-cellular regulators of heart failure
O’Connor et al. Retinoblastoma-binding proteins 4 and 9 are important for human pluripotent stem cell maintenance
Trohatou et al. miR-26a mediates adipogenesis of amniotic fluid mesenchymal stem/stromal cells via PTEN, Cyclin E1, and CDK6
Xu et al. Hsa_circ_0031288/hsa‐miR‐139‐3p/Bcl‐6 regulatory feedback circuit influences the invasion and migration of cervical cancer HeLa cells
Gao et al. miR-499 promotes immature porcine Sertoli cell growth through the PI3K/AKT pathway by targeting the PTEN gene
Zhang et al. A novel lncRNA, lnc403, involved in bovine skeletal muscle myogenesis by mediating KRAS/Myf6
Ando et al. Time-lapse imaging of microRNA activity reveals the kinetics of microRNA activation in single living cells
Daguia Zambe et al. miR‐19b‐3p induces cell proliferation and reduces heterochromatin‐mediated senescence through PLZF in goat male germline stem cells
Dong et al. LINC00511/miRNA-143-3p modulates apoptosis and malignant phenotype of bladder carcinoma cells via PCMT1
Kang et al. MiR‐543 regulates myoblast proliferation and differentiation of C2C12 cells by targeting KLF6
Min et al. A circular intronic RNA ciPVT1 delays endothelial cell senescence by regulating the miR‐24‐3p/CDK4/pRb axis
Xie et al. MircroRNA-10b promotes human embryonic stem cell-derived cardiomyocyte proliferation via novel target gene LATS1
Li et al. Long non-coding RNA FGD5-AS1 enhances osteosarcoma cell proliferation and migration by targeting miR-506-3p/RAB3D axis

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19919298

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2019433418

Country of ref document: AU

Date of ref document: 20190524

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19919298

Country of ref document: EP

Kind code of ref document: A1