Chimeric decoy oligonucleotide for inhibiting activation of Notch and NF-kB signals and application thereof
阅读说明：本技术 一种抑制Notch和NF-κB信号激活的嵌合诱骗寡核苷酸及其应用 (Chimeric decoy oligonucleotide for inhibiting activation of Notch and NF-kB signals and application thereof ) 是由 何飞 李维娜 于 2021-08-03 设计创作，主要内容包括：本发明涉及一种抑制Notch和NF-κB信号激活的嵌合诱骗寡核苷酸及其应用,该嵌合诱骗寡核苷酸是由2个RBPJ特异性识别位点、1个NF-κB特异性识别位点组成的单链,其序列为：5’-TCGTGGGAATTTCCCACGCTAGTTTTTCTAGCGTGGGAAATTCCCACGA-3’,序列两端进行硫代修饰,退火后形成发卡样双链结构。所述嵌合诱骗寡核苷酸进入细胞后能竞争结合Notch信号的关键转录因子RBPJ,抑制Notch信号的激活,同时还能竞争结合p65/NF-κB,抑制NF-κB信号的激活,因此可应用于体外和体内研究Notch/NF-κB信号对细胞增殖、分化和凋亡等的影响,以及治疗Notch和NF-κB信号同时异常激活的急性T淋巴细胞白血病等恶性肿瘤。(The invention relates to a chimeric decoy oligonucleotide for inhibiting Notch and NF-kB signal activation and application thereof, wherein the chimeric decoy oligonucleotide is a single chain consisting of 2RBPJ specific recognition sites and 1 NF-kB specific recognition site, and the sequence of the chimeric decoy oligonucleotide is as follows: 5'-TCGTGGGAATTTCCCACGCTAGTTTTTCTAGCGTGGGAAATTCCCACGA-3', performing thio modification at two ends of the sequence, and annealing to form hairpin-like double-stranded structure. After entering cells, the chimeric decoy oligonucleotide can compete with and bind to a key transcription factor RBPJ of a Notch signal, inhibit the activation of the Notch signal, simultaneously compete with and bind to p 65/NF-kappa B, and inhibit the activation of the NF-kappa B signal, so that the chimeric decoy oligonucleotide can be applied to in vitro and in vivo research on the influence of the Notch/NF-kappa B signal on cell proliferation, differentiation, apoptosis and the like, and treatment of malignant tumors such as acute T lymphocyte leukemia and the like with the abnormal activation of Notch and NF-kappa B signals.)
1. A chimeric decoy oligonucleotide that inhibits Notch and NF- κ B signaling activation, characterized by: the single-stranded DNA is a single strand consisting of 2RBPJ specific recognition sites and 1 kB specific recognition site, a hairpin-like double-stranded structure is formed after annealing, and the sequence of RBPJ/NF-kB chimeric decoy oligonucleotide is shown in SEQ.ID.NO. 1.
2. The chimeric decoy oligonucleotide of claim 1, which inhibits Notch and NF- κ B signaling activation, wherein: the 3 nucleotides at the 5 'and 3' ends of the sequence are modified by sulfo.
3. The chimeric decoy oligonucleotide of claim 1 or 2, which inhibits Notch and NF- κ B signaling activation, wherein: the decoy oligonucleotide can inhibit activation of Notch signal and NF-kB signal.
4. Use of the chimeric decoy oligonucleotide according to any one of claims 1 to 3 for inhibiting Notch and NF- κ B signaling activation in the preparation of a medicament for treating malignant tumor.
5. The use of the chimeric decoy oligonucleotide for inhibiting Notch and NF- κ B signaling activation according to claim 4 in the preparation of anti-malignant tumor medicament, wherein: the malignant tumor is acute T lymphocyte leukemia, breast cancer, melanoma, ovarian cancer or colon cancer.
Technical Field
The invention relates to decoy oligonucleotide and application thereof, in particular to chimeric decoy oligonucleotide for inhibiting Notch and NF-kB signal activation and application thereof, specifically to transcription factor immunoglobulin Kappa J region recombination signal binding protein (RBPJ) and B cell Kappa light chain gene enhancer binding nuclear factor (NF-kB) chimeric decoy oligonucleotide and application thereof in inhibiting Notch and NF-kB signal activation, and belongs to the technical field of medicines.
Background
The Notch signal pathway is a highly conserved signal pathway in evolution, mediates direct signal transduction between adjacent cells, widely participates in cell fate determination, cell proliferation, differentiation, apoptosis and the like, and plays an important role in angiogenesis, tumorigenesis development and the like. The Notch signaling pathway consists of ligands, receptors, downstream signaling molecules, and nuclear response processes. At present, 5 Notch ligands (Jagged1, Jagged2, Delta-like 1, Delta-like 3 and Delta-like4) and 4 Notch receptors (Notch1-4) are commonly found in mammals. After ligand between adjacent cells is combined with receptor, it is cut by gamma secretase complex enzyme to release intracellular segment NICD (active form of Notch), which can enter into nucleus, combine with transcription factor RBPJ to change RBPJ from transcription inhibition state to activation state, recruit co-activator Mastermind-like, combine with specific DNA sequence (C/TGGGAA), activate expression of downstream genes such as Hes1, etc. RBPJ is a common downstream transcription factor for the four Notch receptors, and blockade or inhibition of RBPJ effectively blocks classical Notch signaling (Artavanis-Tsakonas S et al, Science,1999,284: 770-6; Borggrefe T et al, Cell Mol Life Sci,2009,66: 1631-46; High FA et al, Nat Rev Genet,2008,9: 49-61).
NF-kB signals are widely involved in regulating immune and inflammatory responses, cell survival, proliferation, differentiation, apoptosis, etc., and are continuously activated in various diseases, such as tumors, arthritis, asthma, neurodegenerative diseases, heart diseases, etc. Currently, the NF-kappa B family members in mammals mainly comprise RelA (p65), RelB, c-Rel, NF-kappa B1(p50/p105) and NF-kappa B2(p52/p100), wherein a highly conserved Rel homology domain exists at the N terminal, and two identical or different NF-kappa B members form a homodimer or a heterodimer, wherein the heterodimer formed by p65 and p50 is the most common. NF- κ B binds to its suppressor Iκ B, is localized in the cytoplasm in an inactive form, and when cells are stimulated Iκ B is phosphorylated by its kinase (IKK) and ubiquitination-degradation occurs rapidly, NF- κ B dissociates from Iκ B and exposes a nuclear localization signal, enters the nucleus to bind to specific sequence GGGRNWYYCC (R: A/G, W: A/T, Y: C/T) on chromosomal DNA, activating expression of downstream multiple target genes, such as Bcl2, Bcl-Xl, C-Myc, IL8, VEGF, etc. (Zhang Q et al, Cell,2017,168: 37-57; Hayden MS et al, Cell,2008,132: 344-62).
Notch signaling interacts with NF-. kappa.B signaling, and both are often simultaneously activated in a variety of tumors, such as acute T-lymphoblastic leukemia (T-ALL), breast Cancer, melanoma, ovarian Cancer, colon Cancer, pancreatic Cancer, etc., and play an important role in the development and progression of tumors (DiDonato JA et al, Immunol Rev,2012,2461: 379-400; Ntziachristos P et al, Cancer Cell,2014,25: 318-34). In the case of acute T-lymphoblastic leukemia, Weng AP et al found that more than 50% of human T-ALL had Notch 1-activating mutations (Science,2004,306:269-71), and recent studies showed that there was also abnormal activation of NF-. kappa.B signaling in many T-ALL samples, probably by the mechanism that abnormally activated Notch signaling activates NF-. kappa.B signaling by promoting IKK expression, and that the NF-. kappa.B signaling inhibitor bortezomib significantly inhibited T-ALL cell growth (Vilimas T et al, Nat Med,2007,13: 70-7). Espinosa L et al also found that Notch signaling in T-ALL inhibited the expression of Cyld, a negative regulator of NF-. kappa.B signaling, by Hes1, leading to the activation of NF-. kappa.B signaling in T-ALL (Cancer Cell, 2010, 18: 268-81). Xiu et al found that co-activation of the Notch signal and NF-. kappa.B signal promoted malignant transformation of B cells (Blood,2020,135: 108-120).
The principle of the TFD strategy is that oligonucleotides are synthesized in vitro, whose sequence is consistent with the specific recognition site of Transcription Factor, and introduced into cells, which can competitively inhibit the specific Transcription Factor from binding with cis-elements on the genomic DNA sequence, thereby preventing the Transcription of target genes. The transcription factor decoy technology has the following outstanding characteristics: the sequence is short, easy to synthesize and easy to introduce into cells; secondly, the specificity is high, and the target protein is easy to recognize; ③ belongs to DNA sequence, and is more stable compared with Small Interfering RNA (SiRNA); (iv) it exerts inhibitory action before and at the transcriptional level and is potent (Hecker M et al, Biochem Pharmacol,2017,144: 29-34). At present, double-stranded decoy oligonucleotides synthesized aiming at nuclear transcription factors such as E2F, AP1, NF-kB and the like are used as tools for gene function research and have a large number of preclinical and clinical researches, so that a transcription factor decoy strategy has good application prospect. At present, no report of chimeric decoy oligonucleotide aiming at RBPJ and NF-kB is found.
Disclosure of Invention
The invention aims to provide a chimeric decoy oligonucleotide for inhibiting activation of Notch and NF-kB signals, which is a decoy oligonucleotide simultaneously aiming at transcription factors RBPJ and NF-kB, and can compete for binding with a key transcription factor RBPJ of Notch signals, effectively inhibit activation of Notch signals, simultaneously compete for binding with p 65/NF-kB and inhibit activation of NF-kB signals.
The second object of the invention is the use of a chimeric decoy oligonucleotide that inhibits Notch and NF- κ B signaling activation.
In order to achieve the purpose, the invention adopts the following technical scheme: a chimeric decoy oligonucleotide for inhibiting activation of Notch and NF-kB signals is a single chain consisting of 2RBPJ specific recognition sites and 1 kB specific recognition site, and a hairpin-like double-chain structure is formed after annealing, wherein the sequence of the RBPJ/NF-kB chimeric decoy oligonucleotide is shown in SEQ ID No. 1.
Further, 3 nucleotides at the 5 'and 3' ends of the sequence are modified with thio.
Further, the decoy oligonucleotide can inhibit activation of Notch signal and NF-kB signal.
Furthermore, the chimeric decoy oligonucleotide for inhibiting Notch and NF-kB signal activation is applied to preparing the anti-malignant tumor medicament.
Further, the malignant tumor is acute T lymphocyte leukemia, breast cancer, melanoma, ovarian cancer or colon cancer.
Due to the adoption of the technical scheme, the invention has the following beneficial effects:
the RBPJ/NF-kB chimeric decoy oligonucleotide can be used as a candidate molecule for simultaneously inhibiting Notch signal and NF-kB activation, and specifically inhibits the transcriptional expression of all target genes at the downstream of the Notch signal and the downstream of the NF-kB signal;
2. the RBPJ/NF-kB chimeric decoy oligonucleotide is a single-stranded DNA molecule, can be directly annealed to form a hairpin-like double-stranded structure with strong stability, and has strong in-vivo and in-vitro stability due to the fact that two ends of the sequence are subjected to thio-modification;
3. according to the principle of RBPJ/NF-kB chimeric decoy oligonucleotide, the RBPJ/NF-kB chimeric decoy oligonucleotide has application prospect in malignant tumors with abnormal activation of Notch and NF-kB signals, including but not limited to acute T lymphocyte leukemia, breast cancer, melanoma, ovarian cancer, colon cancer, pancreatic cancer and the like.
The foregoing is only an overview of the technical solutions of the present invention, and in order to clearly understand the technical means of the present invention and to implement the technical means according to the content of the description, the present invention will be described in detail with reference to the accompanying drawings, so that the objects and advantages of the present invention will become more apparent.
Drawings
FIG. 1 is a schematic diagram showing the sequence structure and hairpin-like double-stranded structure formation of an RBPJ/NF- κ B chimeric decoy oligonucleotide;
FIG. 2 is a schematic diagram of the mechanism of inhibition of Notch and NF-. kappa.B signaling by RBPJ/NF-. kappa.B chimeric decoy oligonucleotides;
FIG. 3 the effect of RBPJ/NF- κ B chimeric decoy oligonucleotides on Notch reporter genes;
FIG. 4 the effect of RBPJ/NF- κ B chimeric decoy oligonucleotides on NF- κ B reporter genes;
FIG. 5 is a real-time quantitative PCR detection of the effect of RBPJ/NF-kB chimeric decoy oligonucleotides on expression of Notch downstream target genes Hes1 and Hey1, and NF-kB downstream target genes Bcl2 and c-Myc;
FIG. 6 is a immunoblot to examine the effect of RBPJ/NF-kB chimeric decoy oligonucleotides on expression of Notch downstream target genes Hes1 and Hey1, and NF-kB downstream target genes Bcl2 and c-Myc;
FIG. 7 chromatin coprecipitation assay the effect of RBPJ/NF- κ B chimeric decoy oligonucleotides on the binding of RBPJ to the Hes1 promoter region and on the binding of p65 to the Bcl2 promoter region;
FIG. 8 Effect of RBPJ/NF- κ B chimeric decoy oligonucleotides on Jurkat cell growth;
FIG. 9Annexin V/PI staining tests the effect of RBPJ/NF- κ B chimeric decoy oligonucleotides on Jurkat apoptosis;
FIG. 10 immunoblot detection of the expression of the Jurkat apoptotic molecule cleared-Casp 3;
FIG. 11TUNEL assay detects the effect of RBPJ/NF-. kappa.B chimeric decoy oligonucleotides on Jurkat apoptosis.
Detailed Description
According to the transcription factor RBPJ specific recognition sequence C/TGTGGGAA reported in the Research of the literature (Tun T et al. recognition sequence of a high-level conserved DNA binding protein RBP-Jkappa. Nucleic Acids Research,1994,22:965-971), the NF-kappa B specific recognition sequence GGGRNWYYCC (R: A/G, W: A/T, Y: C/T) reported in the review literature (Zhang Q, et al.30Yeast of NF-kappa B: A Blossoming of Human Patholobiology. cell.2017,168:37-57) was designed and synthesized to simultaneously use RBPJ and NF-kappa B as chimeric target spots, and the single-stranded decoy oligonucleotide composed of 2RBPJ specific recognition sites and 1 NF-kappa B specific recognition site was designed and synthesized, and the sequence thereof was: seq.id No. 1: RBPJ/NF-kB chimeric decoy oligonucleotide sequences: 5'-TCGTGGGAATTTCCCACGCTAGTTTTTCTAGCGTGGGAAATTCCCACGA-3', the 5 'and 3' end 3 nucleotides of the sequence are modified by sulfo, and after annealing, a hairpin-like double-stranded structure is formed.
The RBPJ/NF-kappa B chimeric decoy oligonucleotide is used for inhibiting the activation of classical Notch and NF-kappa B signals, and after the chimeric decoy oligonucleotide subjected to thio-modification and annealing is transfected into cells, transcription factors RBPJ and NF-kappa B are competitively inhibited from being combined with corresponding cis-elements on a genome DNA sequence, so that the transcription of downstream target genes of the Notch and NF-kappa B signals is prevented, and the aim of inhibiting the activation of the Notch and NF-kappa B signals is fulfilled.
Examples
Design and Synthesis of RBPJ/NF-. kappa.B Decoy oligonucleotides (hereinafter referred to as Decoy ODN)
RBPJ/NF-kB decoy oligonucleotides are designed according to the specific recognition sequence C/TGTGGGAA of the transcription factor RBPJ and the specific recognition sequence GGGRNWYYCC (R: A/G, W: A/T, Y: C/T) of NF-kB, the specific recognition sequences are mutated to be used as control oligonucleotides (Ctrl), and the high homology with other genes is not found through Genbank search.
Seq.id No. 2: decoy ODN sequence:
5’TCGTGGGAATTTCCCACGCTAGTTTTTCTAGCGTGGGAAATTCCCACGA3’
seq.id No. 3: ctrl sequence:
5’TCGTTTTAATTTAAAACGCTAGTTTTTCTAGCGTTTTAAATTAAAACGA3’
the underlined part in the sequence is a mutant sequence, 3 nucleotides at both ends of the sequence are subjected to sulfo-modification, and the product is synthesized and purified by Beijing Oakco biotechnology limited.
The synthesized oligonucleotide was dissolved in double distilled water to prepare a 20. mu.M concentration, and annealed by boiling in a water bath for 10 minutes, followed by natural cooling and repeating for 3 times to form a hairpin-like double-stranded structure.
2. Cell culture
HEK293 cells: is human embryonic kidney cell line, presented by biochemical research and development room of air force military medical university, in DMEM medium (containing 10% fetal bovine serum and 1% cyan/streptomycin) at 5% CO2And performing conventional subculture in a constant-temperature incubator at 37 ℃.
HUVEC cells: human umbilical vein endothelial cells were fed from the biochemical research laboratory of the university of military medical science and air and cultured in the endothelial cell specific medium ECM (containing 5% fetal bovine serum, 1% penicillin/streptomycin and 1% ECGS) of Sciencell.
Jurkat cells: is an acute T lymphocyte leukemia cell line purchased from Wuhan Prov. Life technologies GmbH and subjected to conventional subculture in RPMI-1640 medium (containing 10% fetal bovine serum and 1% penicillin/streptomycin).
3. Reporter gene assay
Reporter plasmid pGa9816 (promoter region containing 3RBPJ binding sites), pEFBOS-NICD (Notch activated form NICD) plasmid, reporter plasmid 3 xkB-Luc (promoter region containing 3 NF kB binding sites), pCMV-p65 plasmid and reference plasmid phRL-TK were from professor Korean university of air force military university Biochemical textbook.
1) HEK293 cells were seeded 1.5X 10 the day before transfection4In 96-well plates, one set of 4 wells each, were 70% confluent on the day of transfection.
2) Plasmid and oligonucleotide (oligonucleotide configured at 20 μ M concentration) were mixed and DNA was added per well as follows, Notch signaling reporter:
negative control group: pGa 981650 ng, pEFBOS 50ng, phRL-TK5ng
Control group 1: pGa 981650 ng, pEFBOS-NICD 50ng, Ctrl 0.125. mu.L, phRL-TK5ng
Control group 2: pGa 981650 ng, pEFBOS-NICD 50ng, Ctrl 0.25. mu.L, phRL-TK5ng
Control group 3: pGa 981650 ng, pEFBOS-NICD 50ng, Ctrl 0.5. mu.L, phRL-TK5ng
Experimental group 1: pGa 981650 ng, pEFBOS-NICD 50ng, Decoy ODN 0.125 μ L, phRL-TK5ng
Experimental group 2: pGa 981650 ng, pEFBOS-NICD 50ng, Decoy ODN 0.25 μ L, phRL-TK5ng
Experimental group 3: pGa 981650 ng, pEFBOS-NICD 50ng, Decoy ODN 0.5. mu.L, phRL-TK5ng
NF- κ B signal reporter:
negative control group: 3 × κ B-Luc 50ng, pCMV 50ng, phRL-TK5ng
Control group 1:3 Xkappa B-Luc 50ng, pCMV-p 6550 ng, Ctrl 0.125. mu.L, phRL-TK5ng
Control group 2:3 Xkappa B-Luc 50ng, pCMV-p 6550 ng, Ctrl 0.25. mu.L, phRL-TK5ng
Control group 3: 3 Xkappa B-Luc 50ng, pCMV-p 6550 ng, Ctrl 0.5. mu.L, phRL-TK5ng
Experimental group 1:3 Xkappa B-Luc 50ng, pCMV-p 6550 ng, Decoy ODN 0.125. mu.L, phRL-TK5ng
Experimental group 2:3 Xkappa B-Luc 50ng, pCMV-p 6550 ng, Decoy ODN 0.25. mu.L, phRL-TK5ng
Experimental group 3: 3 Xkappa B-Luc 50ng, pCMV-p 6550 ng, Decoy ODN 0.5. mu.L, phRL-TK5ng
3) Serum-free DMEM was added at 0.5. mu.l liposomes per well and incubated for no more than 5 min.
4) Mixing the DNA with the liposomes, incubating at room temperature for 20-30min, and adding the DNA/liposome mixture to the cells.
5) After 24-48h, the medium was discarded, the cells were washed gently with PBS, and the supernatant was discarded.
6) 1 XPassive lysine was prepared, 20. mu.l was added to each well, and the mixture was subjected to shaking lysis at room temperature for 15 min.
7) The lysate was aspirated into a fresh centrifuge tube and centrifuged at 12000rpm for 30s at 4 ℃.
8) 5 μ l of the supernatant was aspirated, and 20 μ l of LARII and 20 μ l of stop & Glo were added to report the gene detector assay.
4. Decoy oligonucleotide cell transfection assay
Jurkat and HUVEC cells were transfected with Hiperfect transfection reagent from QIAGEN.
Jurkat cell transfection: jurkat cells were plated in 24-well plates with 400. mu.l complete medium per well at 2X 105Cells were cultured in 100. mu.l serum-free 1640 medium with 2.5. mu.l decoy oligonucleotide (stock solution concentration 20. mu.M) and 6. mu.l Hiperfect, Vortex mixed, incubated at room temperature for 10min, and added to the cultured cells for 24-48 h.
HUVEC cell transfection and Notch signaling activation: HUVEC cells were seeded in 24-well plates, confluent at 80% on the day of transfection, 400. mu.l complete medium ECM was changed per well, 2.5. mu.l decoy oligonucleotide (stock solution concentration 20. mu.M) and 3. mu.l Hiperfect, Vortex were added to 100. mu.l serum-free ECM medium and mixed well, incubated at room temperature for 10min, and added to the cultured cells and cultured for 24 h. Meanwhile, Dll4-Fc protein (purchased from Sino biology, stock solution concentration 0.25mg/ml, 250. mu.l PBS solution and 1. mu.l Dll4-Fc) was added to another 24-well plate, and incubated at 4 ℃ for 12 hours. After 24h transfection, HUVEC were trypsinized, plated into Dll4-Fc coated culture wells, and cultured for 24h to stimulate Notch signaling activation of HUVEC.
5. Real-time quantitative polymerase chain reaction (Real-time PCR) detection of expression of Notch and NF-kB signal downstream genes
1) Extraction of Total cellular RNA
Total RNA is extracted from cells by the Trizol method. The method comprises the following steps: collecting Jurkat cells or HUVEC cells cultured in 24-well plate, adding 0.5ml TRIzol into each well, and standing at room temperature for 5 min; adding 0.15ml chloroform, shaking vigorously for 15s, standing at room temperature for 3min, centrifuging at 12000rpm at 4 deg.C for 15min, sucking upper water phase, and transferring into new 1.5ml centrifuge tube; adding 0.25ml of isopropanol, shaking and uniformly mixing, standing at room temperature for 10min, centrifuging at 12000rpm at 4 ℃ for 10min, and removing supernatant; adding 0.5ml 75% ethanol (prepared with RNase-free water), washing, centrifuging at 12000rpm at 4 deg.C for 5min, and removing supernatant; dried at room temperature and dissolved in 20. mu.l of DEPC treated water; quantifying with a spectrophotometer; the A260/280 ratio monitors the purity of the RNA.
2) Reverse transcription of RNA
A reverse transcription kit (PrimeScript RT Master Mix Perfect Real Time) was purchased from Takara, Inc., Bow.
20 μ l reaction:
5×PrimeScript RT Master Mix 4μl
RNA 1μg
RNase Free dH2O to 20. mu.l
Reaction conditions are as follows: the reaction was stopped at 37 ℃ for 15min and 85 ℃ for 5 s.
3) Real-time quantitative PCR primer design and synthesis
The real-time quantitative PCR primers were designed and synthesized by Okka in Beijing. Primer sequences are shown in table 1:
4) real-time quantitative PCR
Using Takara TB GreenTM Premix EX TaqTMII (Tli RNaseH plus) kit for real-time quantitative PCR, wherein the 20. mu.l reaction system is as follows:
real-time quantitative PCR reaction conditions: pre-denaturation at 95 ℃ for 30s, and extension at 60 ℃ for 34s for a total of 40 cycles. Triplicate wells of the same sample, with β -actin as the internal reference.
6. Immunoblotting (Western blot)
Cell protein is extracted from RIPA lysate by a conventional method, and the cell protein is quantified by a BCA method. SDS-PAGE electrophoresis was performed, the membrane was stably transferred to a PVDF membrane, the PVDF membrane was blocked with a 5% nonfat dry milk PBST solution, and the membrane was incubated at room temperature for 2 hours. Primary antibody was diluted with blocking solution overnight at 4 ℃. anti-Hes 1 antibody (Cell Signaling Technology), 1:1000 dilution; primary antibody Hey1 (ex proteintech), 1:1000 dilution; anti-cleared-Casp 3 (Cell Signaling Technology), 1:1000 dilution; primary anti-Bcl 2 (available from Cell Signaling Technology), 1:1000 dilution; anti-c-Myc (available from Cell signalling Technology), 1:1000 dilution; primary anti-internal reference anti-beta-Actin antibody 1:5000 dilution (ex proteintech) and primary anti-4 ℃ incubation overnight. PBST membrane washing 3 times, each time 15 min. Secondary antibodies were purchased from proteintech: anti-mouse IgG-HRP 1:5000 dilution and anti-rabbit IgG-HRP 1:5000 dilution. PBST membrane washing 3 times, each time 15 min. ECL color luminescence recording.
7. Chromatin coprecipitation experiment (ChIP)
The relevant experiments were carried out using the enzyme-treated chromosome coprecipitation kit from Cell Signaling Technology, as described in the specification, and the specific steps were as follows:
1) inoculation of 1X 10 seeds per dish7Jurkat cells, using Hiperfect for transfection decoy oligonucleotides (comparably expanding the transfection system);
2) protein and DNA crosslinking: the culture medium of each 10ml needs 270 mul of 37% fresh formaldehyde solution, the mixture is placed for 10 minutes at room temperature, 1ml of 10 Xglycine solution is added and slightly rotated to be mixed uniformly, the mixture is incubated for 5 minutes at room temperature, the fixing reaction is stopped, the fixed cells are collected into a 50ml conical bottom tube, the cells are precipitated by centrifugation at 4 ℃ and 1500rpm for 5 minutes, and then the cells are rinsed twice by ice-cold PBS;
3) nuclear processing and chromosome shearing: each 1 × 107Resuspending the cells in 1ml of 1 Xbuffer A + DTT + PIC, incubating on ice for 10min, mixing them by inversion every 3min, centrifuging at 4 ℃ for 5min to precipitate the nuclei, discarding the supernatant, resuspending in 1ml of ice-cold buffer B + DTT, centrifuging again, discarding the supernatant, resuspending the precipitate with 100. mu.l of buffer B + DTT, adding 0.5. mu.l of Micrococcus nuclease, mixing them by inversion for several times, incubating at 37 ℃ for 20min, mixing them by inversion every 3-5 min, digesting the DNA into a fragment of about 150-and 900bp in length, adding 10. mu.l of 0.5M EDTA to stop digestion, centrifuging at 4 ℃ for 1 min 16000g to precipitate the nuclei, resuspending in 100. mu.l of 1 XChIP buffer + PIC, incubating on iceIncubating for 10min, ultrasonically disrupting cell nuclei (60W, 20 s/time, 3 times), centrifuging at 9400g at 4 deg.C for 10min, removing cell nucleus debris from the sample, and transferring the supernatant to a new tube to obtain a crosslinked chromatin fragment sample;
4) chromatin immunoprecipitation: 5-10. mu.g of chromatin DNA per tube, 500. mu.l of diluted 1 XChIP buffer for each precipitation reaction, 10. mu.l of diluted ChIP chromatin per tube and transfer to a new centrifuge tube as a 2% sample Input control (Input), the corresponding anti-RBPJ antibody or anti-p 65 antibody (available from Cell Signaling Technology, 1:50 dilution) was added to each sample tube, IgG is added into each group of negative control tubes, the rotor at 4 ℃ is incubated overnight, protein G magnetic beads are evenly suspended and mixed, 30 mu l of the mixture is added into each immunoprecipitation reaction, the rotor at 4 ℃ is incubated for 2 hours, placing the centrifuge tube on a magnetic separation frame, adsorbing protein G magnetic beads to the tube wall, carefully sucking the supernatant, adding 1ml of low-salt rinsing solution, rotating at 4 ℃ and incubating for 5 minutes, repeatedly rinsing for 3 times, adding 1ml of high-salt rinsing solution, and rinsing for 1 time;
5) elution and decrosslinking: adding 150 mu.l of 1 XChIP elution buffer solution into each ChIP immunoprecipitate sample, gently shaking (1200rpm) by using a vortex mixer, incubating at 65 ℃ for 30 minutes to elute chromatin from antibody-protein G microspheres, placing the centrifuge tube on a magnetic separation rack to adsorb protein G magnetic beads, carefully transferring the chromatin supernatant eluted from each sample tube into a new centrifuge tube, adding 6 mu.l of 5M NaCl and 2 mu.l of protease K into all tubes including the 2% sample Input control (2% Input) tube in the first step, and incubating at 65 ℃ for 2 hours;
6) purifying DNA by using a centrifugal column: adding 750 ul of DNA binding buffer to each DNA sample, transferring to a centrifugal column, centrifuging for 30 seconds at room temperature of 18500g, discarding the waste solution, adding 750 ul of DNA rinsing buffer to each centrifugal column, centrifuging for 30 seconds at 18500g, discarding the waste solution, centrifuging for 30 seconds at 18500g again, taking out the centrifugal column, inserting into a clean 1.5ml centrifuge tube, adding 50 ul of DNA elution buffer to each centrifugal column, standing for 2 minutes at room temperature, and centrifuging for 30 seconds at 18500g to elute DNA.
7) Performing real-time quantitative PCR reaction
ChIP primer pairs for the Hes1 and Bcl2 promoter regions were synthesized by Okko, Beijing.
The Hes1 sequence is:
Forward：5’-ATTGGCCGCCAGACCTTG-3’(SEQ.ID.NO.14),
Reverse：5’-GCTCGTGTGAAACTTCCCAAAC-3’(SEQ.ID.NO.15)；
the Bcl2 sequence is:
Forward：5’-CTTTAACCTTTCAGCATCACAGAGG-3’(SEQ.ID.NO.16)
Reverse：5’-CTTTGCATTCTTGGACGAGGG-3’(SEQ.ID.NO.17)
8. cell proliferation assay
Taking Jurkat cells in the growth phase, counting and then resuspending 2X 105Cells were transfected in 24-well plates using the decoy oligonucleotide transfection procedure described above, with triplicate wells per group, and viable cells counted after trypan blue staining for 3 consecutive days.
TUNEL method for detecting apoptosis
The TUNEL kit was purchased from Promega corporation and was performed as follows:
1) pretreatment of the glass slide: polylysine (Gibco, 50. mu.g/ml) was incubated at 37 ℃ for 12h, washed 3 times with sterile water and dried;
2) inoculating Jurkat cells conventionally, transfecting according to the transfection step of the decoy oligonucleotide, and culturing for 3 days;
3) soaking in normal saline for 5min, washing with PBS for 5min, soaking in 4% paraformaldehyde for 15min, washing with PBS for 5min, and repeating;
4) diluting proteinase K to 20 μ g/ml with PBS, covering the sample for 8-10min, washing with PBS for 5min, soaking in 4% paraformaldehyde for 5min, and washing with PBS for 5 min;
5)100 μ l Equi Buffer covered the sample for 5-10 min;
6) preparing rTdT Buffer on ice,
50 μ l System 45 μ l Equi Buffer
5μl NucleMix
1μl rTdTE；
7) rTdT Buffer covers the sample, and incubation is carried out for 60min at 37 ℃ in the dark;
8) diluting 20 times SSC with deionized water to obtain 2 times SSC, soaking 40ml of the solution in 2 times SSC in dark for 15 min;
9) PBS wash 3 times, each time for 5 min;
10) diluting with Hochest 1:5000, and dyeing for 15 min;
11) washing with deionized water for 3 times, sealing with 50% glycerol, and collecting image under fluorescence microscope;
detection of apoptosis by Annexin V/PI method
1) Transfecting the cells according to the transfection step of the decoy oligonucleotide, culturing for 3 days, collecting the cells, centrifuging for 5min at 300g, discarding supernatant, washing with PBS, resuspending the cells and counting;
2) take 2X 105Adding 500 mu L of diluted 1 × Annexin V Binding Buffer working solution into the resuspended cells to resuspend the cells;
3) adding 5 mu L Annexin V-APC staining solution and 5 mu L PI cell nucleus DNA staining solution into the cell suspension, mixing the mixture by gentle vortex, and incubating the mixture for 15-20min at room temperature in a dark place;
4) and (4) FACS detection.
11. Statistical treatment
The experiment was repeated 3 more times and the data was analyzed with GraphPad Prism 8 software, and two-by-two comparisons using t-test, P <0.05 had statistical significance, <0.05, <0.01, <0.001, < 0.0001.
The experimental results are further illustrated below with reference to the accompanying drawings.
As shown in FIG. 1, FIG. 1 shows the sequence of RBPJ/NF-. kappa.B chimeric Decoy oligonucleotide (Decoy ODN) containing 2 RBPJ-specific recognition sites (yellow square boxes) and 1 NF-. kappa.B-specific recognition site (green square boxes), hairpin-like structure formation by boiling-annealing with natural cooling, and thio-modification at 3 nucleotide sites at both ends of the sequence.
As shown in FIG. 2, FIG. 2 is a schematic diagram of the mechanism of inhibition of Notch signaling and NF-. kappa.B signaling activation by RBPJ/NF-. kappa.B chimeric decoy oligonucleotides: after the annealed hairpin-like Decoy ODN is transfected into cells, the hairpin-like Decoy ODN can competitively bind a Notch signal key transcription factor RBPJ, inhibit the binding of the RBPJ and chromosomal DNA, and inhibit the transcriptional expression of target genes such as Notch downstream Hes1, Hey1 and the like; the Decoy ODN can competitively bind p 65/NF-kB, inhibit the binding of RBPJ and chromosomal DNA, and inhibit the transcriptional expression of target genes such as Bcl2, c-Myc and the like at the downstream of NF-kB signals.
As shown in FIG. 3, reporter gene experiments were performed to verify whether the RBPJ/NF- κ B chimeric decoy oligonucleotide could inhibit Notch signaling: the reporter gene plasmid pGa9816 promoter region contains 3RBPJ specific binding sites, when a Notch signal is activated (a Notch activated form NICD is transfected), the RBPJ is changed from a suppression state to an activation transition state to activate the expression of luciferase, the expression of the luciferase can be obviously reduced after the Decoy ODN is transfected, and the suppression effect is enhanced along with the increase of the concentration of the Decoy ODN.
As shown in FIG. 4, to verify whether the RBPJ/NF- κ B chimeric decoy oligonucleotide could inhibit NF- κ B signaling, reporter gene experiments were performed: the 3 xkB-Luc promoter region of the reporter gene plasmid contains 3 NF-kB specific binding sites, NF-kB signal activation (transfection activation form p65) can promote the expression of luciferase, the expression of the luciferase can be obviously reduced after the Decoy ODN is transfected, and the inhibition effect is enhanced along with the increase of the concentration of the Decoy ODN.
As shown in FIG. 5, in order to verify whether the RBPJ/NF- κ B chimeric Decoy oligonucleotide can inhibit Notch signals and NF- κ B signals, A) the mRNA levels of target genes Hes1 and Hey1 downstream of the Notch signals were detected by real-time quantitative PCR, Decoy ODN (100nM) was transfected in Jurkat cells to inhibit the expression of Hes1 and Hey1, while hDll4 activated the Notch signals of HUVEC, at which time the Decoy ODN (100nM) was also transfected to reduce the mRNA levels of Hes1 and Hey 1; B) the mRNA levels of target genes Bcl2 and c-Myc downstream of NF-kB signals are detected through real-time quantitative PCR, and the transfection Decoy ODN (100nM) in Jurkat cells can inhibit the expression of Bcl2 and c-Myc thereof.
As shown in FIG. 6, in order to verify whether the RBPJ/NF-kB chimeric Decoy oligonucleotide can inhibit the Notch and NF-kB signals, the expression of proteins downstream of the Notch and NF-kB signals is detected by an immunoblotting method, A) Decoy ODN (50, 100nM) is transfected in HUVEC cells, and the protein levels of Hes1 and Hey1 are reduced; B) transfection of Decoy ODN (50, 100nM) in Jurkat cells reduced protein expression by Hes1, Hey1, Bcl2, and c-Myc.
As shown in FIG. 7, it was determined whether the RBPJ/NF- κ B chimeric Decoy oligonucleotide affected the binding of RBPJ to the Hes1 promoter (A and B) and the binding of NF- κ B to the Bcl2 promoter (C and D) by chromatin co-precipitation, and it was revealed that Decoy ODN significantly attenuated the binding of RBPJ to the RBPJ binding site of the Hes1 promoter region and significantly attenuated the binding of p65 to the NF- κ B binding site of the Bcl2 promoter region.
As shown in FIG. 8, after the transfection of Decoy ODN (50, 100nM) and Ctrl (100nM) into Jurkat cells, cell counting was performed at different times, and it was found that Decoy ODN could inhibit the growth of Jurkat cells, and the cell number of the group with the Decoy ODN concentration of 100nM was the least, suggesting that it has better inhibitory effect on the growth of Jurkat cells than the 50nM group.
As shown in FIG. 9, the annexin V/PI staining of Jurkat cells was detected by FACS, and the results showed that the proportion of annexin V positive in Jurkat cells after transfection of Decoy ODN at 50nM and 100nM was increased, indicating increased apoptosis, and that the apoptosis in the 100nM group was more than that in the 50nM group.
As shown in FIG. 10, the expression of the molecule Cleaved-Casp3 in apoptosis of Jurkat cells was detected by immunoblotting, and it was revealed that the expression of Cleaved-Casp3 was increased after transfection of 50nM and 100nM of Decoy ODN into Jurkat cells, and that the Cleaved-Casp3 in the 100nM group was higher than that in the 50nM group.
As shown in FIG. 11, apoptosis was detected by TUNEL staining, which revealed that Decoy ODN promoted apoptosis of Jurkat cells (green positive), and that the number of apoptosis was the greatest in the 100nM group.
The experimental results show that:
the RBPJ/NF-kB chimeric decoy oligonucleotide can simultaneously inhibit the activation of Notch signals and NF-kB signals;
the RBPJ/NF-kB chimeric decoy oligonucleotide can inhibit the growth of the Jurkat cells cultured in vitro;
the RBPJ/NF-kB chimeric decoy oligonucleotide can promote apoptosis of Jurkat cells cultured in vitro.
After entering cells, the chimeric decoy oligonucleotide can compete with and bind to a key transcription factor RBPJ of a Notch signal, inhibit the activation of the Notch signal, simultaneously compete with and bind to p 65/NF-kappa B, and inhibit the activation of the NF-kappa B signal, so that the chimeric decoy oligonucleotide can be applied to in vitro and in vivo research on the influence of the Notch/NF-kappa B signal on cell proliferation, differentiation, apoptosis and the like, and can be applied to drugs for treating malignant tumors such as acute T lymphocyte leukemia and the like with abnormal activation of Notch and NF-kappa B signals. Malignant tumors include, but are not limited to, acute T-lymphocyte leukemia, breast cancer, melanoma, ovarian cancer, colon cancer, pancreatic cancer, and the like.
The parts of the embodiment not described in detail and the english abbreviations are common general knowledge in the industry and can be searched on the internet, which is not described herein.
Sequence listing
<110> what flies
<120> a chimeric decoy oligonucleotide inhibiting activation of Notch and NF-kB signals
<130> 20210320001
<160> 17
<170> SIPOSequenceListing 1.0
<210> 1
<211> 49
<212> DNA
<213> (Artificial sequence)
<400> 1
tcgtgggaat ttcccacgct agtttttcta gcgtgggaaa ttcccacga 49
<210> 2
<211> 49
<212> DNA
<213> (Artificial sequence)
<400> 2
tcgtgggaat ttcccacgct agtttttcta gcgtgggaaa ttcccacga 49
<210> 3
<211> 49
<212> DNA
<213> (Artificial sequence)
<400> 3
tcgttttaat ttaaaacgct agtttttcta gcgttttaaa ttaaaacga 49
<210> 4
<211> 19
<212> DNA
<213> (Artificial sequence)
<400> 4
tggcacccag cacaatgaa 19
<210> 5
<211> 25
<212> DNA
<213> (Artificial sequence)
<400> 5
ctaagtcata gtccgcctag aagca 25
<210> 6
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 6
tggaaatgac agtgaagcac ctc 23
<210> 7
<211> 21
<212> DNA
<213> (Artificial sequence)
<400> 7
tcgttcatgc actcgctgaa g 21
<210> 8
<211> 24
<212> DNA
<213> (Artificial sequence)
<400> 8
agcaaagcgt tgacaaatca gatg 24
<210> 9
<211> 20
<212> DNA
<213> (Artificial sequence)
<400> 9
ctgcgtagtt gtgctgatgt 20
<210> 10
<211> 19
<212> DNA
<213> (Artificial sequence)
<400> 10
ggctcctggc aaaaggtca 19
<210> 11
<211> 20
<212> DNA
<213> (Artificial sequence)
<400> 11
ctgcgtagtt gtgctgatgt 20
<210> 12
<211> 19
<212> DNA
<213> (Artificial sequence)
<400> 12
ggtggggtca tgtgtgtgg 19
<210> 13
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 13
cggttcaggt actcagtcat cc 22
<210> 14
<211> 18
<212> DNA
<213> (Artificial sequence)
<400> 14
attggccgcc agaccttg 18
<210> 15
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 15
gctcgtgtga aacttcccaa ac 22
<210> 16
<211> 25
<212> DNA
<213> (Artificial sequence)
<400> 16
ctttaacctt tcagcatcac agagg 25
<210> 17
<211> 21
<212> DNA
<213> (Artificial sequence)
<400> 17
ctttgcattc ttggacgagg g 21