CN113648420A - Application of PEG10 gene/protein as target in preparation of anti-skin T cell lymphoma product - Google Patents

Application of PEG10 gene/protein as target in preparation of anti-skin T cell lymphoma product Download PDF

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CN113648420A
CN113648420A CN202110910843.5A CN202110910843A CN113648420A CN 113648420 A CN113648420 A CN 113648420A CN 202110910843 A CN202110910843 A CN 202110910843A CN 113648420 A CN113648420 A CN 113648420A
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汪旸
刘凤洁
孙婧茹
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Abstract

The invention discloses application of PEG10 gene/protein as a target point in preparation of an anti-skin T cell lymphoma product. The nucleotide sequence of the PEG10 gene is 94,656,370 th to 94,665,698 th of genbank accession number ENST 00000612748.1. The embodiment of the invention shows that the PEG10 gene expression in CTCL cells is inhibited, the cells are obviously reduced, the in vitro tumor forming capability is weakened, and the CTCL tumor growth in a xenograft mouse model is inhibited.

Description

Application of PEG10 gene/protein as target in preparation of anti-skin T cell lymphoma product
Technical Field
The invention relates to the technical field of biology, in particular to application of PEG10 gene/protein as a target point in preparation of a product for resisting cutaneous T cell lymphoma.
Background
Cutaneous T Cell Lymphoma (CTCL) is a lymphoma of cutaneous homing, T cell origin, with multiple subtypes, varying in course and prognosis and treatment modality for different subtypes. According to foreign statistics in recent years, the number of the products is about 10 per year6There are 10.2(Korgavkar et al, 2013) to 11.32(Ghazawi et al, 2017) new CTCL cases in humans. Mycosis Fungoides (MF) is the most common type of cutaneous T-cell lymphoma. The incidence of MF has increased rapidly in recent years, outside the nodesNon-hodgkin lymphoma has risen to the second place (Criscione and Weinstock, 2007). The tumor cells of MF are derived from peripheral CD4+ memory T cells, and are T cell lymphomas mainly caused by proliferation of small and medium cells. MF is hidden and progressive, and its course is chronic. Early patients manifest as patches and plaques of the skin (stage T1/T2), with progressive disease to form skin tumors (stage T3) and may invade the lymph nodes or blood system (Hwang, 2008). Although most of MF presents an inert, occult course, some patients progress faster, the tumor cells of these patients histologically switch from small to large cells, 4-fold greater than normal small lymphocytes, and the proportion of large cells exceeds 25% or forms nodules (Salhany et al, 1988), known as Large Cell Transformation (LCT) of MF. The disease course of patients with MF-LCT is often invasive, generalized and rapidly growing nodules and lumps appear, lymph nodes, peripheral blood, bone marrow and internal organs are more easily invaded, and the survival rate and median survival time of the patients are obviously reduced. Although LCTs can occur at various stages early to late in MF, the vast majority of LCTs occur during MF progression (about 25% for IIB and about 50% for IV). Statistically, patients with LCT at an earlier stage tend to have a poorer prognosis. (Agar et al, 2010, Herrmann and Hughey, 2012, Pulitzer et al, 2014). The reason for LCT of MF and the specific molecular mechanism are not clear, and no effective targeted therapeutic means can be aimed at MF-LCT patients at present. Patients in the progressive phase or in which Large Cell Transformation (LCT) occurs are invasive in their course and resistant to conventional therapies and chemotherapy. The disease progress of how to control MF has been a big problem in MF treatment.
Disclosure of Invention
An object of the present invention is to provide a substance inhibiting the expression of PEG10 gene or a substance inhibiting or reducing the activity and/or content of protein expressed by PEG10 gene.
The invention provides an application of a substance inhibiting PEG10 gene expression or a substance inhibiting or reducing the activity and/or content of PEG10 gene expression protein, wherein the application is any one of the following substances:
(1) the application in preparing products for resisting cutaneous T cell lymphoma;
(2) the application in preparing products for improving the sensitivity of T cell lymphoma cells of skin to anti-tumor drugs;
(3) the application of the composition in preparing products for reducing adverse symptoms caused by the drug resistance of skin T cell lymphoma cells to anti-tumor drugs;
(4) the application in preparing products for inducing apoptosis of T cell lymphoma cells of skin;
(5) the application in preparing products for inhibiting the growth of T cell lymphoma cells of skin.
The cutaneous T-cell lymphoma cells may be selected from at least one of HH, Hut78, Myla, MJ, PB2B, H9, Sz4, stably knocked-down HH cells of PEG10, and Myla and Hut78 cells stably overexpressed PEG10 of different fragments. The cutaneous T-cell lymphoma may be selected from at least one of mycosis fungoides and szary syndrome. For example, the case of cutaneous T cell lymphoma is a patient with advanced cutaneous T cell lymphoma.
The nucleotide sequence of the PEG10 gene can be 94,656,370 th to 94,665,698 th of genbank accession number ENST 00000612748.1.
Optionally, according to the above application, the substance inhibiting PEG10 gene expression is PEG10 gene-related biomaterial, and the PEG10 gene-related biomaterial is any one of the following:
b1) nucleic acid molecules for inhibiting the expression of PEG10 gene, such as b1) the nucleic acid molecule sequence is shown as SEQ ID No.5 and/or SEQ ID No. 6;
b2) an expression cassette comprising the nucleic acid molecule of b 1);
b3) a recombinant vector comprising the nucleic acid molecule of b1) or a recombinant vector comprising the expression cassette of b 2);
b4) a recombinant microorganism containing b1) the nucleic acid molecule, or a recombinant microorganism containing b2) the expression cassette, or a recombinant microorganism containing b3) the recombinant vector.
In the above biological material, the expression cassette is a DNA capable of expressing the nucleic acid molecule of b1) in a host cell, and the DNA may include not only a promoter for initiating gene transcription but also a terminator for terminating gene transcription. Further, the expression cassette may also include an enhancer sequence.
The inhibition of PEG10 gene expression can be achieved by gene knock-out or by gene silencing.
The gene knock-out (gene knock-out) is inactivation of a specific target gene by alteration of the DNA sequence.
The gene silencing refers to the phenomenon that a gene is not expressed or is under expression under the condition of not damaging the original DNA. Gene silencing is premised on no change in DNA sequence, resulting in no or low expression of the gene. Gene silencing can occur at two levels, one at the transcriptional level due to DNA methylation, differential staining, and positional effects, and the other post-transcriptional gene silencing, i.e., inactivation of a gene at the post-transcriptional level by specific inhibition of a target RNA, including antisense RNA, co-suppression (co-suppression), gene suppression (quelling), RNA interference (RNAi), and micro-RNA (mirna) -mediated translational suppression, among others.
In the above biological material, the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be an RNA, such as an mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.
Optionally, the agent that reduces the expression of PEG10 gene is HLM006474 according to the above-described use. HLM006474 has a CAS number of 353519-63-8.
The chemical structure of HLM006474 is as follows:
Figure BDA0003201913270000031
optionally, the PEG10 gene expression protein is selected from at least one of RF1a protein, RF1b protein, RF1a/2 protein, RF1b/2 protein and CNF fragment according to the above-mentioned application.
In the above application, the anti-tumor drug may be an HDAC inhibitor and/or a proteasome inhibitor, the HDAC inhibitor may include romidepsin and/or SAHA, and the proteasome inhibitor may include bortezomib and/or carfilzomib.
The PEG10 gene related biomaterial also belongs to the protection scope of the invention.
The invention also provides a product which comprises the substance for reducing the expression of the PEG10 gene and/or the substance for inhibiting or reducing the activity and/or content of the protein coded by the PEG10 gene.
The above products may further comprise HDAC inhibitors and/or proteasome inhibitors. The HDAC inhibitor can include romidepsin and/or SAHA. The proteasome inhibitor may include bortezomib and/or carfilzomib.
The product functions are any one of the following:
(1) anti-cutaneous T cell lymphoma;
(2) improving the sensitivity of T cell lymphoma cells of the skin to anti-tumor drugs;
(3) reducing adverse symptoms caused by the drug resistance of the skin T cell lymphoma cells to the anti-tumor drugs;
(4) inducing apoptosis of cutaneous T cell lymphoma cells;
(5) inhibiting the growth of T cell lymphoma cells of skin.
The invention also provides an application of the substance A and the substance B, wherein the application is any one of the following:
(1) the application in preparing products for resisting cutaneous T cell lymphoma;
(2) the application in preparing products for improving the sensitivity of T cell lymphoma cells of skin to anti-tumor drugs;
(3) the application of the composition in preparing products for reducing adverse symptoms caused by the drug resistance of skin T cell lymphoma cells to anti-tumor drugs;
(4) the application in preparing products for inducing apoptosis of T cell lymphoma cells of skin;
(5) the application in preparing products for inhibiting the growth of T cell lymphoma cells of skin;
the substance A is the substance for reducing the expression of the PEG10 gene and/or the substance for inhibiting or reducing the activity and/or content of the protein coded by the PEG10 gene, and the substance B is an HDAC inhibitor and/or a proteasome inhibitor.
The invention also provides application of the PEG10 gene or PEG10 gene expression protein in screening of an anti-skin T cell lymphoma drug model.
The embodiment of the invention shows that the PEG10 gene expression in CTCL cells is inhibited, the cells are obviously reduced, the in vitro tumor forming capability is weakened, and the CTCL tumor growth in a xenograft mouse model is inhibited; over-expressing PEG10 gene in CTCL cell, the in vitro tumor forming ability of the cell is enhanced, and the in vitro and in vivo growth advantages of CTCL are enhanced.
The examples of the present invention also show that PEG10 gene silencing can promote sensitivity of CTCL cells to HDAC inhibitors and/or proteasome inhibitors, and that PEG10 gene overexpression can promote resistance of CTCL cells to HDAC inhibitors and/or proteasome inhibitors.
The embodiment of the invention also proves that the target PEG10 expression through HLM006474 can specifically inhibit the survival of primary CTCL cells in vitro and in vivo and in patients; HLM006474 synergistically kills CTCL cells with HDAC inhibitors and/or proteasome inhibitors, suggesting that the application of HLM006474 may be a potential therapeutic approach for late CTCL.
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FIG. 1 shows the results of Western blotting experiment in example 1.
FIG. 2 shows the results of the flow cytometry detection of RNA interfering lentivirus transfected cells of example 1.
FIG. 3 shows the results of successful transfection in example 1 using fluorescence microscopy.
FIG. 4 shows the results of colony formation experiments for HH-Ctrl, HH-shPEG10-1, and HH-shPEG10-2 in example 2.
FIG. 5 shows the results of colony formation experiments in examples 2Hut78-RF1b, Hut78-RF1b/2, Hut78-RF1b/2+ CNF, Hut 78-vector.
FIG. 6 shows the results of the cell colony formation experiments of Myla-RF1b, Myla-RF1b/2, Myla-RF1b/2+ CNF, Myla-vector of example 2.
FIG. 7 shows the results of the experiments in mice of the group HH-shPEG10 and the group HH-Ctrl in example 3.
FIG. 8 shows the experimental results of mice in the Hut78-RF1b group and the Hut78-vector group of example 3.
Fig. 9 is a statistics of cell viability values and drug-induced specific apoptosis rates of HH-shPEG10 cells treated with HDAC inhibitors of example 4.
FIG. 10 is a statistics of cell viability values and drug-induced specific apoptosis rates of HH-shPEG10 cells treated with proteasome inhibitors of example 4.
FIG. 11 is a summary of cell viability values and drug-induced specific apoptosis rates of example 4HDAC inhibitor treated Hut78-RF1b cells.
FIG. 12 is a statistics of cell viability values and drug-induced specific apoptosis rates of example 4 proteasome inhibitor treated Hut78-RF1b cells.
FIG. 13 is a statistic of GFP + reduction rate of Myla-RF1b cells treated with HDAC inhibitors, proteasome inhibitors, in example 4.
Fig. 14 shows the results of immunoblotting experiments of HH-shPEG10 cells treated with HDAC inhibitors of example 4.
Fig. 15 is the results of immunoblotting experiments of HH-shPEG10 cells treated with the proteasome inhibitor of example 4.
FIG. 16 shows the results of immunoblot experiments of example 4HDAC inhibitor treated Hut78-RF1b cells.
FIG. 17 is the results of immunoblot experiments of proteasome inhibitor treated Hut78-RF1b cells of example 4.
FIG. 18 shows the results of immunoblotting experiments for example 5HLM006474 treatment of cells HH and SZ 4.
FIG. 19 is a summary of cell viability values and drug-induced specific apoptosis rates of example 5HLM006474 treated cell HH-shPEG 10.
FIG. 20 is a photograph of the mouse and the tumor in example 6.
FIG. 21 is a graph showing statistics of tumor volume and body weight of mice according to the number of treatments in example 6.
Figure 22 is a statistic of specific apoptosis rates of example 7HLM006474 treated PBMCs.
Fig. 23 is a statistics of cell viability values and drug-induced specific apoptosis rates of example 8HLM006474 in combination with HDAC inhibitor treatment of CTCL cells.
Fig. 24 is statistics of cell viability values and drug-induced specific apoptosis rates of the HLM006474 in combination with proteasome inhibitors treatment of CTCL cells of example 8.
Wherein p represents < 0.05; p < 0.01; nM represents nmol/L; μ M means μmol/L.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The quantitative data of the experiment comes from 3 independent experiments, GraphPad Prism 6.0 statistical software is adopted to process the data, the experimental result is expressed by mean value +/-standard deviation, and t test is adopted.
The main reagents, sources of biological material, used in the following examples are as follows.
Lentiviral cloning vector GL 404: from Shanghai and Meta Biotech Co
Lentiviral cloning vector GV 409: from Kyork Biotech, Shanghai
HH (CTCL cell line): a gift from the university of British Columbia dermatology and Young professor of the department of dermatology, described in the event of an environmental role of aberration TOX activation in the clinical laboratory T-cell lymphoma PMID: 25548321, respectively; DOI: 10.1182/blood-2014-05-571778
Hut78(CTCL cell line): purchased from cooperative cell banks
Myla (CTCL cell line): a gift from the university of British Columbia dermatology and Young professor of the department of dermatology, described in the event of an environmental role of aberration TOX activation in the clinical laboratory T-cell lymphoma PMID: 25548321, respectively; DOI: 10.1182/blood-2014-05-571778
SZ4(CTCL cell line): a gift from the university of British Columbia dermatology and Young professor of the department of dermatology, described in the event of an environmental role of aberration TOX activation in the clinical laboratory T-cell lymphoma PMID: 25548321, respectively; DOI: 10.1182/blood-2014-05-571778
RPMI1640 medium: from Hyclone Laboratories
RPMI1640 medium with 10% volume FBS: FBS was purchased from Gibco, and RPMI1640 medium was purchased from HyClone Laboratories
Polybrene: from Shanghai and Meta Biotech Co
IMDM medium: from Sigma-Aldrich
Romidepsin (Romidepsin): seleck, configure concentration gradient SAHA with DMSO (Sigma-Aldrich): Sigma-Aldrich, using DMSO (Sigma-Aldrich) to configure the concentration gradient
Bortezomib (Bortezomib): selleck, concentration gradient Carfilzomib (Carfilzomib) with DMSO (Sigma-Aldrich): seleck, configured with a concentration gradient of 20% sulfobutylcyclodextrin in DMSO (Sigma-Aldrich): from Sigma-Aldrich
HLM 006474: MedChemexpress, DMSO (Sigma-Aldrich) is adopted to configure concentration gradient
The cells used in the following examples were cultured in RPMI-1640 basic medium + 10% fetal bovine serum + 1% penicillin (100U/ml) -streptomycin (0.1mg/ml) double antibody. All CTCL cell lines grow in suspension in culture medium at constant temperature of 37 deg.C and relative humidity of 100% and 5% CO2The cell culture box carries out liquid changing and passage according to the growth condition, and cells used for experiments are all in logarithmic growth phase.
The specific method of western blotting of the following examples is as follows:
(1) suspension cell whole protein extraction
The suspension cell holoprotein is extracted by adopting a Kaikyi holoprotein extraction kit (the product number is KGP250, purchased from Nanjing Kaikyi).
(2) Protein content detection
By using PierceTMBCA Protein Assay Kit (cat # 23227, available from Thermo Fisher) calculationThe whole protein concentration of the suspension cells extracted above.
(3) Western blot
1) Preparation of SDS-PAGE gels
Figure BDA0003201913270000061
Figure BDA0003201913270000071
And cleaning the glass plate of the glue filling device, naturally airing, sticking and fixing the glass plate to a glue clamping frame, and vertically placing the glass plate on a glue making bracket. Firstly, pouring separation glue (about 4.5mL is needed for each piece of glue) into a 1mL gun head, stopping when the liquid level rises to about 0.3cm below a short plate, quickly pressing the glue by using isopropanol or 500 mu L of water to prevent residual bubbles, keeping the liquid level flat, and being beneficial to isolating air to promote gel polymerization. The remaining separated glue can be set aside for evaluation of the degree of setting of the glue in the glass plate. The mixture can be solidified after being placed at room temperature for 45 minutes to 1 hour.
After the separation gel is solidified, pouring off isopropanol, washing with deionized water, sucking water with absorbent paper, pouring 6% lamination gel (each gel needs about 2.5mL) with a 1mL gun head until the gel is filled, vertically inserting a sample comb, and standing at room temperature for about 40-50 minutes. After the gel is solidified, the glass clamping plate filled with the gel is arranged in a main core (the inner part of the glass plate is short and the outer part of the glass plate is long) of an electrophoresis tank, 1 Xelectrophoresis buffer solution is filled in the main core, after the main core is checked to have no leakage, the main core is arranged in the electrophoresis tank filled with the 1 Xelectrophoresis buffer solution, and a comb is carefully removed and the sample is loaded. If the solidified glue is not used temporarily, the prepared glue can be tightly wrapped by soaked toilet paper and then sealed by a preservative film, and the glue is placed in a refrigerator at 4 ℃ for 1 month.
2) Sample denaturation and SDS polyacrylamide gel electrophoresis (SDS-PAGE):
calculating the volume of the Protein according to the amount of the Protein to be loaded, adding 6 Xprotein Loading Buffer according to the volume, supplementing 1 Xprotein Loading Buffer in a proper amount to 30 mu L, uniformly mixing, performing denaturation for 8 minutes at 100 ℃ in a metal bath, and performing short centrifugation. The sample was again mixed and loaded at 30. mu.L per lane, and 6-10. mu.L Protein Marker loaded (empty wells around which 1 Xprotein Loading Buffer could be filled to 30. mu.L). And (3) switching on a power supply, setting the electrophoresis apparatus to be in a constant voltage state, adjusting the voltage to 80V, raising the voltage to 120V when the sample reaches the junction of the lamination glue and the separation glue (about half an hour), and ending the electrophoresis until a Loading Buffer indicating line runs to the bottom green plate.
3) Room temperature rapid film transfer (wet process):
preparing 1 × rapid membrane transfer liquid, pouring the rapid membrane transfer liquid into a square tray, taking out the gel after electrophoresis, properly trimming, and placing the gel into the square tray for later use. At the same time, the PVDF membrane was cut to a size comparable to the gel size. The PVDF membrane should be soaked in 100% methanol for 1 minute for activation. Loading sample glue, a film and the like into a film transferring clamping plate in a wet environment of a rapid film transferring liquid, and sequentially placing various materials from a negative electrode to a positive electrode: transfer film splint negative pole (blackboard) → sponge pad → 3-5 layers of filter paper → sample glue → PVDF film → 3-5 layers of filter paper → sponge pad → transfer film splint positive pole (transparent plate). During the process, a glass rod is used for removing bubbles completely, glue is placed in the direction according to the sample loading sequence of the samples, the clamping plate is fastened after the completion, the clamping plate is placed in an electric rotary groove, the anode and the cathode are noticed, all the rapid film transfer liquid is poured into the electric rotary groove, and two ice boxes placed at normal temperature are added to enable the liquid level to reach the corresponding level. The film is correctly connected and is switched on, and the film is rotated for 30 to 40 minutes at the room temperature of 400mA under the constant current state.
4) And (3) sealing:
after the film transfer is finished, taking out the PVDF film carefully (the front surface is the surface which is directly contacted with the glue and faces upwards), cutting according to the purpose and the position of the internal reference strip, respectively putting the cut films into containers with corresponding sizes, washing with 1 × TBST (90-110rpm) for 5 minutes and 3 times to remove the residual rapid film transfer liquid. After completion, the TBST was decanted, 5% skim milk powder solution was added and shaken slowly (60-70rpm) on a shaker at room temperature for 1-2 hours.
5) Antibody incubation:
pouring the skimmed milk powder solution after sealing, respectively adding specific antibodies of the to-be-detected and internal references diluted by 5% skimmed milk powder solution at corresponding concentrations, placing in a shaker at 4 ℃, and incubating the primary antibodies overnight at 40 rpm. The next day, after recovering the primary antibody (the primary antibody can be recycled 3-4 times), the primary antibody is placed on a shaker and washed with 1 × TBST (90-110rpm) for 3 times, the remaining primary antibody for 10 minutes. After completion, a secondary antibody of appropriate concentration was added and incubated slowly (60-70rpm) on a shaker at room temperature for 1 hour. Residual secondary antibody was washed, as was the same antibody.
6) Chemiluminescence developing and imaging:
chemiluminescent solutions were prepared according to the easy se Western Blot Kit (TransGen Biotech) instructions: the volumes of the solution A and the solution B are equal, and the volume of the solution C is (A + B)/1000. And (3) uniformly covering the surface of the PVDF membrane with the luminous liquid, incubating for half a minute in a dark place at room temperature, and performing imaging analysis by using a SYNGENE gel image analysis system.
Cell viability assays for the following examples were performed by the tetrazolium colorimetric method using CellTiter
Figure BDA0003201913270000081
The AQueous One Solution Cell Proliferation Assay kit (MTS Assay) (Promega). The method for calculating the cell viability value specifically comprises the following steps:
relative cell viability was the original OD/(OD0h × OD0 μ M) upon drug treatment,
cell growth curves, without drug treatment, relative cell viability as raw OD/OD0h,
OD0h refers to the OD value of a well cell in a 6-well plate measured at 0 hour, and OD 0. mu.M refers to the OD value of a negative control cell at the same time point.
Apoptosis Detection in the following examples apoptosis was detected using Annexin V-PE/APC Detection Kit (BD Pharmingen, San Diego, Calif.) for cell stain labeling and flow cytometry FACS Calibur (BD Biosciences, San Diego, Calif.). Data collected was analyzed using flowjo7.6software to calculate drug-specific apoptosis rate, formula: drug-induced specific apoptosis rate ═ (percentage of apoptotic cells-percentage of natural apoptosis of cells at the time of drug treatment)/(percentage of natural apoptosis of cells 100) × 100%.
The drug-induced GFP + reduction rate formula of the following examples: drug-induced GFP + decrease rate (solvent control treated cell GFP + percent-drug treated cell GFP + percent)/(solvent control treated cell GFP + percent) x 100%.
By cross-comparing differentially expressed genes between patients with MF-LCT (mycosis fungoides with large cell transformation) and MF-NLCT (granuloma fungoides without large cell transformation), and genes in the MF-LCT-enriched 7q gene set, a significant upregulation in PEG10 gene expression at the 7q21.3 site was found. The nucleotide sequence of the PEG10 gene is 94,656,370 th to 94,665,698 th of genbank accession number ENST00000612748.1 (14 th at 2021), and the expression product thereof comprises RF1a protein (amino acid sequence is from 1 st to 325 th at the genbank accession number Q86TG7-2 (18 th at 2008 3 month), RF1B protein (amino acid sequence is from 1 st to 401 th at the genbank accession number B4DSP0-1 (23 th at 2008 11 month), RF1a/2 protein (amino acid sequence is from 1 st to 708 th at the genbank accession number Q86TG7-1 (18 th at 2008 month 18) and RF1B/2 protein (amino acid sequence is from 1 st to 783 th at the genbank accession number A0A087WX23-1 (29 th at 2014 year 29 th) and CNF fragment (RF1a/2 or RF1B/2 fragment of RF 1/2 protein).
Example 1 inhibition or increase of PEG10 expression in cells
RNA interference lentivirus cloning
The experimental procedures for preparing RNA interference lentivirus clone are as follows:
1) fragments of interest Ctrl, shPEG10-1 and shPEG10-2 were prepared. The specific sequence of the fragment of interest is as follows:
Ctrl：5′-TTCTCCGAACGTGTCACGT-3′(SEQ ID No.1)
PEG10-1：5′-CCCAGTGCCAGATCTTCAT-3′(SEQ ID No.2)
PEG10-2：5′-AAAGCTGGAGCGCTCCCACTA-3′(SEQ ID No.3)。
2) the target fragments prepared above were inserted into the AgeI and EcoRI recognition sites of lentiviral cloning vector GL404, respectively, to obtain recombinant plasmids.
3) And (3) transforming the recombinant plasmid into competent cells, selecting a single clone in a plate for colony PCR identification, and sending the positive clone bacterial liquid to a sequencing company for sequencing verification. The recombinant plasmids in the positive clone bacterial liquid which are verified to be correct are named as GL404-Ctrl, GL404-PEG10-1 and GL404-PEG10-2 respectively.
The recombinant plasmid GL404-Ctrl is a recombinant plasmid obtained by replacing a small fragment between AgeI and EcoRI recognition sites of the vector GL404 with the target fragment Ctrl. The recombinant plasmid GL404-PEG10-1 is a recombinant plasmid obtained by replacing a small fragment between AgeI and EcoRI recognition sites of the vector GL404 with the objective fragment shPEG 10-1. The recombinant plasmid GL404-PEG10-2 is a recombinant plasmid obtained by replacing a small fragment between AgeI and EcoRI recognition sites of the vector GL404 with the objective fragment shPEG 10-2.
4) The clone bacterial liquid containing the recombinant plasmids GL404-Ctrl, GL404-PEG10-1 and GL404-PEG10-2 is respectively subjected to amplification culture and plasmid extraction to obtain high-purity recombinant plasmids GL404-Ctrl, GL404-PEG10-1 and GL404-PEG 10-2.
5) High-purity recombinant plasmids GL404-Ctrl, GL404-PEG10-1 and GL404-PEG10-2 and virus-coated Helper plasmids (Gickac company, Helper 1.0 and Helper 2.0) are respectively transferred into 293T cells, and after transfection is completed, virus harvesting (namely unpurified virus supernatant) is carried out for 48-72 hours to obtain lentivirus, and then centrifugal concentration and filtration purification of the virus are carried out to obtain the lentivirus Ctrl, shPEG10-1 and shPEG 10-2.
The slow virus Ctrl expresses a target fragment Ctrl, an expression product is shRNA Ctrl, and the sequence of the shRNA Ctrl is shown as SEQ ID No. 4. The target segment shPEG10-1 is expressed by the lentivirus shPEG10-1, the expression product is shRNA shPEG10-1, and the sequence of shRNA shPEG10-1 is shown in SEQ ID No. 5. The target segment shPEG10-2 is expressed by the lentivirus shPEG10-2, the expression product is shRNA shPEG10-2, and the sequence of shRNA shPEG10-2 is shown in SEQ ID No. 6.
The obtained lentivirus titer was Ctrl: 1.02E +09IU/mL, shPEG 10-1: 1.17E +09IU/mL, shPEG 10-2: 4.77E +08 IU/mL.
Second, overexpression of lentivirus clones
The experimental procedure for the preparation of the overexpressed lentiviral clones was as follows:
1) the target fragments PEG10-RF1b (shown in SEQ ID No. 7), PEG10-RF1b/2 (shown in SEQ ID No. 8) and PEG10-RF1b/2+ CNF (shown in SEQ ID No. 9) were prepared.
2) The target fragments prepared above were inserted between the BamHI and AgeI recognition sites of the lentiviral cloning vector GV409 to obtain recombinant plasmids.
3) And (3) directly transforming the recombinant product into competent cells, selecting a single clone in a plate for colony PCR identification, and sending the positive clone bacterial liquid to a sequencing company for sequencing verification. The recombinant plasmids in the positive clone bacterial liquid which are verified to be correct are named as GV409-PEG10-RF1b, GV409-PEG10-RF1b/2 and GV409-PEG10-RF1b/2+ CNF respectively. The recombinant plasmid GV409-PEG10-RF1b is a recombinant plasmid obtained by replacing a small segment between the BamHI and AgeI recognition sites of the vector GV409 with the desired fragment PEG10-RF1 b. The recombinant plasmid GV409-PEG10-RF1b/2 is a recombinant plasmid obtained by replacing a small segment between the BamHI and AgeI recognition sites of the vector GV409 with the desired fragment PEG10-RF1 b/2. The recombinant plasmid GV409-PEG10-RF1b/2+ CNF is a recombinant plasmid obtained by replacing a small segment between BamHI and AgeI recognition sites of the vector GV409 with the target fragment PEG10-RF1b/2+ CNF.
4) The clone bacterial liquid containing the recombinant plasmids GV409-PEG10-RF1b, GV409-PEG10-RF1b/2 or GV409-PEG10-RF1b/2+ CNF is subjected to amplification culture and plasmid extraction to respectively obtain high-purity recombinant plasmids GV409-PEG10-RF1b, GV409-PEG10-RF1b/2 and GV409-PEG10-RF1b/2+ CNF.
5) The obtained high-purity recombinant plasmid and lentiviral cloning vector GV409 are respectively transferred into 293T cells together with a virus-encapsidated Helper plasmid (Gicky corporation, Helper 1.0, Helper 2.0), virus harvesting (namely unpurified virus supernatant) is carried out 48-72 hours after transfection is finished to obtain lentiviruses, and then centrifugal concentration and filtration purification of the viruses are carried out to obtain lentiviruses PEG10-RF1b, PEG10-RF1b/2, PEG10-RF1b/2+ CNF and an empty vector. The target fragment PEG10-RF1b is expressed in cells by lentivirus PEG10-RF1b, and the expression product is RF1b protein. The lentivirus PEG10-RF1b/2 expresses a target fragment PEG10-RF1b/2 in cells, and compared with a PEG10 gene, the target fragment PEG10-RF1b/2 has the 'tggcaatc' substitution at the 1056-1063 position to replace 'gggaaac', so that a ribosome cannot generate-1 frameshift and has one more base, the first tag stop codon cannot appear to continue the backward translation, and the synthesis sequence of the following amino acid is not changed; the substitution of aspartic protease site 1211, a, with c, results in the aspartic protease not being able to recognize this site and thus not inducing spontaneous cleavage by RF1b/2 to produce the CNF fragment. Therefore, the expression product of the target fragment PEG10-RF1b/2 is RF1b/2 protein. The lentivirus PEG10-RF1b/2+ CNF expresses a target fragment PEG10-RF1b/2+ CNF in cells, compared with a PEG10 gene, the 'tggcaatc' at the 1056-1063 position in the target fragment PEG10-RF1b/2+ CNF replaces 'gggaaac', so that a ribosome cannot carry out-1 frameshift and has one more base, the first tag stop codon cannot appear so as to continue to translate backwards, and the synthesis sequence of the following amino acids is not changed, so that the expression products are the RF1b/2 protein and the CNF fragment.
The obtained lentivirus titers were empty vector: 8.0E +08 IU/mL; RF1 b: 7.0E +08 IU/mL; RF1 b/2: 6.0E +08 IU/mL: RF1b/2+ CNF: 3.0E +08 IU/mL.
RNA interference lentivirus transfection
1) Taking HH cells of logarithmic growth phase, counting, adjusting to cell density of 2 × 10 with RPMI1640 medium containing 10% FBS5mu.L of cell fluid was used (2X 10 cells per transfection)5One);
2) polybrene was diluted to 10 × (50 μ g/mL) with RPMI1640 medium for use at a final concentration of 5 μ g/mL;
3) the volume of virus fluid required for each transfection was calculated based on the virus titer and the multiplicity of infection (MOI), defined as the ratio of virus to cell number, i.e., MOI (virus titer. times. virus fluid volume)/2X 10, as 1005；
4) The following components were added to each well in a 24-well plate in order: 10 XPolybrene 50. mu.L, virus liquid (i.e. the prepared lentivirus Ctrl or lentivirus shPEG10-1 or shPEG10-2) and cell liquid 400. mu.L prepared in step 1) (the cell liquid covers the virus and Polybrene mixed liquid, and does not need to be mixed evenly), and RPMI1640 culture medium is used for supplementing the total volume of 500. mu.L;
5) constant temperature of 37 ℃, 100% relative humidity and 5% CO2Culturing for 24 hr, centrifuging to remove supernatant, removing the culture medium containing transfection mixture, and culturing in RPMI1640 medium containing 10% FBS;
6) detecting fluorescence positive cells by adopting a flow cytometer 72 hours after transfection, wherein the fluorescence positive cells are cells successfully transfected by the lentivirus and are respectively named as HH-Ctrl, HH-shPEG10-1 and HH-shPEG 10-2;
7) the average cell area of cells successfully transfected (GFP +) collected by fluorescence microscopy was calculated by flow cytometry detection of the forward scattered light of the cells (Rathmell et al, 2003) and ImageJ (Cai et al, 2009); and (3) carrying out gene expression level verification on the cells successfully transfected by adopting Western immunoblotting (Western blot).
Four, RNA overexpression lentivirus transfection
1) The logarithmic growth phase of Hut78, Myla cells were counted and adjusted to a cell density of 2X 10 in RPMI1640 medium containing 10% FBS5mu.L of cell fluid was used (2X 10 cells per transfection)5One);
2) polybrene was diluted to 10 × (50 μ g/mL) with RPMI1640 medium for use at a final concentration of 5 μ g/mL;
3) the volume of virus fluid required for each transfection was calculated based on the virus titer and the multiplicity of infection index (MOI) of 100. The re-infection index is defined as the ratio of virus to cell number, i.e. MOI ═ virus titer × virus fluid volume)/2 × 105；
4) The following components were added to each well in a 24-well plate in order: 10 XPolybrene 50. mu.L, viral fluid (i.e. lentivirus PEG10-RF1b, PEG10-RF1b/2, PEG10-RF1b/2+ CNF or empty vector (vector) prepared previously), 400. mu.L of cell fluid prepared in step 1 (cell fluid is covered on the viral Polybrene mixture without mixing), and RPMI1640 culture medium is used to make up for 500. mu.L of total volume;
5) constant temperature of 37 ℃, 100% relative humidity and 5% CO2Culturing for 24 hr, centrifuging to remove supernatant, removing the medium containing transfection mixture, and culturing in RPMI1640 complete medium containing 10% FBS;
6) 72 hours after transfection, the cells successfully transfected by the lentivirus express GFP tag protein, and a flow cytometer is adopted to detect fluorescence positive cells, wherein the fluorescence positive cells are the cells successfully transfected by the lentivirus and are respectively named Hut78-RF1b, Hut78-RF1b/2, Hut78-RF1b/2+ CNF, Hut78-vector, Myla-RF1b, Myla-RF1b/2, Myla-RF1b/2+ CNF and Myla-vector;
7) the average cell area of cells successfully transfected (GFP +) collected by a fluorescence microscope was calculated by flow cytometry (Rathmell et al, 2003) and ImageJ (Cai et al, 2009), and the cells successfully transfected were subjected to gene expression level verification using Western blotting (antibody anti-PEG10 (Novus; NBP 2-13749)).
The results of the Western blot are shown in FIG. 1, in which lanes Ctrl, shPEG10-1, shPEG10-2, vector, RF1b, RF1b/2 and PEG10-RF1b/2+ CNF indicate transfection with the corresponding lentiviruses. In a CTCL cell line (HH) with high PEG10 expression, the expression of a PEG10 gene is inhibited by using shRNA transfected by lentivirus, and in a CTCL cell line (Hut78 and Myla) with low PEG10 expression, different forms of PEG10 protein isomers are respectively overexpressed: hut78-RF1b and Myla-RF1b express only RF1b, Hut78-RF1b/2 and Myla-RF1b/2 express only RF1b/2, Hut78-RF1b/2+ CNF and Myla-RF1b/2+ CNF express both RF1b/2 and CNF.
The results of flow cytometry for RNA interference in lentivirus transfected cells are shown in FIG. 2, the left panel shows the forward scattered light (FSC) signal at linear level for PEG10 silenced cells (HH-shPEG10-1, shPEG10 in the figure) and control HH cells (HH-Ctrl, Crtl in the figure), and the right panel shows the average signal intensity.
The results of successful transfection collected by fluorescence microscope are shown in fig. 3, the left Image is the Image of HH-shPEG10-1(shPEG10) and HH-Ctrl (Ctrl) collected by fluorescence microscope, and the right Image is the average cell area calculated by Image J, and it can be seen that the cell area of HH-shPEG10 is reduced compared with HH-Ctrl, which indicates that the cells are obviously reduced after knocking down PEG10 in HH, and the growth of tumor cells is inhibited.
Example 2 cell colony formation assay (CFC)
When a single cell is propagated for more than 6 generations in vitro, the cell population formed by the descendants becomes a colony or a clone. Each clone contained more than 50 cells, ranging in size from 0.3 to 1.0 mm. The tumor cell colony formation rate is indicative of the in vitro tumorigenic capacity of the cells.
The test cells used in this example were the cells HH-Ctrl, HH-shPEG10-1, HH-shPEG10-2, Hut78-RF1b, Hut78-RF1b/2, Hut78-RF1b/2+ CNF, Hut78-vector, Myla-RF1b, Myla-RF1b/2, Myla-RF1b/2+ CNF, and Myla-vector prepared in example 1. This example uses methylcellulose semisolid medium (H4230, Stem Cell Technologies Vancouver Canada), and the procedure is performed with reference to the reagent instructions.
(1) Complete methylcellulose medium preparation:
1) h4230 medium (incomplete medium, 80mL) was thawed on a shaker at 4 ℃ overnight.
2) 20mL of IMDM medium was added to H4230 medium after thawing at 1)4 degrees overnight to make up to 100mL to obtain complete medium.
3) The complete medium obtained in step 2) was vigorously shaken using a shaker for about 1-2 minutes and allowed to stand for 5-10 minutes until air bubbles were discharged to the top.
4) 10mL of the complete medium mixed in step 3) was aspirated sequentially through a 10mL needle cannula + a 16 gauge blunt needle.
5) And (3) sequentially subpackaging 10mL of complete culture medium pumped in the step 4) into 3 round-flat-bottom flow type tubes with the volume of 3 mL/tube, namely the complete methyl cellulose culture medium used in the subsequent experiment.
6) The completely cultured medium can be used for subsequent experiments, and the rest is stored in a refrigerator at-80 deg.C.
(2) Preparation of the experiment:
1) preparing a culture dish: placing two 35mm small culture dishes and 1 small culture dish without a cover, which is added with 2-3mL of sterilized water for injection, in a culture dish with the diameter of 100 mm;
2) cell preparation: after the cells to be tested are centrifuged, the cells are diluted by IMDM + 2% FBS and then counted, and finally the cells are adjusted to the density of 3x103Mixing the cell suspension with the cell suspension solution of one/mL, and adding 200 mu L of the cell suspension solution into 3mL of completely-packaged methylcellulose culture medium;
3) forcibly swirling the mixture of the cell suspension and the culture medium in the step 2) for at least 4 seconds to fully and uniformly mix the culture medium and the cells, standing for at least 5 minutes to discharge bubbles to the top to obtain a uniformly mixed cell culture solution;
4) extracting the cell culture solution uniformly mixed in the step 4) by using a 16- # blunt needle and a 5mL needle tube, respectively punching 1.2mL of the cell culture solution slightly above the center distance between the two 35mm small culture dishes in the step 1) and the bottom surface, preventing the needle tip from touching the bottom of the culture dish, and finally slowly and uniformly shaking the culture dish to uniformly spread the culture solution to the whole small culture dish;
5) covering the two small culture dishes with covers, placing in a 100mm big dish, adding a small culture dish without cover, adding 2-3mL sterile water for injection, placing at 37 deg.C and 5% CO2And culturing in a cell culture box with 100% humidity for 12-14 days;
6) the diameter is measured by a scale under the microscope visual field to judge the small, medium and large colonies; drawing a cross at the bottom of the culture dish, and respectively counting the size and the number of colonies in four quadrants; finally, a low power microscope is used for photographing.
The results of colony formation experiments for HH-Ctrl, HH-shPEG10-1, and HH-shPEG10-2 are shown in FIG. 4, in which the upper graph is a statistical graph of the number of small, medium, and large colonies, and the lower graph is a low power microscope photograph. The number of colonies formed in the semisolid medium after PEG10 silencing in HH cells, i.e., HH-shPEG10-1(shPEG 10-1 in the figure) and HH-shPEG10-2 (shPEG 10-2 in the figure), was significantly reduced by more than 2-fold compared to HH-ctrl (ctrl).
The results of cell colony formation experiments of Hut78-RF1b, Hut78-RF1b/2, Hut78-RF1b/2+ CNF and Hut78-vector are shown in FIG. 5, wherein the upper graph is a statistical graph of the numbers of small, medium and large colonies, and the lower graph is a low power microscope photograph. The number of colonies formed in the semisolid medium after PEG10-RF1b overexpression in Hut78 cells (i.e., Hut78-RF1b (RF1b in the figure)), was significantly increased, whereas overexpression of RF1b/2, RF1b/2+ CNF (i.e., Hut78-RF1b/2 (RF 1b/2 in the figure) and Hut78-RF1b/2+ CNF (RF 1b/2+ CNF in the figure) caused only a slight increase in the number of colonies.
The results of Myla-RF1b, Myla-RF1b/2, Myla-RF1b/2+ CNF, Myla-vector cell colony formation experiments are shown in FIG. 6, wherein the upper graph is a statistical graph of the number of small, medium and large colonies, and the lower graph is a low power microscope photograph. The number of colonies formed in semi-solid medium following PEG10-RF1b overexpression in Myla78 cells (i.e., Myla78-RF1b (RF1b in the figure)) was significantly increased, whereas overexpression of RF1b/2, RF1b/2+ CNF (i.e., Myla78-RF1b/2 (RF 1b/2 in the figure) and Myla78-RF1b/2+ CNF (RF 1b/2+ CNF in the figure) caused only a weak increase in the number of colonies.
The in vitro tumor forming ability of the CTCL cell is weakened and the in vitro growth of the CTCL cell is inhibited after the PEG10 gene is knocked down, and the in vitro tumor forming ability of the CTCL cell is enhanced after the PEG10 is over-expressed, so that the in vitro growth advantage of the CTCL cell is enhanced.
Example 3 construction of a CTCL mouse xenograft model
This example uses the NSG (NOD/scid Interleukin-2 receptor gamma-chain-deficient non-obese diabetic/severe combined immunodeficiency mouse model, 6 weeks old, weighing 18-20g, purchased from Witongli.
(1) Tumor cell culture:
the tumor cells used in this example were the cells prepared in example 1, HH-shPEG10-1, HH-Ctrl, Hut78-RF1b and Hut 78-vector. Tumor cells were cultured at 37 ℃ with 5% CO2The culture medium of (1) was RPMI1640 medium containing 10% inactivated fetal bovine serum and 1% streptomycin, and the cells were passaged 1 time a day.
(2) Inoculation of tumor cells
The groups were divided into four experimental groups of HH-shPEG10, HH-Ctrl, Hut78-RF1b, and Hut78-vector, three NSG mice per group. PBS resuspended corresponding tumor cells at 5X 106One/100. mu.L of the cells were inoculated into the subcutaneous/right axilla of the right flank of NSG mice at a concentration of 5X 106Each was resuspended in 100ul PBS. Obtaining the CTCL mouse xenograft model.
(3) Detecting the index
1) Tumor volume:
tumor volumes were measured 3 times per week using a vernier caliper, and the long (L) and short (W) diameters of the tumors were measured, and the volume was calculated as: tumor volume of 0.5 × L × W2。
2) And (3) weight detection:
animal weights were weighed before inoculation, 3 times per week during observation, and before euthanasia.
3) General observations were:
the adaptive feeding period and the experimental period are observed for 1 time every day, and the observation contents comprise tumor nodule ulceration conditions, animal mental states, diet conditions and the like.
4) Tumor weight photographing:
at the end of the experiment, euthanized animals and tumors were photographed and recorded, and mice were stripped of tumors and weighed and photographed and recorded.
(4) Human terminal point of experimental animal
In the course of the experiment, animals should be euthanized if any one or more of the following conditions occur:
when the tumor bearing volume of a single animal exceeds 3000mm3Or the average tumor volume of the whole group exceeds 2000mm3；
Ulceration, necrosis or infection of the tumor;
abnormal movement or paralysis of the animal;
the animal weight was reduced by more than 20% of the weight at the start of the drug treatment.
(5) Death by peace and happiness
At the end of the experiment or at the end of the humanitarian end point, CO is used2Animal euthanization by inhalation
The experimental results of mice of the HH-shPEG10 group and HH-Ctrl group are shown in fig. 7, in which the left side is a photograph of the mice 45 days after injection of tumor cells, the middle is a photograph of the tumors of the mice of the HH-shPEG10 group and HH-Ctrl group, and the right side is a statistical plot of the change in tumor volume with time of the mice of the HH-shPEG10 group and HH-Ctrl group, and it can be seen that the tumor formation rate and volume of the mice injected with HH-shPEG10 are significantly lower than those of the mice injected with HH-Ctrl group, indicating that the silencing of PEG10 gene in HH cells can inhibit the growth of CTCL tumors in the xenograft mouse model.
The experimental results of mice in the Hut78-RF1b group and the Hut78-vector group are shown in FIG. 8, wherein the left graph is a photograph of the mice 40 days after being injected with tumor cells, the middle graph is a photograph of the tumors of the mice in the Hut78-RF1b group and the Hut78-vector group, and the right graph is a statistical plot of the tumor volumes of the mice in the Hut78-RF1b group and the Hut78-vector group over time, so that the tumor formation rate and the tumor volume of the mice injected with Hut78-RF1b are higher than those of the mice injected with Hut78-vector, indicating that the overexpression of PEG10 gene in the Hut78 cells can promote CTCL tumor growth in a xenograft mouse model.
In conclusion, the PEG10 gene silencing can inhibit CTCL tumor growth in a xenograft mouse model, and the PEG10 gene overexpression can enhance the in vitro and in vivo growth advantages of CTCL.
Example 4 treatment of CTCL cells with HDAC inhibitors, proteasome inhibitors
HDAC inhibitor treatment of HH-shPEG10 cells
Cells HH-shPEG10-1 and HH-Ctrl prepared in example 1 were treated with the HDAC inhibitors romidepsin (0mol/L, 1nmol/L, 2nmol/L, and 4nmol/L) and SAHA (0 μmol/L, 0.25 μmol/L, 0.5 μmol/L, and 0.75 μmol/L) in concentration gradients for MTS detection and Annexin V apoptosis detection at 48 hour time points, and the resulting data were used to calculate relative cell viability values and drug-induced specific apoptosis rates.
After centrifugation, the cells were resuspended in complete medium and counted in 12-well plates at 1X105Plating at a cell density of 2 mL/mL, and adding DMSO or a corresponding volume of a prepared drug to ensure that the final concentration is the drug concentration. The cell culture conditions are constant temperature of 37 ℃, 100% relative humidity and 5% CO2。
Immunoblot experiments examined the extent of cleavage of Caspase-3 enzyme (antibodies anti-Caspase3(CST, #9662), anti-cleared Caspase3(CST, # 9664)).
The results of the experiment are shown in FIGS. 9 and 14. The upper graph in fig. 9 is a statistical graph of cell viability values, the lower graph is a drug-induced specific apoptosis rate, and the cell viability of the PEG 10-silenced cell (HH-shPEG10) is significantly lower than that of the control cell group (HH-Ctrl) after applying romidepsin and SAHA, and the specific apoptosis is significantly higher than that of the control cell group, which indicates that PEG10 silencing can promote the sensitivity of HH cells to HDAC inhibitors. FIG. 14 is a result of immunoblot experiments showing that SAHA-induced cleavage of Caspase-3 was higher in PEG10 silenced HH cells (HH-shPEG10) than in control cells (HH-Ctrl), indicating that PEG10 silenced HH cells were more apoptotic and more sensitive to drugs.
Secondly, treating Hut78-RF1b cells by proteasome inhibitor
Cells HH-shPEG10-1 and HH-Ctrl prepared in example 1 were treated with the proteasome inhibitors bortezomib (0nmol/L, 12.5nmol/L, 25nmol/L and 50nmol/L) and carfilzomib (0nmol/L, 2nmol/L, 4nmol/L and 6nmol/L) in a concentration gradient, and MTS detection and Annexin V apoptosis detection were performed at 48 hour time points, and the resulting data were used to calculate relative cell viability values and drug-induced specific apoptosis rates.
The specific treatment method refers to 'one', and is different in that the romidepsin and SAHA in concentration gradient are replaced by bortezomib and carfilzomib in concentration gradient.
Immunoblotting experiments were performed to detect the degree of cleavage of Caspase-3 enzyme.
The results of the experiment are shown in FIGS. 10 and 15. The upper graph in fig. 10 is a statistical graph of cell viability values, the lower graph is a drug-induced specific apoptosis rate, and when bortezomib and carfilzomib are applied to a cell with silencing PEG10 (HH-shPEG10), the specific apoptosis is obviously lower than that of a control cell group (HH-Ctrl), and the specific apoptosis is obviously higher than that of the control cell group, which indicates that silencing of PEG10 can promote sensitivity of HH cells to proteasome inhibitors. FIG. 15 is a result of immunoblot experiments showing that bortezomib-induced cleavage of Caspase-3 was higher in PEG 10-silenced HH cells (HH-shPEG10) than in control cells (HH-Ctrl), indicating that PEG 10-silenced HH cells were more apoptotic and more sensitive to drugs.
Thirdly, HDAC inhibitor treatment of Hut78-RF1b cells
Cells Hut78-RF1b and Hut78-vector prepared in example 1 were treated with Romidepsin (0nmol/L, 1nmol/L, 2nmol/L and 4nmol/L) and SAHA (0. mu. mol/L, 0.25. mu. mol/L, 0.5. mu. mol/L and 1. mu. mol/L) in concentration gradients for MTS detection and Annexin V apoptosis detection at the 48 hour time point, with the resulting data used to calculate relative cell viability values and drug-induced specific apoptosis rates.
The specific treatment method refers to 'one', except that cells HH-shPEG10-1 and HH-Ctrl are replaced by cells Hut78-RF1b and Hut 78-vector.
Immunoblotting experiments were performed to detect the degree of cleavage of Caspase-3 enzyme.
The results of the experiment are shown in FIGS. 11 and 16. The upper graph in fig. 11 is a statistical graph of cell viability values, the lower graph is a specific apoptosis rate induced by drugs, the cell viability of the cells (Hut78-RF1b) over-expressed by PEG10-RF1b is obviously higher than that of the control cell group (Hut78-vector) after romidepsin and SAHA are applied, the specific apoptosis is obviously lower than that of the control cell group, and the over-expression of PEG10 can promote the resistance of the Hut78 cells to HDAC inhibitors. FIG. 16 is a result of immunoblot experiments showing that romidepsin-induced cleavage of Caspase-3 was lower in Hut78 cells over-expressed by PEG10-RF1b (Hut78-RF1b) than in control cells (Hut78-vector), indicating that Hut78 cells over-expressed by PEG10-RF1b were less apoptotic and less sensitive to drugs.
Fourthly, treating Hut78-RF1b cells by proteasome inhibitor
Cells Hut78-RF1b and Hut78-vector prepared in example 1 were treated with bortezomib (0nmol/L, 25nmol/L, 50nmol/L and 75nmol/L) and carfilzomib (0nmol/L, 2nmol/L, 4nmol/L and 6nmol/L) in concentration gradients for MTS detection and Annexin V apoptosis detection at 48 hours, with the resulting data used to calculate relative cell viability values and drug-induced specific apoptosis rates.
The specific treatment method refers to 'one', except that cells HH-shPEG10-1 and HH-Ctrl are replaced by cells Hut78-RF1b and Hut78-vector, and romidepsin and SAHA in concentration gradient are replaced by bortezomib and carfilzomib in concentration gradient.
Immunoblotting experiments were performed to detect the degree of cleavage of Caspase-3 enzyme.
The results of the experiment are shown in FIGS. 12 and 17. The upper graph in fig. 12 is a statistical graph of cell viability values, the lower graph is a specific apoptosis rate induced by drugs, the cell viability of the cell over-expressed by PEG10-RF1b (Hut78-RF1b) after bortezomib and carfilzomib application is obviously higher than that of a control cell group (Hut78-vector), the specific apoptosis is obviously lower than that of the control cell group, and the over-expression of PEG10 can promote the resistance of the Hut78 cell to proteasome inhibitors. FIG. 17 is a result of immunoblot experiments showing that bortezomib-induced cleavage of Caspase-3 was lower in Hut78 cells over-expressed by PEG10-RF1b (Hut78-RF1b) than in control cells (Hut78-vector), indicating that Hut78 cells over-expressed by PEG10-RF1b were less apoptotic and less sensitive to drugs.
Fifthly, treating Myla-RF1b cells by using HDAC inhibitor and proteasome inhibitor
The cells Myla-RF1b and Myla-vector prepared in example 1 were treated with 2nmol/L romidepsin, 2.5. mu. mol/L SAHA, 25nmol/L bortezomib, and 7nmol/L carfilzomib, respectively, and at 48 hours, the percentage of GFP + in the cells was measured by flow cytometry and the drug-induced GFP + reduction rate was calculated, and the GFP + reduction rates of the PEG10-RF1b overexpression group (Myla-RF1b) and the control group (Myla-vector) were compared to obtain relative values.
The specific treatment method refers to 'one', except that cells HH-shPEG10-1 and HH-Ctrl are replaced by cells Myla-RF1b and Myla-vector, and Romidepsin and SAHA in concentration gradient are replaced by 2nmol/L Romidepsin, 2.5. mu. mol/L SAHA, 25nmol/L bortezomib, and 7nmol/L Carfilzomib.
The results, see figure 13, indicate that the rate of GFP + decline of the cell Myla-RF1b (RF1b) after drug treatment is significantly lower than that of the cell Myla-vector (vector), indicating that PEG10 overexpression promotes increased resistance of Myla cells to two HDAC inhibitors (romidepsin and SAHA) and two proteasome inhibitors (bortezomib and carfilzomib).
Example 5 treatment of CTCL cells with HLM006474
Transcription factors E2F1 and E2F4 have proved to be capable of directly binding to the promoter region of PEG10 to regulate the transcription expression, and a small molecule compound HLM006474 can inhibit the DNA binding capability of E2F4 and part of E2F 1. This example is used to demonstrate whether HLM006474 is able to effectively inhibit the transcriptional regulatory link of PEG 10.
HLM006474 treatment of cells HH and SZ4
The CTCL cell lines HH and SZ4 with high PEG10 expression were treated with HLM006474 (0. mu.M, 5. mu.M, 10. mu.M, 15. mu.M and 20. mu.M) in concentration gradient and tested for the expression of PEG10 gene and KLF2 gene after 72 hours using immunoblotting. KLF2 is a key PEG10 negative regulator gene. The antibody used was anti-PEG10(Novus, NBP 2-13749); anti-KLF2(Abcam, ab 236507).
The specific treatment method was as described in "one" of example 4, except that cells HH-shPEG10 and HH-Ctrl were replaced with cells HH and SZ4, and romidepsin and SAHA in concentration gradient were replaced with HLM006474 in concentration gradient.
The experimental results are shown in fig. 18, and HH and SZ4 showed that the PEG10 expression level was inhibited and accompanied by an increase in KLF2 expression level, both concentration gradient-dependent, 72 hours after HLM006474 treatment.
II, HLM006474 treatment of cells HH-shPEG10
Cells prepared in example 1, HH-shPEG10-1 and HH-Ctrl, were treated with HLM006474(0 μ M, 10 μ M, 15 μ M, 20 μ M and 30 μ M) in concentration gradient, MTS assay and Annexin V apoptosis assay were performed at 48 hour time point, and the resulting data were used to calculate relative cell viability values and drug-induced specific apoptosis rates.
The specific treatment method was as in "one" of example 4, except that romidepsin and SAHA in concentration gradient were replaced with HLM006474 in concentration gradient.
The experimental results are shown in figure 19, compared with PEG10 silenced HH cell (HH-shPEG10-1, shown as HH-shPEG10), the control cell (HH-Ctrl) has stronger sensitivity to HLM006474, shows lower cell viability and higher specific apoptosis after the same concentration of drug treatment, and can see that HLM006474 concentration gradient dependency inhibits the cell viability and induces apoptosis of the two cells, the cell viability of the HH-Ctrl is obviously lower than that of the HH-shPEG10-1, and the specific apoptosis is higher than that of the HH-shPEG 10-1.
This example shows that PEG10 is an important target for the action of HLM006474, which inhibits the expression of PEG10 gene.
Example 6 in vivo antitumor Effect test of HLM006474
The mouse used in this example was a CTCL mouse xenograft model prepared using HH, which was prepared by referring to example 3, except that HH-shPEG10-1 was replaced with HH when the average tumor volume reached about 100mm3In the process, mice with moderate individual tumor volume are selected and grouped, the mice are randomly distributed into each experimental group according to the tumor volume, 4 mice in each group are divided into groups, and the administration is started on the same day, and the specific administration scheme is shown in table 1.
Table 1 dosing schedule
Figure BDA0003201913270000191
Note: a: the administration volume is calculated according to the body weight of the experimental animal according to 15 mu L/g; b: day1, 3, 5/week refers to 3 doses per week for 2 weeks for 6 total doses.
(1) Observation of experimental animals after drug withdrawal
After the last administration, the body weight and tumor growth status of the experimental animals were continuously observed for 3 days. During the observation period, tumor volumes and animal body weights were measured 3 times per week and recorded. After the observation was completed, the experiment was ended.
(2) Human terminal point of experimental animal
In the course of the experiment, animals should be euthanized if any one or more of the following conditions occur:
when the tumor bearing volume of a single animal exceeds 3000mm3Or the average tumor volume of the whole group exceeds 2000mm3；
Ulceration, necrosis or infection of the tumor;
abnormal movement or paralysis of the animal;
the animal weight was reduced by more than 20% of the weight at the start of the drug treatment.
(3) Death by peace and happiness
At the end of the experiment or at the end of the humanitarian end point, CO is used2Animal euthanization by inhalation
(4) Detecting the index
1) Tumor volume:
tumor volumes were measured 3 times per week using a vernier caliper, and the long (L) and short (W) diameters of the tumors were measured, and the volume was calculated as: tumor volume of 0.5 × L × W2。
2) And (3) weight detection:
animals were weighed prior to vaccination, group (i.e., prior to first dose), 3 times per week during dosing/observation, and before euthanasia.
3) General observations were:
the adaptive feeding period and the experimental period are observed for 1 time every day, and the observation contents comprise tumor nodule ulceration conditions, animal mental states, diet conditions and the like.
4) Tumor weight photographing:
at the end of the experiment, euthanized animals and tumors were photographed and recorded, and mice were stripped of tumors and weighed and photographed and recorded.
The results are shown in FIGS. 20-21. The left panel in fig. 20 is a photograph of a mouse after the end of the experiment, and the right panel is a photograph of a mouse tumor. FIG. 21 is a statistical analysis of tumor volume and body weight of mice as a function of treatment frequency. In an in-vivo mouse xenograft model, the HLM006474 can obviously reduce the tumor formation capacity of CTCL cells, and the tumor growth is gradually inhibited in a Control group (Control) average tumor volume, and the test drug HLM006474 has obvious tumor inhibition effect at a dose of 30 mg/kg. The mice have good activity and eating state during administration, and the weight mean value of animals of an administration group is not significantly different (p is more than 0.05) compared with that of a control group, which indicates that HLM00647 has relatively low toxic and side effects in vivo and better tolerance of the mice.
Example 7HLM006474 treatment of PBMCs
Peripheral Blood Mononuclear Cells (PBMCs) from 2 blood-affected CTCL patients (L-CTCL) and 2 healthy subjects (Normal) were extracted and subjected to a concentration gradient HLM006474 treatment, and apoptosis was detected after 24 hours.
First, PBMC is extracted
(1) Uniformly mixing 8mL of EDTA anticoagulated peripheral blood with 8mL of calcium-free and magnesium ion Phosphate Buffer Solution (PBS) to obtain 16 mL;
(2) prepare 4 tubes of 15mL and add 3mL of Ficoll-Paque PLUS (GE Healthcare, Pittsburgh, Pennsylvania, USA);
(3) inclining the centrifuge tube, slowly adding 4mL of the uniformly mixed peripheral blood on 3mL of Ficoll-Paque PLUS, wherein the height ratio of the two forms 3: 4, and the total amount is 4 tubes;
(4) centrifuge as soon as possible, 400g at room temperature for 40 min, carefully remove the upper light yellow serum;
(5) sucking out the white membrane of the Ficoll-Paque PLUS and the serum interface layer, namely the peripheral blood mononuclear cells, adding 6-8mL of PBS, centrifuging at room temperature for 10 minutes at 80g, repeating the steps, and counting. Cryopreserved with 10% DMSO + 90% Fetal Bovine Serum (FBS).
Two, HLM006474 treatment
The PBMCs prepared above were treated with HLM006474 in concentration gradient (0. mu.M, 2.5. mu.M, 5. mu.M, 10. mu.M, 20. mu.M and 40. mu.M) and Annexin V apoptosis assay was performed at 24 hour time point, and the data was used to calculate drug-induced specific apoptosis rate.
The specific treatment method was as in "one" of example 4, except that cells HH-shPEG10-1 and HH-Ctrl were replaced with PBMC prepared as described above, and romidepsin and SAHA in concentration gradient were replaced with HLM006474 in concentration gradient.
The results are shown in fig. 22, which indicates that HLM006474 can significantly induce apoptosis of neoplastic CTCL cells (blood invading CTCL-1 and blood invading-CTCL-2), and can induce apoptosis at lower concentrations, while PBMCs from normal control sources (normal control-1 and normal control-2) are insensitive to HLM006474, and do not induce significant apoptosis even at high concentrations, indicating that HLM 006474-induced apoptosis occurs specifically in neoplastic T cells.
Example 8 treatment of CTCL cells with HLM006474 in combination with HDAC and proteasome inhibitors, respectively
The cells HH and CTCL primary cells (PBMC cells obtained by separating peripheral blood of volunteer patients with skin lymphoma outpatient of the first hospital of Beijing university, wherein the volunteer patients are patients with advanced-stage skin T cell lymphoma) are respectively mixed at 1x105mL and 1X106The density of (2) was seeded in 24-well plates and 3-5 concentration gradients of single dose drug and combination drug were added for 48 hours.
Treatment of CTCL cells with HLM006474 in combination with HDAC inhibitors
1. Administration of drugs
Cell HH and CTCL primary cells were treated with concentration-graded SAHA (0.25 μ M, 0.5 μ M, 1 μ M, 2 μ M), concentration-graded HLM006474(2.5 μ M, 5 μ M, 10 μ M, 20 μ M), and concentration-graded HLM006474(2.5 μ M, 5 μ M, 10 μ M, 20 μ M), respectively, in combination with the same volume of DMSO-treated cells, and Annexin V apoptosis assays were performed at 48 hour time points, with the results data used to calculate drug-induced specific apoptosis rate.
Cell HH was treated with romidepsin (0.5 μ M, 1 μ M, 2 μ M, 4 μ M) in concentration gradient, HLM006474(2.5 μ M, 5 μ M, 10 μ M, 20 μ M) in concentration gradient, and HLM006474(2.5 μ M, 1 μ M, 2 μ M, 4 μ M) in concentration gradient in combination with DMSO in concentration gradient, negative controls were DMSO treated cells with Annexin V apoptosis assay at 48 hours time point and the results data were used to calculate drug-induced specific apoptosis rate.
The specific treatment method was as in "one" of example 4, except that cells HH-shPEG10-1 and HH-Ctrl were replaced with cells HH and CTCL primary cells, and romidepsin and SAHA in concentration gradient were replaced with corresponding SAHA and romidepsin.
2. Evaluation of synergistic Effect
The synergistic effect of the combined application of two drugs, i.e. the synergy index (CI), was evaluated by the CompuSyn software, which performs the calculation of drug synergistic effect based on the mode of quantitative analysis of the multi-drug dose-effect relationship by Chou-Talalay (Chou, 2010). Drug-induced specific apoptosis rate was taken as the drug effect score (Fa). The concentrations of the single dose of the drug and the combination drug and the corresponding cell-specific apoptosis rates are respectively input into the software, and the software fits a curve, namely a CI-Fa graph, which shows CI values under different Fas.
Evaluation criteria: CI is 1: the two medicines produce a superposition effect; CI is less than 1: the two medicines produce synergistic effect; CI is greater than I: the two drugs produce an antagonistic effect. Calculation of synergistic effect of drug killing of tumor cells requires judgment at a higher drug effect score (e.g., Fa ═ 0.9).
The results are shown in fig. 23, where SAHA was treated with SAHA single drug, HLM was treated with HLM006474 single drug, SAHA + HLM was treated with HLM006474 in combination with SAHA, Romi was treated with romidepsin single drug, Romi + HLM was treated with HLM006474 in combination with romidepsin, HH (top, bottom) on the left and CTCL primary cells (middle panel) showed specific apoptosis rates 48 hours after HLM006474 single drug, two HDAC inhibitor (SAHA/romidepsin) single drug, HLM006474 in combination with HDAC inhibitor, respectively, and comp syn software was used to fit synergy index (CI) curves generated by HLM006474 in combination with HDAC inhibitor on the right.
II, HLM006474 combined with proteasome inhibitor to treat CTCL cells
1. Administration of drugs
Cell HH and CTCL primary cells were treated with bortezomib (6.25 μ M, 12.5 μ M, 25 μ M, 50 μ M) in concentration gradient, HLM006474(2.5 μ M, 5 μ M, 10 μ M, 20 μ M) in concentration gradient, and a combination of bortezomib (6.25 μ M, 12.5 μ M, 25 μ M, 50 μ M) in concentration gradient and HLM006474(2.5 μ M, 5 μ M, 10 μ M, 20 μ M) in concentration gradient, respectively, with the negative control being the same volume of DMSO-treated cells, with Annexin V apoptosis detection at the 48 hour time point, and the results data used to calculate drug-induced specific apoptosis rate.
Cell HH was treated with a concentration gradient of carfilzomib (1 μ M, 2 μ M, 4 μ M, 8 μ M), HLM006474(2.5 μ M, 5 μ M, 10 μ M, 20 μ M), and a combination of carfilzomib (1 μ M, 2 μ M, 4 μ M, 8 μ M) and HLM006474(2.5 μ M, 5 μ M, 10 μ M, 20 μ M), negative controls were DMSO treated in the same volume, and Annexin V apoptosis assays were performed at 48 hours time points and the results data were used to calculate drug-induced specific apoptosis rates.
The specific treatment method was as in "one" of example 4, except that cells HH-shPEG10-1 and HH-Ctrl were replaced with cells HH and CTCL primary cells, and romidepsin and SAHA in concentration gradient were replaced with corresponding bortezomib and carfilzomib.
2. Evaluation of synergistic Effect
The synergistic effect of the combined application of two drugs, i.e. the synergy index (CI), was evaluated by the CompuSyn software, which performs the calculation of drug synergistic effect based on the mode of quantitative analysis of the multi-drug dose-effect relationship by Chou-Talalay (Chou, 2010). Drug-induced specific apoptosis rate was taken as the drug effect score (Fa). The concentrations of the single dose of the drug and the combination drug and the corresponding cell-specific apoptosis rates are respectively input into the software, and the software fits a curve, namely a CI-Fa graph, which shows CI values under different Fas.
Evaluation criteria: CI is 1: the two medicines produce a superposition effect; CI is less than 1: the two medicines produce synergistic effect; CI is more than 1: the two drugs produce an antagonistic effect. Calculation of synergistic effect of drug killing of tumor cells requires judgment at a higher drug effect score (e.g., Fa ═ 0.9).
The experimental results are shown in fig. 24, wherein BZ is treated with bortezomib as a single drug, HL is treated with MHLM006474 as a single drug, BZ + HLM is treated with HLM006474 in combination with bortezomib, CZ is treated with carfilzomib, CZ + HLM is treated with HLM006474 in combination with carfilzomib, HH (top and bottom) and CTCL primary cells (middle) are shown in the left side, the specific apoptosis rates of HH (top and bottom) and CTCL primary cells after 48 hours of treatment with HLM006474 as a single drug, two proteasome inhibitors (bortezomib/carfilzomib), and HLM006474 in combination with proteasome inhibitors, respectively, and the right side is shown in a synergy index (CI) curve generated by fitting HLM006474 in combination with proteasome inhibitors by using Compusyn software.
The above results indicate that HLM006474 can produce a medium-strong synergistic effect with both HDAC inhibitors (SAHA, romidepsin) and protease inhibitors (bortezomib, carfilzomib) both in CTCL cell lines and primary cells, indicating that HLM006474 can enhance the sensitivity of CTCL cells to both inhibitors.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Sequence listing
<110> first Hospital of Beijing university
<120> application of PEG10 gene/protein as target point in preparation of anti-skin T cell lymphoma product
<130> 211187
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ttctccgaac gtgtcacgt 19
<210> 2
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
cccagtgcca gatcttcat 19
<210> 3
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
aaagctggag cgctcccact a 21
<210> 4
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
uucuccgaac gugucacgu 19
<210> 5
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
cccagugcca gaucuucau 19
<210> 6
<211> 21
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
aaagcuggag cgcucccacu a 21
<210> 7
<211> 1080
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
ctgggtcccg actgcccacc tcctcctcct ccccctcccc ccaacaacaa caacaacaac 60
aactccaagc acaccggcca taagagtgcg tgtgtcccca acatgaccga acgaagaagg 120
gacgagctct ctgaagagat caacaactta agagagaagg tcatgaagca gtcggaggag 180
aacaacaacc tgcagagcca ggtgcagaag ctcacagagg agaacaccac ccttcgagag 240
caagtggaac ccacccctga ggatgaggat gatgacatcg agctccgcgg tgctgcagca 300
gctgctgccc caccccctcc aatagaggaa gagtgcccag aagacctccc agagaagttc 360
gatggcaacc cagacatgct ggctcctttc atggcccagt gccagatctt catggaaaag 420
agcaccaggg atttctcagt tgatcgtgtc cgtgtctgct tcgtgacaag catgatgacc 480
ggccgtgctg cccgttgggc ctcagcaaag ctggagcgct cccactacct gatgcacaac 540
tacccagctt tcatgatgga aatgaagcat gtctttgaag accctcagag gcgagaggtt 600
gccaaacgca agatcagacg cctgcgccaa ggcatggggt ctgtcatcga ctactccaat 660
gctttccaga tgattgccca ggacctggat tggaacgagc ctgcgctgat tgaccagtac 720
cacgagggcc tcagcgacca cattcaggag gagctctccc acctcgaggt cgccaagtcg 780
ctgtctgctc tgattgggca gtgcattcac attgagagaa ggctggccag ggctgctgca 840
gctcgcaagc cacgctcgcc accccgggcg ctggtgttgc ctcacattgc aagccaccac 900
caggtagatc caaccgagcc ggtgggaggt gcccgcatgc gcctgacgca ggaagaaaaa 960
gaaagacgca gaaagctgaa cctgtgcctc tactgtggaa caggaggtca ctacgctgac 1020
aattgtcctg ccaaggcctc aaagtcttcg ccggcgggaa actccccggc cccgctgtag 1080
<210> 8
<211> 2229
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ctgggtcccg actgcccacc tcctcctcct ccccctcccc ccaacaacaa caacaacaac 60
aactccaagc acaccggcca taagagtgcg tgtgtcccca acatgaccga acgaagaagg 120
gacgagctct ctgaagagat caacaactta agagagaagg tcatgaagca gtcggaggag 180
aacaacaacc tgcagagcca ggtgcagaag ctcacagagg agaacaccac ccttcgagag 240
caagtggaac ccacccctga ggatgaggat gatgacatcg agctccgcgg tgctgcagca 300
gctgctgccc caccccctcc aatagaggaa gagtgcccag aagacctccc agagaagttc 360
gatggcaacc cagacatgct ggctcctttc atggcccagt gccagatctt catggaaaag 420
agcaccaggg atttctcagt tgatcgtgtc cgtgtctgct tcgtgacaag catgatgacc 480
ggccgtgctg cccgttgggc ctcagcaaag ctggagcgct cccactacct gatgcacaac 540
tacccagctt tcatgatgga aatgaagcat gtctttgaag accctcagag gcgagaggtt 600
gccaaacgca agatcagacg cctgcgccaa ggcatggggt ctgtcatcga ctactccaat 660
gctttccaga tgattgccca ggacctggat tggaacgagc ctgcgctgat tgaccagtac 720
cacgagggcc tcagcgacca cattcaggag gagctctccc acctcgaggt cgccaagtcg 780
ctgtctgctc tgattgggca gtgcattcac attgagagaa ggctggccag ggctgctgca 840
gctcgcaagc cacgctcgcc accccgggcg ctggtgttgc ctcacattgc aagccaccac 900
caggtagatc caaccgagcc ggtgggaggt gcccgcatgc gcctgacgca ggaagaaaaa 960
gaaagacgca gaaagctgaa cctgtgcctc tactgtggaa caggaggtca ctacgctgac 1020
aattgtcctg ccaaggcctc aaagtcttcg ccggctggca atctccccgg ccccgctgta 1080
gagggacctt cagcgaccgg gccagaaata ataaggtccc cacaagatga tgcctcatct 1140
ccacacttgc aagtgatgct ccagattcat cttccgggca gacacaccct gttcgtccga 1200
gccatgatcg cttctggtgc ttctggcaac ttcattgatc acgaatatgt tgctcaaaat 1260
ggaattcctc taagaatcaa ggactggcca atacttgtgg aagcaattga tgggcgcccc 1320
atagcatcgg gcccagttgt ccacgaaact cacgacctga tagttgacct gggagatcac 1380
cgagaggtgc tgtcatttga tgtgactcag tctccattct tccctgtcgt cctaggggtt 1440
cgctggctga gcacacatga tcccaatatc acatggagca ctcgatctat cgtctttgat 1500
tctgaatact gccgctacca ctgccggatg tattctccaa taccaccatc gctcccacca 1560
ccagcaccac aaccgccact ctattatcca gtagatggat acagagttta ccaaccagtg 1620
aggtattact atgtccagaa tgtgtacact ccagtagatg agcacgtcta cccagatcac 1680
cgcctggttg accctcacat agaaatgata cctggagcac acagtattcc cagtggacat 1740
gtgtattcac tgtccgaacc tgaaatggca gctcttcgag attttgtggc aagaaatgta 1800
aaagatgggc taattactcc aacgattgca cctaatggag cccaagttct ccaggtgaag 1860
agggggtgga aactgcaagt ttcttatgat tgccgagctc caaacaattt tactatccag 1920
aatcagtatc ctcgcctatc tattccaaat ttagaagacc aagcacacct ggcaacgtac 1980
actgaattcg tacctcaaat acctggatac caaacatacc ccacatatgc cgcgtacccg 2040
acctacccag taggattcgc ctggtaccca gtgggacgag acggacaagg aagatcacta 2100
tatgtacctg tgatgatcac ttggaatcca cactggtacc gccagcctcc ggtaccacag 2160
tacccgccgc cacagccgcc gcctccacca ccaccaccgc cgccgcctcc atcttacagt 2220
accctgtaa 2229
<210> 9
<211> 2229
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
ctgggtcccg actgcccacc tcctcctcct ccccctcccc ccaacaacaa caacaacaac 60
aactccaagc acaccggcca taagagtgcg tgtgtcccca acatgaccga acgaagaagg 120
gacgagctct ctgaagagat caacaactta agagagaagg tcatgaagca gtcggaggag 180
aacaacaacc tgcagagcca ggtgcagaag ctcacagagg agaacaccac ccttcgagag 240
caagtggaac ccacccctga ggatgaggat gatgacatcg agctccgcgg tgctgcagca 300
gctgctgccc caccccctcc aatagaggaa gagtgcccag aagacctccc agagaagttc 360
gatggcaacc cagacatgct ggctcctttc atggcccagt gccagatctt catggaaaag 420
agcaccaggg atttctcagt tgatcgtgtc cgtgtctgct tcgtgacaag catgatgacc 480
ggccgtgctg cccgttgggc ctcagcaaag ctggagcgct cccactacct gatgcacaac 540
tacccagctt tcatgatgga aatgaagcat gtctttgaag accctcagag gcgagaggtt 600
gccaaacgca agatcagacg cctgcgccaa ggcatggggt ctgtcatcga ctactccaat 660
gctttccaga tgattgccca ggacctggat tggaacgagc ctgcgctgat tgaccagtac 720
cacgagggcc tcagcgacca cattcaggag gagctctccc acctcgaggt cgccaagtcg 780
ctgtctgctc tgattgggca gtgcattcac attgagagaa ggctggccag ggctgctgca 840
gctcgcaagc cacgctcgcc accccgggcg ctggtgttgc ctcacattgc aagccaccac 900
caggtagatc caaccgagcc ggtgggaggt gcccgcatgc gcctgacgca ggaagaaaaa 960
gaaagacgca gaaagctgaa cctgtgcctc tactgtggaa caggaggtca ctacgctgac 1020
aattgtcctg ccaaggcctc aaagtcttcg ccggctggca atctccccgg ccccgctgta 1080
gagggacctt cagcgaccgg gccagaaata ataaggtccc cacaagatga tgcctcatct 1140
ccacacttgc aagtgatgct ccagattcat cttccgggca gacacaccct gttcgtccga 1200
gccatgatcg attctgatgc ttctggcaac ttcattgatc acgaatatgt tgctcaaaat 1260
ggaattcctc taagaatcaa ggactggcca atacttgtgg aagcaattga tgggcgcccc 1320
atagcatcgg gcccagttgt ccacgaaact cacgacctga tagttgacct gggagatcac 1380
cgagaggtgc tgtcatttga tgtgactcag tctccattct tccctgtcgt cctaggggtt 1440
cgctggctga gcacacatga tcccaatatc acatggagca ctcgatctat cgtctttgat 1500
tctgaatact gccgctacca ctgccggatg tattctccaa taccaccatc gctcccacca 1560
ccagcaccac aaccgccact ctattatcca gtagatggat acagagttta ccaaccagtg 1620
aggtattact atgtccagaa tgtgtacact ccagtagatg agcacgtcta cccagatcac 1680
cgcctggttg accctcacat agaaatgata cctggagcac acagtattcc cagtggacat 1740
gtgtattcac tgtccgaacc tgaaatggca gctcttcgag attttgtggc aagaaatgta 1800
aaagatgggc taattactcc aacgattgca cctaatggag cccaagttct ccaggtgaag 1860
agggggtgga aactgcaagt ttcttatgat tgccgagctc caaacaattt tactatccag 1920
aatcagtatc ctcgcctatc tattccaaat ttagaagacc aagcacacct ggcaacgtac 1980
actgaattcg tacctcaaat acctggatac caaacatacc ccacatatgc cgcgtacccg 2040
acctacccag taggattcgc ctggtaccca gtgggacgag acggacaagg aagatcacta 2100
tatgtacctg tgatgatcac ttggaatcca cactggtacc gccagcctcc ggtaccacag 2160
tacccgccgc cacagccgcc gcctccacca ccaccaccgc cgccgcctcc atcttacagt 2220
accctgtaa 2229

Claims (10)

1. The application of a substance for inhibiting the expression of PEG10 gene or a substance for reducing or inhibiting the activity and/or content of protein expressed by PEG10 gene is any one of the following substances:
(1) the application in preparing products for resisting cutaneous T cell lymphoma;
(2) the application in preparing products for improving the sensitivity of T cell lymphoma cells of skin to anti-tumor drugs;
(3) the application of the composition in preparing products for reducing adverse symptoms caused by the drug resistance of skin T cell lymphoma cells to anti-tumor drugs;
(4) the application in preparing products for inducing apoptosis of T cell lymphoma cells of skin;
(5) the application in preparing products for inhibiting the growth of T cell lymphoma cells of skin.
2. Use according to claim 1, characterized in that: the nucleotide sequence of the PEG10 gene is 94,656,370 th to 94,665,698 th of genbank accession number ENST 00000612748.1.
3. Use according to claim 1 or 2, characterized in that: the substance for inhibiting the expression of the PEG10 gene is PEG10 gene-related biomaterial, and the PEG10 gene-related biomaterial is any one of the following:
b1) a nucleic acid molecule that inhibits expression of PEG10 gene;
b2) an expression cassette comprising the nucleic acid molecule of b 1);
b3) a recombinant vector comprising the nucleic acid molecule of b1) or a recombinant vector comprising the expression cassette of b 2);
b4) a recombinant microorganism containing b1) the nucleic acid molecule, or a recombinant microorganism containing b2) the expression cassette, or a recombinant microorganism containing b3) the recombinant vector.
4. Use according to claim 1 or 2, characterized in that: the substance for inhibiting the expression of the PEG10 gene is HLM 006474.
5. Use according to claim 1, characterized in that: the PEG10 gene expression protein is at least one selected from RF1a protein, RF1b protein, RF1a/2 protein, RF1b/2 protein and CNF fragment.
6. The PEG10 gene related biomaterial of claim 3.
7. A product, characterized in that: comprising the substance inhibiting the expression of PEG10 gene and/or the substance reducing or inhibiting the activity and/or content of protein encoded by PEG10 gene of claims 1-5.
8. The product of claim 7, wherein: also included are HDAC inhibitors and/or proteasome inhibitors,
the product functions are any one of the following:
(1) anti-cutaneous T cell lymphoma;
(2) improving the sensitivity of T cell lymphoma cells of the skin to anti-tumor drugs;
(3) reducing adverse symptoms caused by the drug resistance of the skin T cell lymphoma cells to the anti-tumor drugs;
(4) inducing apoptosis of cutaneous T cell lymphoma cells;
(5) inhibiting the growth of T cell lymphoma cells of skin.
9. Use of substance a and substance B, said use being any one of:
(1) the application in preparing products for resisting cutaneous T cell lymphoma;
(2) the application in preparing products for improving the sensitivity of T cell lymphoma cells of skin to anti-tumor drugs;
(3) the application of the composition in preparing products for reducing adverse symptoms caused by the drug resistance of skin T cell lymphoma cells to anti-tumor drugs;
(4) the application in preparing products for inducing apoptosis of T cell lymphoma cells of skin;
(5) the application in preparing products for inhibiting the growth of T cell lymphoma cells of skin;
the substance A is the substance inhibiting the expression of the PEG10 gene and/or the substance reducing or inhibiting the activity and/or content of the protein encoded by the PEG10 gene according to claims 1 to 5, and the substance B is an HDAC inhibitor and/or a proteasome inhibitor.
The application of the PEG10 gene or PEG10 gene expression protein in screening anti-skin T cell lymphoma drug models.
CN202110910843.5A 2021-08-09 2021-08-09 Application of PEG10 gene/protein as target in preparation of anti-skin T cell lymphoma product Pending CN113648420A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1962863A (en) * 2005-11-08 2007-05-16 上海人类基因组研究中心 PEG10-siRNA inhibiting gene expression of human PEG10 and its pharmaceutical use
US20170189376A1 (en) * 2016-01-04 2017-07-06 Mor Research Applications Ltd. Butyroyloxymethyl diethyl phosphate compounds and uses thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1962863A (en) * 2005-11-08 2007-05-16 上海人类基因组研究中心 PEG10-siRNA inhibiting gene expression of human PEG10 and its pharmaceutical use
US20170189376A1 (en) * 2016-01-04 2017-07-06 Mor Research Applications Ltd. Butyroyloxymethyl diethyl phosphate compounds and uses thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LIU FENGJIE ET AL.: "138 Copy number gain at chromosome 7q21 potentiates the large cell transformation in cutaneous T cell lymphoma", 《JOURNAL OF INVESTIGATIVE DERMATOLOGY》 *
ZHAO JIN ET AL.: "Long Non-Coding RNA Paternally Expressed Imprinted Gene 10 (PEG10) Elevates Diffuse Large B-Cell Lymphoma Progression by Regulating Kinesin Family Member 2A (KIF2A) via Targeting MiR-101-3p", 《MEDICAL SCIENCE MONITOR》 *
龚守良: "《肿瘤基因放射治疗学基础》", 30 September 2013, 人民军医出版社 *

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Application publication date: 20211116