WO2024125546A1 - Use of pirin gene and protein in preparing anti-tumor targeted drug - Google Patents
Use of pirin gene and protein in preparing anti-tumor targeted drug Download PDFInfo
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- WO2024125546A1 WO2024125546A1 PCT/CN2023/138386 CN2023138386W WO2024125546A1 WO 2024125546 A1 WO2024125546 A1 WO 2024125546A1 CN 2023138386 W CN2023138386 W CN 2023138386W WO 2024125546 A1 WO2024125546 A1 WO 2024125546A1
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- tumor
- pirin
- protein
- pir
- cancer
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4703—Regulators; Modulating activity
Definitions
- the present invention belongs to the field of molecular biology and biomedicine technology, and in particular relates to the application of a new target Pirin (PIR) gene and protein for resisting Pirin high-expressing tumors in the preparation of a new anti-tumor and immunosensitization target drug.
- PIR target Pirin
- Cancer is a disease caused by abnormal cell proliferation.
- the body's immune system has an immune surveillance function.
- the immune system can recognize and activate the apoptosis pathway to eliminate these abnormal cells.
- tumor cells acquire the ability to resist apoptosis and escape the attack of the immune system, which is also one of the main reasons for the failure of tumor treatment (Igney, F.H., Krammer, P.H. Death and anti-death: tumor resistance to apoptosis [J]. Nat Rev Cancer, 2002, 2 (4): 277-88). Therefore, in-depth research on the new mechanism of tumor cell resistance to apoptosis caused by immune cells has important theoretical and practical significance for discovering new tumor treatment targets and treatment strategies.
- FAS (CD95) is a typical apoptosis-inducing receptor in the tumor necrosis factor receptor (TNFR) superfamily
- FASL (CD95L) is its ligand (Griffith, T.S., Brunner, T., Fletcher, S.M., Green, D.R., Ferguson, T.A. Fas ligand-induced apoptosis as a mechanism of immune privilege[J]. Science, 1995, 270(5239): 1189-92).
- FAS When FAS is overexpressed or binds to FASL secreted by immune cells, FAS will automatically transform into a trimer form, thereby triggering a signaling pathway cascade reaction, recruiting and activating signaling proteins such as FADD, caspase8, BID and caspase3, and ultimately leading to cell apoptosis (Kaufmann, T., Strasser, A., Jost, P.J. Fas death receptor signaling: roles of Bid and XIAP[J]. Cell Death Differ, 2012, 19(1):42-50).
- the apoptosis pathway mediated by the death receptor FAS is one of the most important ways for immune cells to kill tumors and an important way to inhibit tumor occurrence.
- Pirin proteins are members of the cupin superfamily and are encoded by PIR genes. Highly conserved in nuclear organisms (Aguayo, FP-DDC-BRBJPMGL-CUUGMCaFRole of Pirin, an Oxidative Stress Sensor Protein, in Epithelial Carcinogenesis[J]. Prepirints, 2020.). Its mRNA and protein are lowly expressed in all normal human tissues (Wendler, WM, Kremmer, E., Forster, R., Winnacker, ELIdentification of pirin, a novel highly conserved nuclear protein[J].
- Cupin superfamily is considered to be one of the most functionally diverse protein superfamilies, and its proteins are characterized by containing a conserved ⁇ -barrel structure and a unique metal ion binding sequence (Dunwell, JM Cupins: a new superfamily of functionally diverse proteins that include germins and plant storage proteins[J].
- Pirin gene and protein can be used as an anti-tumor target, and its abnormally high expression is associated with the occurrence and development of various cancers such as breast cancer, colon cancer, and prostate cancer.
- the first purpose of the present invention is to provide an application of Pirin gene and protein as anti-tumor drug targets.
- the inventors of the present application found in their research that Pirin gene is highly expressed in a variety of tumors, and its expression is positively correlated with poor prognosis, suggesting that Pirin can be used as a new anti-tumor therapeutic target.
- Pirin gene and protein as targets, new anti-tumor drugs can be screened, and Pirin can also be used as a detection index to evaluate the efficacy of anti-tumor drugs.
- the second purpose of the present invention is to provide an application of targeting Pirin gene and protein as an immunotherapy sensitizer.
- the inventors of the present application found in their research that targeting Pirin can promote the activation of the FAS apoptosis pathway and significantly enhance the tumor killing effect of immune cells and FAS-activating monoclonal antibodies (which can activate the FAS apoptosis pathway by causing FAS to form trimers), suggesting that targeting Pirin can be used as a new immunosensitizer, which can be further combined with existing clinical tumor immunotherapy drugs for tumor treatment and drug efficacy evaluation.
- the present invention provides the application of Pirin gene/protein as an anti-tumor target.
- the Pirin gene is Ensembl-ENSG00000087842.
- the Pirin protein is UniProtKB-000625, and its amino acid sequence is (SEQ NO 1):
- the tumor is a tumor with high expression of Pirin, including but not limited to colorectal cancer, liver cancer, lung cancer, breast cancer, pancreatic cancer, prostate cancer, cervical cancer, kidney cancer, gastric cancer, glioma, melanoma, endometrial cancer, etc.
- the present invention also provides the use of Pirin inhibitors in anti-tumor applications, or the use of Pirin inhibitors in the preparation of anti-tumor drugs.
- Pirin inhibitors include Pirin gene inhibitors and Pirin protein inhibitors.
- the present invention also provides a method for tumor diagnosis/tumor screening/immunotherapy/prognosis judgment, which comprises detecting the Pirin content.
- the present invention also provides the use of a drug targeting Pirin gene or protein as a tumor immunosensitizer.
- the application of the tumor immunosensitizer includes using Pirin inhibitor as a component to enhance immune function, or using Pirin Inhibitors are used in combination with immunotherapy drugs for anti-tumor applications.
- the application of the immunosensitizer includes the application of the Pirin content detection reagent for tumor screening/immunotherapy/prognosis judgment.
- Pirin gene/protein as a target for tumor therapy/tumor immunosensitization.
- the application of the tumor treatment/immunosensitization target includes using the Pirin inhibitor as an anti-tumor active ingredient, or using the Pirin inhibitor in the preparation of anti-tumor drugs.
- the application as an anti-tumor active ingredient refers to a tumor prevention and treatment drug used to regulate Pirin function, and/or downregulate Pirin expression level, and/or reduce Pirin protein activity. Further preferred is a drug that can target and regulate Pirin-related functions to treat tumors; specifically: the drug targets Pirin to cause activation of the FAS apoptosis pathway, thereby inhibiting tumor cell growth.
- the application of the anti-tumor drug preparation includes the application of the Pirin content detection reagent to a tumor diagnosis/prognosis reagent, or the application of preparing a diagnosis/prognosis kit.
- the application of the tumor treatment/immunosensitization target includes the application of the Pirin content detection reagent to the tumor screening/prognosis judgment reagent, or the application of the preparation of the efficacy/prognosis judgment kit.
- the Pirin gene is highly expressed in a variety of tumors.
- the present invention provides application of a drug targeting Pirin gene or protein as a tumor treatment/tumor immunosensitizer.
- the application of tumor treatment includes the application of Pirin inhibitor as a tumor treatment drug.
- the application of the immunosensitizer includes using the Pirin inhibitor as a component for enhancing immune function, or combining the Pirin inhibitor with an immunotherapy drug for anti-tumor application.
- the immune enhancement function refers to targeting Pirin to cause activation of the FAS apoptosis pathway, thereby promoting immune cells to kill tumor cells through the FASL/FAS pathway or FAS-activated monoclonal antibodies to kill tumor cells through the FAS pathway, thereby inhibiting tumor growth.
- the application of the tumor treatment/immunosensitizer includes the application of the Pirin content detection reagent in tumor screening/prognosis judgment.
- the present invention also provides a method for screening anti-tumor drugs, which is performed by using Pirin protein as a drug screening target.
- the present invention knocks down PIR through RNA interference technology and then restores the expression of PIR protein.
- Flow cytometry analysis shows that tumor cell apoptosis caused by PIR knockdown can be rescued by restoring PIR.
- RNA-Seq analysis found that the FAS-mediated apoptosis pathway was enriched after PIR knockdown.
- Knocking down PIR can significantly increase the expression level of FAS protein.
- Knocking down PIR protein after knocking down FAS in advance no longer causes tumor cell death, indicating that knocking down PIR mainly causes cell apoptosis by upregulating FAS, and PIR maintains cell survival by inhibiting FAS expression.
- HCT 116 cells with different degrees of PIR knockdown were treated with FAS monoclonal antibody (FAS monoclonal antibody can promote FAS aggregation on the cell membrane and activate the FAS apoptosis pathway) and activated CD8 + T cells.
- FAS monoclonal antibody can promote FAS aggregation on the cell membrane and activate the FAS apoptosis pathway
- CD8 + T cells activated CD8 + T cells.
- the present invention provides the use of Pirin (PIR) gene/protein as a target for tumor treatment/tumor immunosensitization, and the use of a drug targeting the PIR gene/protein as a tumor treatment drug/immunosensitizer.
- Pirin PIR
- FIG. 1 Expression of PIR protein in various tumor tissues and their adjacent adjacent tissues.
- FIG. 1 Expression levels of PIR proteins in different tumor cells and normal cells.
- Figure 3 Analysis results of PIR mRNA levels in various tissues in the TCGA clinical database.
- Figure 4 Analysis results of the correlation between PIR expression and prognosis in the TCGA clinical database.
- Figure 5 Statistical graph of cell survival rate after knocking down PIR protein in various tumor cells.
- Figure 6 Cell apoptosis after knocking down or restoring PIR protein expression levels.
- FIG. 8 Immunoblotting results of key regulatory proteins in the FAS signaling pathway after knocking down PIR protein levels.
- Figure 9 Survival rate of tumor cells after knocking down FAS protein and then knocking down PIR protein.
- FIG. 10 Survival rate of cells with different PIR protein levels treated with FAS monoclonal antibody.
- FIG. 11 Survival rate of CD8 + T cells treated with cells with different PIR protein levels and FAS protein level knockdown.
- FIG. 12 Cell survival after treatment with PIR protein inhibitors TphA, CCG1423 or CCG203911.
- FIG. 13 Cell survival after treatment with different concentrations of PIR protein inhibitor TphA.
- Figure 14 Tumor development in nude mouse xenograft model treated with PIR protein inhibitor CCG1423.
- Figure 15 High-throughput screening method for anti-tumor drugs using PIR protein as drug target.
- the present invention provides the application of Pirin gene/protein as a target for tumor treatment/tumor immunosensitization.
- the Pirin protein is UniProtKB-000625, and its amino acid sequence and nucleotide sequence are shown in the sequence table.
- the tumor treatment/immunosensitization target includes using Pirin inhibitor as an anti-tumor active ingredient, or using Pirin inhibitor in the preparation of anti-tumor drugs.
- the application of the anti-tumor active ingredient refers to a tumor prevention and treatment drug used to regulate Pirin function, and/or downregulate Pirin expression level, and/or reduce Pirin protein activity. Further preferred is a drug that can target and regulate Pirin-related functions to treat tumors; specifically: the drug targets Pirin to activate the FAS apoptosis pathway, thereby inhibiting tumor cell growth.
- the application of the tumor treatment/immunosensitization target includes the application of the Pirin content detection reagent to the tumor screening/prognosis judgment reagent, or the application of the preparation of the efficacy/prognosis judgment kit.
- the Pirin gene is highly expressed in a variety of tumors.
- the application of tumor treatment includes the application of Pirin inhibitor as a tumor treatment drug.
- the application of the immunosensitizer includes using the Pirin inhibitor as a component for enhancing immune function, or combining the Pirin inhibitor with an immunotherapy drug for anti-tumor application.
- the immune enhancement function refers to targeting Pirin to cause activation of the FAS apoptosis pathway, thereby promoting immune cells to kill tumor cells through the FASL/FAS pathway or FAS-activated monoclonal antibodies to kill tumor cells through the FAS pathway, thereby inhibiting tumor growth.
- the application of the tumor treatment/immunosensitizer includes the application of the Pirin content detection reagent in tumor screening/prognosis judgment.
- the tissue chip was hydrated with gradient ethanol.
- the hydration order was: xylene I--xylene II--anhydrous ethanol I--anhydrous ethanol II--90% ethanol--80% ethanol--70% ethanol--60% ethanol--50% ethanol--distilled water, 5min each.
- the transcriptome data and clinical data tables of colorectal cancer were downloaded from the TCGA database, the Pirin gene expression was analyzed using R language, and the Kaplan-Meier survival curve was drawn.
- the cells were digested with trypsin, washed twice with PBS buffer, centrifuged at 500 rpm to remove the supernatant, 50 ⁇ L each of Annexin V and PI dye were added, incubated in the dark for 10 min, 900 ⁇ L of stop solution was added, and then detected by flow cytometry.
- mice The age of nude mice was 6-7 weeks. Each mouse was injected with 1 ⁇ 10 7 HCT 116 cells subcutaneously at the posterior thigh and the back of the nude mouse. One week after the injection, CCG1423 (10 mg/kg) was injected into the tail vein. Three times a week for three consecutive weeks, when the tumor grew to an appropriate size, the mice were killed by cervical dislocation, and the tumors were photographed and dissected to obtain the weight.
- PIR Pirin gene or protein.
- FAS mAb FAS activating monoclonal antibody.
- Figure 1 shows the expression of PIR protein in various tumor tissues and their adjacent tissues. IHC staining results of PIR protein in tumor tissue and adjacent tissues of colon cancer tissue microarray (A left) and the corresponding statistical analysis graph (A right). IHC staining of PIR protein in tumor tissue and adjacent tissues of cervical cancer (B), breast cancer (C), liver cancer (D), pancreatic cancer (E), and gastric cancer (F). Colon: colon; Cervix: cervix; Breast: breast; Liver: liver; Pancreas: pancreas; Gastro: stomach.
- Figure 2 shows the expression level of PIR protein in different tumor cells and normal cells.
- the above cells were cultured for 24 hours and proteins were extracted, and western blot analysis was performed using anti-PIR and anti- ⁇ -actin (control) antibodies.
- HeLa human cervical cancer cell line
- SW620 human colon cancer cell line
- CT26.WT mouse colon cancer cell line
- HCT 116 human colon cancer cell line
- Hep G2 human liver cancer cell line
- L-929 mouse fibroblast cell line
- sMPNST human sporadic malignant peripheral nerve sheath tumor cell line
- MCF7 human breast cancer cell line
- HFF-1 human embryonic fibroblast cell line
- 293T (HEK293T) human embryonic kidney cell line
- 786-O human renal clear cell adenocarcinoma
- OVCAR3 human ovarian cancer cell line
- A549 human lung adenocarcinoma cell line
- MHCC97H human hepatoma cell line
- LLC mouse Lewis lung
- FIG. 3 shows the results of PIR mRNA level analysis in various cancers in the TCGA clinical database.
- COAD colon adenocarcinoma
- DLBC diffuse large B-cell lymphoma
- UCEC endometrial carcinoma
- GBM glioblastoma multiforme
- KIRC renal clear cell carcinoma
- KIRP renal papillary cell carcinoma
- LIHC hepatocellular carcinoma
- LUSC lung squamous cell carcinoma
- PAAD pancreatic adenocarcinoma
- READ rectal adenocarcinoma
- SKCM cutaneous melanoma
- THYM thymoma.
- Figure 4 shows the analysis results of the correlation between PIR expression level and prognosis in the TCGA clinical database.
- the relationship between PIR expression level in the TCGA clinical database and the prognosis of patients with glioblastoma (A), liver cancer (B), breast cancer (C) and kidney cancer (D) was analyzed.
- GBM glioblastoma multiforme
- LIHC hepatocellular carcinoma
- BRCA breast cancer
- KIRC renal clear cell carcinoma.
- Figure 5 is a statistical graph of various tumor cells stained with PI 60 hours after infection with a virus expressing shPIR#1, and cell survival rates were detected by flow cytometry. Data represent mean ⁇ standard deviation, three independent experiments (unpaired t test, ***p ⁇ 0.001), CTRL: Control.
- HCT 116 human colon cancer cells; HeLa: human cervical cancer cells; MCF7: human breast cancer cells; LLC: mouse Lewis lung cancer cells; sMPNST: human sporadic malignant peripheral nerve sheath tumor cells; MV3: human melanoma cells; OVCAR3: human ovarian cancer cells; A549: human lung adenocarcinoma cells; 786-O: human renal clear cell adenocarcinoma cells.
- CTRL Control.
- Figure 6 shows the cell apoptosis after knocking down or replenishing the expression level of PIR protein.
- HCT 116 cells were first infected with viruses expressing rHA-PIR or CTRL for 24 hours, and then infected with viruses expressing shPIR#1 for 72 hours. After further staining with Annexin V-PI, cell apoptosis was analyzed by flow cytometry (left). The corresponding PIR protein was expressed by immunofluorescence staining. Detection by immunoblotting (right).
- rHA-PIR rescuing HA-PIR, i.e., rescue expression vector of PIR with HA tag.
- Figure 7 shows the enrichment analysis of RNA-Seq results after PIR protein knockdown.
- Total RNA was extracted from HCT 116 cells 60 hours after infection with a virus expressing shPIR, and RNA-Seq experiments were performed, and gene enrichment analysis was further performed.
- Figure 8 shows the immunoblotting results of key regulatory proteins in the FAS signaling pathway after knocking down the PIR protein level.
- HCT 116 cells were first infected with a virus expressing rHA-PIR or CTRL for 24 hours, and then infected with a virus expressing shPIR#1 or shPIR#2 for 60 hours, and the changes in key regulatory proteins in the FAS signaling pathway were detected by immunoblotting experiments.
- Casp8 Caspase 8;
- Casp3 Caspase 3.
- Figure 9 shows the survival rate of tumor cells after knocking down FAS protein and then knocking down PIR protein.
- HCT 116 cells were first infected with a virus expressing shFAS or CTRL for 24 hours and then infected with a virus expressing shPIR#1 for 48 hours. Cells were stained with PI and survival analysis was performed by flow cytometry (top). PIR and FAS protein immunoblotting results (bottom). Data represent mean ⁇ SD, five independent experiments (unpaired t test, ***p ⁇ 0.001).
- Figure 10 shows the survival rate of cells with different PIR protein levels treated with FAS mAb.
- HCT 116 cells were infected with viruses expressing shPIR in gradient volume for 24 hours, and the cells were treated with 2.5 g mL -1 FAS activating mAb (FAS mAb) for 24 hours and then stained with PI.
- the above cells were further analyzed for survival by flow cytometry (upper), and the corresponding PIR protein levels were analyzed by immunoblotting (lower). Data represent mean ⁇ standard deviation, three independent experiments (unpaired t test, **p ⁇ 0.01).
- Figure 11 shows the survival rate of cells with different PIR protein levels and FAS protein levels knocked down by CD8 + T cells.
- HCT 116 cells were infected with viruses expressing shPIR in gradient volumes for 24 hours, and HCT 116 cells were incubated with pre-activated human effector CD8 + T cells for 20 hours and then stained with PI. The above cells were further analyzed for survival by flow cytometry (top), and PIR and FAS protein levels were detected by immunoblotting (bottom). The data represent mean ⁇ standard deviation, three independent experiments (unpaired t test, ***p ⁇ 0.001, ns: no significant difference).
- FIG. 12 shows the survival of cells after treatment with PIR protein inhibitors TphA, CCG1423 or CCG203971.
- HCT 116, HeLa, sMPNST and HFF-1 cells were treated with DMSO (CTRL) or PIR protein inhibitors TphA (100 ⁇ M), CCG1423 (10 ⁇ M), CCG203971 (10 ⁇ M), and photographed with a microscope 48 hours later and stained with PI, and further survival analysis was performed by flow cytometry. Data represent mean ⁇ standard deviation, three independent experiments (unpaired t test, ***p ⁇ 0.001, ns: no significant difference).
- Figure 13 shows the survival of cells after treatment with different concentrations of PIR protein inhibitor TphA.
- A549, MCF7, SN12-PM6, HeLa, U118, PANC-1, and PC-3 cells were treated with DMSO (CTRL), 50 ⁇ M TphA, or 150 ⁇ M TphA. After 48 hours, the cells were photographed under a microscope and stained with PI. Flow cytometry was used for survival analysis. The data represent mean ⁇ standard deviation, three independent experiments (unpaired t test, ***p ⁇ 0.001).
- A549 human lung adenocarcinoma cell line
- MCF7 human breast cancer cell line
- SN12-PM6 human kidney cancer cell line
- HeLa human cervical cancer cell line
- U118 human brain astrocytoblastoma cell line
- PANC-1 human pancreatic cancer cell line
- PC-3 human prostate cancer cell line.
- Figure 14 shows the tumor development of nude mouse transplanted tumor model treated with PIR protein inhibitor CCG1423.
- 1 ⁇ 10 7 HCT 116 cells were inoculated subcutaneously into nude mice, and PBS (control group) or PIR protein inhibitor CCG1423 (10 mg kg -1 CCG1423 was subcutaneously injected three times a week) was injected into the nude mice one week later. After one month, the nude mice were euthanized, and the tumors were removed and weighed by dissection.
- FIG15 is a high-throughput screening method for anti-tumor drugs using PIR protein as a drug target.
- This screening method uses the quercetin 2,3-dioxygenase activity of PIR to evaluate drug efficacy.
- the compounds to be screened whose rate was significantly lower than that of the negative control (only without adding PIR protein) were selected as candidate compounds (Candidates) for other confirmation experiments such as ITC (isothermal titration calorimetry) and MST (microthermophoresis technology).
- the present invention detected the PIR protein level in tumor tissues and cells ( Figures 1-2) and combined with the TCGA database analysis ( Figure 3) to find that PIR is highly expressed in a variety of tumors, and the PIR expression level is negatively correlated with prognosis ( Figure 4).
- Figure 5 After knocking down PIR by RNA interference technology, it was found that tumor cell death increased significantly ( Figure 5), and after knocking down PIR by RNA interference technology and re-expressing PIR protein, flow cytometry analysis found that tumor cell apoptosis caused by PIR knockdown can be rescued by re-expressing PIR ( Figure 6), indicating that PIR is a key anti-apoptotic protein.
- RNA-Seq analysis found that the FAS-mediated apoptosis pathway was enriched after PIR knockdown (Figure 7). Knocking down PIR can significantly increase the expression level of FAS protein (Figure 8). Knocking down PIR protein after knocking down FAS in advance no longer causes tumor cell death ( Figure 9), indicating that knocking down PIR mainly causes cell apoptosis by up-regulating FAS, and PIR maintains cell survival by inhibiting FAS expression.
- HCT 116, HeLa, sMPNST and HFF-1 cells were treated with existing PIR inhibitors TphA, CCG1423 or CCG203971 (Figure 12). It was found that PIR inhibitor treatment would cause cell death with high PIR protein expression levels, but no effective cytotoxicity was produced for cells with low PIR protein expression levels. The higher the concentration of PIR inhibitor TphA treatment, the more obvious the cell death ( Figure 13). When CCG1423 was used to treat mice with transplanted tumors ( Figure 14), it was found that CCG1423 had a significant inhibitory effect on the proliferation of transplanted tumors. In addition, we provide a high-throughput screening method for anti-tumor drugs using PIR protein as drug target ( FIG. 15 ).
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Abstract
The present disclosure relates to the technical field of molecular biology and biological pharmaceuticals, and particularly, to use of the Pirin gene and protein in preparing an anti-tumor targeted drug. As new anti-tumor targets, the Pirin gene and protein can be used as the targets for tumor therapies (including but not limited to immunotherapy sensitization), and inhibitors targeting Pirin can effectively enhance the ability of immune cells to kill tumor cells and inhibit the development and progression of tumors. The therapeutic effect and prognosis of patients can be predicted by means of determining Pirin expression level, and drugs targeting Pirin can serve as a new means of tumor-targeted therapy alone or in combination with immunotherapy. The present invention lays a foundation for the combination of tumor-targeted chemotherapeutics with immunotherapies, the enhancement of sensitivity to tumor immunotherapy, and the improvement of immunotherapeutic effects, thereby possessing great prospects.
Description
相关申请的交叉引用CROSS-REFERENCE TO RELATED APPLICATIONS
本申请要求于2022年12月14日递交的申请号为CN202211605658.6的中国专利申请的优先权，在此引用上述中国专利申请的内容全文以作为本申请的一部分。This application claims priority to Chinese patent application No. CN202211605658.6 filed on December 14, 2022. The full text of the above-mentioned Chinese patent application is hereby cited as part of this application.
本发明属于分子生物学和生物医药技术领域，尤其是涉及一种抗Pirin高表达肿瘤的新靶点Pirin(PIR)基因及蛋白在制备一种新的抗肿瘤及免疫增敏作用靶点药物中的应用。The present invention belongs to the field of molecular biology and biomedicine technology, and in particular relates to the application of a new target Pirin (PIR) gene and protein for resisting Pirin high-expressing tumors in the preparation of a new anti-tumor and immunosensitization target drug.
癌症是细胞异常增生引起的一种疾病。正常情况下，机体免疫系统具有免疫监视功能，当体内细胞出现异常增生时，免疫系统能够识别并激活凋亡通路从而清除这些异常细胞。然而，在肿瘤发生发展过程中，肿瘤细胞会获得抗凋亡的能力，逃避免疫系统的攻击，这也是导致肿瘤治疗失败的主要原因之一(Igney,F.H.,Krammer,P.H.Death and anti-death:tumour resistance to apoptosis[J].Nat Rev Cancer,2002,2(4):277-88)。因此，深入研究肿瘤细胞抵抗免疫细胞所致凋亡的新机理对发现新的肿瘤治疗靶点和治疗策略具有重要的理论和现实意义。Cancer is a disease caused by abnormal cell proliferation. Under normal circumstances, the body's immune system has an immune surveillance function. When cells in the body proliferate abnormally, the immune system can recognize and activate the apoptosis pathway to eliminate these abnormal cells. However, during the development of tumors, tumor cells acquire the ability to resist apoptosis and escape the attack of the immune system, which is also one of the main reasons for the failure of tumor treatment (Igney, F.H., Krammer, P.H. Death and anti-death: tumor resistance to apoptosis [J]. Nat Rev Cancer, 2002, 2 (4): 277-88). Therefore, in-depth research on the new mechanism of tumor cell resistance to apoptosis caused by immune cells has important theoretical and practical significance for discovering new tumor treatment targets and treatment strategies.
FAS(CD95)是肿瘤坏死因子受体(TNFR)超家族中一种典型的凋亡诱导受体，FASL(CD95L)是其配体(Griffith,T.S.,Brunner,T.,Fletcher,S.M.,Green,D.R.,Ferguson,T.A.Fas ligand-induced apoptosis as a mechanism of immune privilege[J].Science,1995,270(5239):1189-92)。当FAS过度表达或与免疫细胞分泌的FASL结合后，FAS会自动转变成三聚体形式，从而引发信号通路级联反应，募集并激活FADD、caspase8、BID和caspase3等信号蛋白，最终导致细胞凋亡(Kaufmann,T.,Strasser,A.,Jost,P.J.Fas death receptor signalling:roles of Bid and XIAP[J].Cell Death Differ,2012,19(1):42-50)。死亡受体FAS介导的凋亡通路是免疫细胞杀伤肿瘤最重要的方式之一，是抑制肿瘤发生的重要途径，而其在肿瘤细胞中常呈现抑制状态(Ivanov,V.N.,Bhoumik,A.,Krasilnikov,M.,Raz,R.,Owen-Schaub,L.B.,Levy,D.,Horvath,C.M.,Ronai,Z.Cooperation between STAT3 and c-jun suppresses Fas transcription[J].Mol Cell,2001,7(3):517-28)。FAS (CD95) is a typical apoptosis-inducing receptor in the tumor necrosis factor receptor (TNFR) superfamily, and FASL (CD95L) is its ligand (Griffith, T.S., Brunner, T., Fletcher, S.M., Green, D.R., Ferguson, T.A. Fas ligand-induced apoptosis as a mechanism of immune privilege[J]. Science, 1995, 270(5239): 1189-92). When FAS is overexpressed or binds to FASL secreted by immune cells, FAS will automatically transform into a trimer form, thereby triggering a signaling pathway cascade reaction, recruiting and activating signaling proteins such as FADD, caspase8, BID and caspase3, and ultimately leading to cell apoptosis (Kaufmann, T., Strasser, A., Jost, P.J. Fas death receptor signaling: roles of Bid and XIAP[J]. Cell Death Differ, 2012, 19(1):42-50). The apoptosis pathway mediated by the death receptor FAS is one of the most important ways for immune cells to kill tumors and an important way to inhibit tumor occurrence. It is often in an inhibitory state in tumor cells (Ivanov, V.N., Bhoumik, A., Krasilnikov, M., Raz, R., Owen-Schaub, L.B., Levy, D., Horvath, C.M., Ronai, Z. Cooperation between STAT3 and c-jun suppresses Fas transcription[J]. Mol Cell, 2001, 7(3): 517-28).
Pirin蛋白是cupin超家族的成员，由PIR基因编码，在哺乳动物、植物、真菌和原
核生物中高度保守(Aguayo,F.P.-D.D.C.-B.R.B.J.P.M.G.L.-C.U.U.G.M.C.a.F.Role of Pirin,an Oxidative Stress Sensor Protein,in Epithelial Carcinogenesis[J].Prepirints,2020.)。其mRNA及蛋白在人类所有正常组织中均低表达(Wendler,W.M.,Kremmer,E.,Forster,R.,Winnacker,E.L.Identification of pirin,a novel highly conserved nuclear protein[J].J Biol Chem,1997,272(13):8482-9)，而在结肠癌、乳腺癌、肺癌和黑色素瘤等多种肿瘤中高表达，提示其可能与肿瘤发生有关(Licciulli,S.,Luise,C.,Zanardi,A.,Giorgetti,L.,Viale,G.,Lanfrancone,L.,Carbone,R.,Alcalay,M.Pirin delocalization in melanoma progression identified by high content immuno-detection based approaches[J].BMC Cell Biol,2010,11:5.；Li,T.,Fu,J.,Zeng,Z.,Cohen,D.,Li,J.,Chen,Q.,Li,B.,Liu,X.S.TIMER2.0 for analysis of tumor-infiltrating immune cells[J].Nucleic Acids Res,2020,48(W1):W509-W514)。Cupin超家族被认为是功能最为多样化的蛋白超家族之一，其蛋白质的特点是含有保守的β桶状结构和独特金属离子结合序列(Dunwell,J.M.Cupins:a new superfamily of functionally diverse proteins that include germins and plant storage proteins[J].Biotechnol Genet Eng Rev,1998,15:1-32)。研究表明，cupin超家族成员具有双加氧酶、异构酶、蛋白结合以及转录因子等功能，与人类疾病密切相关(Schmidt,H.R.,Zheng,S.,Gurpinar,E.,Koehl,A.,Manglik,A.,Kruse,A.C.Crystal structure of the human sigma1receptor[J].Nature,2016,532(7600):527-30)。Pirin proteins are members of the cupin superfamily and are encoded by PIR genes. Highly conserved in nuclear organisms (Aguayo, FP-DDC-BRBJPMGL-CUUGMCaFRole of Pirin, an Oxidative Stress Sensor Protein, in Epithelial Carcinogenesis[J]. Prepirints, 2020.). Its mRNA and protein are lowly expressed in all normal human tissues (Wendler, WM, Kremmer, E., Forster, R., Winnacker, ELIdentification of pirin, a novel highly conserved nuclear protein[J]. J Biol Chem, 1997, 272(13): 8482-9), but highly expressed in various tumors such as colon cancer, breast cancer, lung cancer and melanoma, suggesting that it may be related to tumorigenesis (Licciulli, S., Luise, C., Zanardi, A., Giorgetti, L., Viale, G., Lanfrancone, L., Carbone, R., Alcalay, M. Pirin delocalization in melanoma progression identified by high content immuno-detection based approaches[J]. BMC Cell Biol, 2010, 11: 5.; Li, T., Fu, J., Zeng, Z., Cohen, D., Li, J., Chen, Q., Li, B., Liu, XSTIMER2.0 for analysis of tumor-infiltrating immune cells[J]. Nucleic Acids Res, 2020, 48(W1): W509-W514). The Cupin superfamily is considered to be one of the most functionally diverse protein superfamilies, and its proteins are characterized by containing a conserved β-barrel structure and a unique metal ion binding sequence (Dunwell, JM Cupins: a new superfamily of functionally diverse proteins that include germins and plant storage proteins[J]. Biotechnol Genet Eng Rev, 1998, 15: 1-32). Studies have shown that members of the cupin superfamily have functions such as dioxygenase, isomerase, protein binding and transcription factor, and are closely related to human diseases (Schmidt, HR, Zheng, S., Gurpinar, E., Koehl, A., Manglik, A., Kruse, A. Crystal structure of the human sigma1 receptor[J]. Nature, 2016, 532(7600): 527-30).
发明内容Summary of the invention
我们的研究发现Pirin基因和蛋白可以作为一种抗肿瘤靶点，其异常高表达与乳腺癌、结肠癌、前列腺癌等多种癌症的发生发展相关。Our research found that Pirin gene and protein can be used as an anti-tumor target, and its abnormally high expression is associated with the occurrence and development of various cancers such as breast cancer, colon cancer, and prostate cancer.
本发明的第一目的在于提供一种将Pirin基因及蛋白作为抗肿瘤药物靶点的应用。本申请发明人在研究中发现Pirin基因在多种肿瘤中高表达，且其表达与不良预后正相关，提示Pirin可作为一种全新的抗肿瘤治疗靶点。以Pirin基因及蛋白作为靶点，可筛选新型抗肿瘤药物，同时，也可利用Pirin作为检测指标评价抗肿瘤药物的疗效。The first purpose of the present invention is to provide an application of Pirin gene and protein as anti-tumor drug targets. The inventors of the present application found in their research that Pirin gene is highly expressed in a variety of tumors, and its expression is positively correlated with poor prognosis, suggesting that Pirin can be used as a new anti-tumor therapeutic target. Using Pirin gene and protein as targets, new anti-tumor drugs can be screened, and Pirin can also be used as a detection index to evaluate the efficacy of anti-tumor drugs.
本发明的第二目的在于提供一种将靶向Pirin基因及蛋白作为免疫治疗增敏剂的应用。本申请发明人在研究中发现靶向Pirin可促进FAS凋亡通路的激活并显著增强免疫细胞和FAS激活单抗(能通过引起FAS形成三聚体而激活FAS凋亡通路)的肿瘤杀伤效果，提示靶向Pirin可以作为一种全新的免疫增敏剂，可进一步联合现有的肿瘤免疫治疗临床药物用于肿瘤治疗和药物疗效评价。The second purpose of the present invention is to provide an application of targeting Pirin gene and protein as an immunotherapy sensitizer. The inventors of the present application found in their research that targeting Pirin can promote the activation of the FAS apoptosis pathway and significantly enhance the tumor killing effect of immune cells and FAS-activating monoclonal antibodies (which can activate the FAS apoptosis pathway by causing FAS to form trimers), suggesting that targeting Pirin can be used as a new immunosensitizer, which can be further combined with existing clinical tumor immunotherapy drugs for tumor treatment and drug efficacy evaluation.
本发明所采用的技术方案为：The technical solution adopted by the present invention is:
本发明提供Pirin基因/蛋白可作为抗肿瘤靶点的应用。
The present invention provides the application of Pirin gene/protein as an anti-tumor target.
所述Pirin基因即Ensembl-ENSG00000087842。The Pirin gene is Ensembl-ENSG00000087842.
所述Pirin蛋白即UniProtKB-O00625，其氨基酸序列为(SEQ NO 1)：
The Pirin protein is UniProtKB-000625, and its amino acid sequence is (SEQ NO 1):
The Pirin protein is UniProtKB-000625, and its amino acid sequence is (SEQ NO 1):
其核苷酸序列为(SEQ NO 2)：
Its nucleotide sequence is (SEQ NO 2):
Its nucleotide sequence is (SEQ NO 2):
所述肿瘤为Pirin高表达的肿瘤，包括但不限于结直肠癌、肝癌、肺癌、乳腺癌、胰腺癌、前列腺癌、宫颈癌、肾癌、胃癌、胶质瘤、黑色素瘤、子宫内膜癌等。The tumor is a tumor with high expression of Pirin, including but not limited to colorectal cancer, liver cancer, lung cancer, breast cancer, pancreatic cancer, prostate cancer, cervical cancer, kidney cancer, gastric cancer, glioma, melanoma, endometrial cancer, etc.
本发明还提供Pirin抑制剂在抗肿瘤中的应用，或Pirin抑制剂在制备抗肿瘤药物中的应用。Pirin抑制剂包括Pirin基因抑制剂和Pirin蛋白抑制剂。The present invention also provides the use of Pirin inhibitors in anti-tumor applications, or the use of Pirin inhibitors in the preparation of anti-tumor drugs. Pirin inhibitors include Pirin gene inhibitors and Pirin protein inhibitors.
本发明还提供一种肿瘤诊断/肿瘤筛查/免疫治疗/预后判断的方法，包括检测Pirin含量。The present invention also provides a method for tumor diagnosis/tumor screening/immunotherapy/prognosis judgment, which comprises detecting the Pirin content.
本发明还提供靶向Pirin基因或蛋白的药物作为肿瘤免疫增敏剂的应用。The present invention also provides the use of a drug targeting Pirin gene or protein as a tumor immunosensitizer.
所述肿瘤免疫增敏剂的应用包括将Pirin抑制剂作为增强免疫功能的成分，或将Pirin
抑制剂与免疫治疗药物联合应用于抗肿瘤的应用。The application of the tumor immunosensitizer includes using Pirin inhibitor as a component to enhance immune function, or using Pirin Inhibitors are used in combination with immunotherapy drugs for anti-tumor applications.
所述免疫增敏剂的应用包括将Pirin含量检测试剂用于肿瘤筛查/免疫治疗/预后判断的应用。The application of the immunosensitizer includes the application of the Pirin content detection reagent for tumor screening/immunotherapy/prognosis judgment.
Pirin基因/蛋白作为肿瘤治疗/肿瘤免疫增敏靶点的应用。Application of Pirin gene/protein as a target for tumor therapy/tumor immunosensitization.
所述肿瘤治疗/免疫增敏靶点的应用包括将Pirin抑制剂作为抗肿瘤活性成分，或将Pirin抑制剂应用于抗肿瘤药物制备的应用。The application of the tumor treatment/immunosensitization target includes using the Pirin inhibitor as an anti-tumor active ingredient, or using the Pirin inhibitor in the preparation of anti-tumor drugs.
所述作为抗肿瘤活性成分的应用指用于调控Pirin功能、和/或下调Pirin表达水平、和/或降低Pirin蛋白活性的肿瘤防治药物。更进一步优选的为可靶向调控Pirin相关的功能从而治疗肿瘤的药物；具体为：药物靶向Pirin引起FAS凋亡通路的激活，从而抑制肿瘤细胞生长。The application as an anti-tumor active ingredient refers to a tumor prevention and treatment drug used to regulate Pirin function, and/or downregulate Pirin expression level, and/or reduce Pirin protein activity. Further preferred is a drug that can target and regulate Pirin-related functions to treat tumors; specifically: the drug targets Pirin to cause activation of the FAS apoptosis pathway, thereby inhibiting tumor cell growth.
所述抗肿瘤药物制备的应用包括将Pirin含量检测试剂应用于肿瘤诊断/预后判断试剂的应用，或制备诊断/预后判断试剂盒的应用。The application of the anti-tumor drug preparation includes the application of the Pirin content detection reagent to a tumor diagnosis/prognosis reagent, or the application of preparing a diagnosis/prognosis kit.
所述肿瘤治疗/免疫增敏靶点的应用包括将Pirin含量检测试剂应用于肿瘤筛查/预后判断试剂的应用，或疗效/预后判断试剂盒制备的应用。The application of the tumor treatment/immunosensitization target includes the application of the Pirin content detection reagent to the tumor screening/prognosis judgment reagent, or the application of the preparation of the efficacy/prognosis judgment kit.
所述Pirin基因在多种肿瘤中高表达。The Pirin gene is highly expressed in a variety of tumors.
本发明提供靶向Pirin基因或蛋白的药物作为肿瘤治疗/肿瘤免疫增敏剂的应用。The present invention provides application of a drug targeting Pirin gene or protein as a tumor treatment/tumor immunosensitizer.
所述肿瘤治疗的应用包括将Pirin抑制剂作为肿瘤治疗药物的应用。The application of tumor treatment includes the application of Pirin inhibitor as a tumor treatment drug.
所述免疫增敏剂的应用包括将Pirin抑制剂作为增强免疫功能的成分，或将Pirin抑制剂与免疫治疗药物联合用于抗肿瘤的应用。The application of the immunosensitizer includes using the Pirin inhibitor as a component for enhancing immune function, or combining the Pirin inhibitor with an immunotherapy drug for anti-tumor application.
所述免疫增强功能指用于靶向Pirin引起FAS凋亡通路的激活，从而促进免疫细胞通过FASL/FAS通路或FAS激活单抗通过FAS通路杀伤肿瘤细胞，抑制肿瘤生长。The immune enhancement function refers to targeting Pirin to cause activation of the FAS apoptosis pathway, thereby promoting immune cells to kill tumor cells through the FASL/FAS pathway or FAS-activated monoclonal antibodies to kill tumor cells through the FAS pathway, thereby inhibiting tumor growth.
所述肿瘤治疗/免疫增敏剂的应用包括将Pirin含量检测试剂应用于肿瘤筛查/预后判断的应用。The application of the tumor treatment/immunosensitizer includes the application of the Pirin content detection reagent in tumor screening/prognosis judgment.
本发明还提供一种筛选抗肿瘤药物的方法，通过使用Pirin蛋白作为药物筛选靶点进行筛选。The present invention also provides a method for screening anti-tumor drugs, which is performed by using Pirin protein as a drug screening target.
本发明通过RNA干扰技术敲低PIR并回补表达PIR蛋白后流式分析发现PIR敲低引起的肿瘤细胞凋亡能被回补PIR所挽救。RNA-Seq分析发现PIR敲低后FAS介导的凋亡通路被富集。敲低PIR能显著升高FAS蛋白的表达水平，提前将FAS敲低后再敲低PIR蛋白不再引起肿瘤细胞死亡，说明敲低PIR主要通过上调FAS引起细胞凋亡，PIR是通过抑制FAS表达维持细胞生存。利用FAS单抗(FAS单抗能促进FAS在细胞膜上聚集并激活FAS凋亡通路)和激活的CD8+T细胞处理PIR不同程度敲低的HCT 116细
胞和PIR敲除的MEF细胞，发现PIR敲低的细胞对FAS单抗和CD8+T细胞的敏感性显著增强。实验表明，Pirin是极具潜力的肿瘤免疫增敏剂。The present invention knocks down PIR through RNA interference technology and then restores the expression of PIR protein. Flow cytometry analysis shows that tumor cell apoptosis caused by PIR knockdown can be rescued by restoring PIR. RNA-Seq analysis found that the FAS-mediated apoptosis pathway was enriched after PIR knockdown. Knocking down PIR can significantly increase the expression level of FAS protein. Knocking down PIR protein after knocking down FAS in advance no longer causes tumor cell death, indicating that knocking down PIR mainly causes cell apoptosis by upregulating FAS, and PIR maintains cell survival by inhibiting FAS expression. HCT 116 cells with different degrees of PIR knockdown were treated with FAS monoclonal antibody (FAS monoclonal antibody can promote FAS aggregation on the cell membrane and activate the FAS apoptosis pathway) and activated CD8 + T cells. The researchers used Pirin to detect and detect PIR-knockout MEF cells and found that PIR-knockout cells were significantly more sensitive to FAS monoclonal antibody and CD8 + T cells. The experiment showed that Pirin is a highly potential tumor immunosensitizer.
本发明优点：Advantages of the present invention:
本发明提供Pirin(PIR)基因/蛋白可作为肿瘤治疗/肿瘤免疫增敏靶点的应用，以及靶向PIR基因/蛋白的药物作为肿瘤治疗药物/免疫增敏剂的应用。The present invention provides the use of Pirin (PIR) gene/protein as a target for tumor treatment/tumor immunosensitization, and the use of a drug targeting the PIR gene/protein as a tumor treatment drug/immunosensitizer.
本申请的其他特征和优点将在随后的具体实施方式部分予以详细说明。Other features and advantages of the present application will be described in detail in the subsequent specific implementation section.
图1：多种肿瘤组织及其癌旁组织中PIR蛋白的表达情况。Figure 1: Expression of PIR protein in various tumor tissues and their adjacent adjacent tissues.
图2：不同肿瘤细胞和正常细胞中PIR蛋白的表达水平。Figure 2: Expression levels of PIR proteins in different tumor cells and normal cells.
图3：TCGA临床数据库PIR mRNA在多种组织中的水平分析结果。Figure 3: Analysis results of PIR mRNA levels in various tissues in the TCGA clinical database.
图4：TCGA临床数据库PIR表达与预后相关性分析结果。Figure 4: Analysis results of the correlation between PIR expression and prognosis in the TCGA clinical database.
图5：多种肿瘤细胞敲低PIR蛋白后细胞存活率统计图。Figure 5: Statistical graph of cell survival rate after knocking down PIR protein in various tumor cells.
图6：敲低或回补PIR蛋白表达水平后细胞凋亡情况。Figure 6: Cell apoptosis after knocking down or restoring PIR protein expression levels.
图7：PIR蛋白敲低后的RNA-Seq结果富集分析。Figure 7: Enrichment analysis of RNA-Seq results after PIR protein knockdown.
图8：敲低PIR蛋白水平后FAS信号通路中的关键调控蛋白的免疫印迹结果图。Figure 8: Immunoblotting results of key regulatory proteins in the FAS signaling pathway after knocking down PIR protein levels.
图9：敲低FAS蛋白后再敲低PIR蛋白的肿瘤细胞的存活率。Figure 9: Survival rate of tumor cells after knocking down FAS protein and then knocking down PIR protein.
图10：FAS单抗处理不同PIR蛋白水平的细胞的存活率。Figure 10: Survival rate of cells with different PIR protein levels treated with FAS monoclonal antibody.
图11：CD8+T细胞处理不同PIR蛋白水平和FAS蛋白水平敲低的细胞的存活率。FIG. 11 : Survival rate of CD8 + T cells treated with cells with different PIR protein levels and FAS protein level knockdown.
图12：PIR蛋白抑制剂TphA、CCG1423或CCG203911处理后细胞的存活情况。Figure 12: Cell survival after treatment with PIR protein inhibitors TphA, CCG1423 or CCG203911.
图13：不同浓度PIR蛋白抑制剂TphA处理后细胞的存活情况。Figure 13: Cell survival after treatment with different concentrations of PIR protein inhibitor TphA.
图14：PIR蛋白抑制剂CCG1423处理裸小鼠移植瘤模型的肿瘤发展情况。Figure 14: Tumor development in nude mouse xenograft model treated with PIR protein inhibitor CCG1423.
图15：以PIR蛋白作为药物靶点的抗肿瘤药物的高通量筛选方法。Figure 15: High-throughput screening method for anti-tumor drugs using PIR protein as drug target.
以下结合附图对本申请的具体实施方式进行详细说明。应当理解的是，此处所描述
的具体实施方式仅用于说明和解释本申请，并不用于限制本申请。The specific implementation of the present application is described in detail below with reference to the accompanying drawings. It should be understood that the The specific implementation methods are only used to illustrate and explain the present application and are not used to limit the present application.
以下实施例将结合附图对本发明作进一步的说明。The following embodiments will further illustrate the present invention in conjunction with the accompanying drawings.
本发明提供Pirin基因/蛋白作为肿瘤治疗/肿瘤免疫增敏靶点的应用。所述Pirin蛋白即UniProtKB-O00625，其氨基酸序列和核苷酸序列如序列表所示。The present invention provides the application of Pirin gene/protein as a target for tumor treatment/tumor immunosensitization. The Pirin protein is UniProtKB-000625, and its amino acid sequence and nucleotide sequence are shown in the sequence table.
所述肿瘤治疗/免疫增敏靶点包括将Pirin抑制剂作为抗肿瘤活性成分，或将Pirin抑制剂应用于抗肿瘤药物制备的应用。The tumor treatment/immunosensitization target includes using Pirin inhibitor as an anti-tumor active ingredient, or using Pirin inhibitor in the preparation of anti-tumor drugs.
所述抗肿瘤活性成分的应用指用于调控Pirin功能、和/或下调Pirin表达水平、和/或降低Pirin蛋白活性的肿瘤防治药物。更进一步优选的为可靶向调控Pirin相关的功能从而治疗肿瘤的药物；具体为：药物靶向Pirin引起FAS凋亡通路的激活，从而抑制肿瘤细胞生长。The application of the anti-tumor active ingredient refers to a tumor prevention and treatment drug used to regulate Pirin function, and/or downregulate Pirin expression level, and/or reduce Pirin protein activity. Further preferred is a drug that can target and regulate Pirin-related functions to treat tumors; specifically: the drug targets Pirin to activate the FAS apoptosis pathway, thereby inhibiting tumor cell growth.
所述肿瘤治疗/免疫增敏靶点的应用包括将Pirin含量检测试剂应用于肿瘤筛查/预后判断试剂的应用，或疗效/预后判断试剂盒制备的应用。The application of the tumor treatment/immunosensitization target includes the application of the Pirin content detection reagent to the tumor screening/prognosis judgment reagent, or the application of the preparation of the efficacy/prognosis judgment kit.
所述Pirin基因在多种肿瘤中高表达。The Pirin gene is highly expressed in a variety of tumors.
靶向Pirin基因或蛋白的药物作为肿瘤治疗/肿瘤免疫增敏剂的应用。Application of drugs targeting Pirin gene or protein as tumor treatment/tumor immunosensitizer.
所述肿瘤治疗的应用包括将Pirin抑制剂作为肿瘤治疗药物的应用。The application of tumor treatment includes the application of Pirin inhibitor as a tumor treatment drug.
所述免疫增敏剂的应用包括将Pirin抑制剂作为增强免疫功能的成分，或将Pirin抑制剂与免疫治疗药物联合用于抗肿瘤的应用。The application of the immunosensitizer includes using the Pirin inhibitor as a component for enhancing immune function, or combining the Pirin inhibitor with an immunotherapy drug for anti-tumor application.
所述免疫增强功能指用于靶向Pirin引起FAS凋亡通路的激活，从而促进免疫细胞通过FASL/FAS通路或FAS激活单抗通过FAS通路杀伤肿瘤细胞，抑制肿瘤生长。The immune enhancement function refers to targeting Pirin to cause activation of the FAS apoptosis pathway, thereby promoting immune cells to kill tumor cells through the FASL/FAS pathway or FAS-activated monoclonal antibodies to kill tumor cells through the FAS pathway, thereby inhibiting tumor growth.
所述肿瘤治疗/免疫增敏剂的应用包括将Pirin含量检测试剂应用于肿瘤筛查/预后判断的应用。The application of the tumor treatment/immunosensitizer includes the application of the Pirin content detection reagent in tumor screening/prognosis judgment.
材料与方法如下：Materials and methods are as follows:
1、常用药品和试剂：1. Commonly used drugs and reagents:
本说明使用的大部分药品与试剂分别购自Sigma公司、上海生工(Sangon)生物工程公司和MedChemExpress(MCE)公司。用于Western Blot分析的抗体购自Sigma,Cell Signaling Technology(CST)和Proteintech公司。用于分离和活化CD8+T细胞的试剂盒
购自BioLegend公司。FAS激活单抗购自Merck Millipore公司。Most of the drugs and reagents used in this description were purchased from Sigma, Shanghai Sangon Bioengineering Co., Ltd., and MedChemExpress (MCE). Antibodies used for Western Blot analysis were purchased from Sigma, Cell Signaling Technology (CST), and Proteintech. Kits for isolation and activation of CD8 + T cells The FAS-activated monoclonal antibody was purchased from Merck Millipore.
2、组织芯片免疫组化分析：2. Tissue chip immunohistochemistry analysis:
组织芯片65℃脱腊1h后进行梯度乙醇水化，水化顺序依次为：二甲苯I--二甲苯II--无水乙醇I--无水乙醇II--90％乙醇--80％乙醇--70％乙醇--60％乙醇--50％乙醇--蒸馏水，各5min。将组织芯片置于柠檬酸钠(pH＝6.0)修复液中通过沸水浴进行抗原修复20min。自然冷却，经PBS缓冲液清洗后加入1:100稀释的Pirin抗体,4℃孵育过夜，二抗常温孵育30min，最后DAB显色。After dewaxing at 65℃ for 1h, the tissue chip was hydrated with gradient ethanol. The hydration order was: xylene I--xylene II--anhydrous ethanol I--anhydrous ethanol II--90% ethanol--80% ethanol--70% ethanol--60% ethanol--50% ethanol--distilled water, 5min each. The tissue chip was placed in sodium citrate (pH=6.0) repair solution and antigen repaired in a boiling water bath for 20min. After natural cooling and washing with PBS buffer, a 1:100 dilution of Pirin antibody was added, incubated at 4℃ overnight, the secondary antibody was incubated at room temperature for 30min, and finally DAB color was developed.
3、TCGA数据库分析Pirin mRNA表达情况和生存曲线分析：3. Analysis of Pirin mRNA expression and survival curve using TCGA database:
从TCGA数据库中下载结直肠癌转录组数据和临床数据表格，通过R语言分析Pirin基因表达情况，绘制Kaplan-Meier生存曲线。The transcriptome data and clinical data tables of colorectal cancer were downloaded from the TCGA database, the Pirin gene expression was analyzed using R language, and the Kaplan-Meier survival curve was drawn.
4、流式细胞术分析细胞凋亡：4. Analysis of cell apoptosis by flow cytometry:
细胞培养后用胰蛋白酶消化，PBS缓冲液洗细胞两次，500rpm离心去上清，加入Annexin V和PI染料各50μL，避光孵育10min，加中止液900μL，随后流式细胞仪检测。After cell culture, the cells were digested with trypsin, washed twice with PBS buffer, centrifuged at 500 rpm to remove the supernatant, 50 μL each of Annexin V and PI dye were added, incubated in the dark for 10 min, 900 μL of stop solution was added, and then detected by flow cytometry.
5、裸鼠肿瘤移植实验：5. Nude mouse tumor transplantation experiment:
裸鼠的年龄为6-7周。每只小鼠注射1×107个HCT 116细胞于裸鼠的后侧大腿与背部相邻的皮下位置。注射1周后，尾静脉注射CCG1423(10mg/kg)。每周三次，连续三周，肿瘤长至合适大小，断颈法处死小鼠，拍照并解剖取得肿瘤称重。The age of nude mice was 6-7 weeks. Each mouse was injected with 1×10 7 HCT 116 cells subcutaneously at the posterior thigh and the back of the nude mouse. One week after the injection, CCG1423 (10 mg/kg) was injected into the tail vein. Three times a week for three consecutive weeks, when the tumor grew to an appropriate size, the mice were killed by cervical dislocation, and the tumors were photographed and dissected to obtain the weight.
以下给出有关缩写：The following are the relevant abbreviations:
1、PIR：Pirin基因或蛋白。1. PIR: Pirin gene or protein.
2、FAS mAb:FAS激活单抗。2. FAS mAb: FAS activating monoclonal antibody.
图1为多种肿瘤组织及其癌旁组织中PIR蛋白的表达情况。结肠癌组织芯片(A左)的肿瘤组织及其癌旁组织中PIR蛋白的IHC染色结果与对应的统计学分析图(A右)。宫颈癌(B)、乳腺癌(C)、肝癌(D)、胰腺癌(E)、胃癌(F)的肿瘤组织及其癌旁组织中PIR蛋白的IHC染色。Colon：结肠；Cervix：子宫颈；Breast：乳腺；Liver：肝脏；Pancreas：胰腺；Gastro：胃。
Figure 1 shows the expression of PIR protein in various tumor tissues and their adjacent tissues. IHC staining results of PIR protein in tumor tissue and adjacent tissues of colon cancer tissue microarray (A left) and the corresponding statistical analysis graph (A right). IHC staining of PIR protein in tumor tissue and adjacent tissues of cervical cancer (B), breast cancer (C), liver cancer (D), pancreatic cancer (E), and gastric cancer (F). Colon: colon; Cervix: cervix; Breast: breast; Liver: liver; Pancreas: pancreas; Gastro: stomach.
图2为不同肿瘤细胞和正常细胞中PIR蛋白的表达水平。将上述细胞培养24小时并提取蛋白质，用抗PIR和抗β-actin(对照)的抗体进行蛋白印迹分析。HeLa：人类宫颈癌细胞系；SW620：人类结肠癌细胞系；CT26.WT：小鼠结肠癌细胞系；HCT 116：人类结肠癌细胞系；Hep G2：人类肝癌细胞系；L-929：小鼠成纤维细胞系；sMPNST：人类散发性恶性周围神经鞘膜瘤细胞系；MCF7：人类乳腺癌细胞系；HFF-1：人类胚胎成纤维细胞系；293T(HEK293T)：人胚胎肾细胞系；786-O：人类肾透明细胞腺癌；OVCAR3：人类卵巢癌细胞系；A549：人类肺腺癌细胞系；MHCC97H：人类肝癌细胞系；LLC：小鼠Lewis肺癌细胞系；MDA-MB-231：人类乳腺癌细胞系；MCA205：小鼠纤维肉瘤细胞系；MV3：人类黑色素瘤细胞系；B16-F10：小鼠黑色素瘤细胞系；SKOV3：人类卵巢癌细胞系；T/G HA-VSMC：人类血管平滑肌细胞系；HEB：人脑星型胶质正常细胞；H1299：人类非小细胞肺癌细胞系；B104：大鼠神经母细胞瘤系；LO2：人正常肝细胞系；NIH 3T3：小鼠成纤维细胞系。Figure 2 shows the expression level of PIR protein in different tumor cells and normal cells. The above cells were cultured for 24 hours and proteins were extracted, and western blot analysis was performed using anti-PIR and anti-β-actin (control) antibodies. HeLa: human cervical cancer cell line; SW620: human colon cancer cell line; CT26.WT: mouse colon cancer cell line; HCT 116: human colon cancer cell line; Hep G2: human liver cancer cell line; L-929: mouse fibroblast cell line; sMPNST: human sporadic malignant peripheral nerve sheath tumor cell line; MCF7: human breast cancer cell line; HFF-1: human embryonic fibroblast cell line; 293T (HEK293T): human embryonic kidney cell line; 786-O: human renal clear cell adenocarcinoma; OVCAR3: human ovarian cancer cell line; A549: human lung adenocarcinoma cell line; MHCC97H: human hepatoma cell line; LLC: mouse Lewis lung cancer cell line; MDA-MB-231: human breast cancer cell line; MCA205: mouse fibrosarcoma cell line; MV3: human melanoma cell line; B16-F10: mouse melanoma cell line; SKOV3: human ovarian cancer cell line; T/G HA-VSMC: human vascular smooth muscle cell line; HEB: human brain astrocyte normal cells; H1299: human non-small cell lung cancer cell line; B104: rat neuroblastoma line; LO2: human normal liver cell line; NIH 3T3: mouse fibroblast cell line.
图3为TCGA临床数据库多种癌症中PIR mRNA水平分析结果。COAD：结肠腺癌；DLBC：弥漫性大B细胞淋巴瘤；UCEC：子宫内膜癌；GBM：多形性胶质母细胞瘤；KIRC：肾透明细胞癌；KIRP：肾乳头状细胞癌；LIHC：肝细胞肝癌；LUSC：肺鳞状细胞癌；PAAD：胰腺腺癌；READ：直肠腺癌；SKCM：皮肤黑色素瘤；THYM：胸腺瘤。Figure 3 shows the results of PIR mRNA level analysis in various cancers in the TCGA clinical database. COAD: colon adenocarcinoma; DLBC: diffuse large B-cell lymphoma; UCEC: endometrial carcinoma; GBM: glioblastoma multiforme; KIRC: renal clear cell carcinoma; KIRP: renal papillary cell carcinoma; LIHC: hepatocellular carcinoma; LUSC: lung squamous cell carcinoma; PAAD: pancreatic adenocarcinoma; READ: rectal adenocarcinoma; SKCM: cutaneous melanoma; THYM: thymoma.
图4为TCGA临床数据库PIR表达水平与预后相关性的分析结果。分析TCGA临床数据库PIR表达水平与胶质母细胞瘤(A)、肝癌(B)、乳腺癌(C)及肾癌(D)患者预后关系。GBM：多形性胶质母细胞瘤；LIHC：肝细胞肝癌；BRCA：乳腺癌；KIRC：肾透明细胞癌。Figure 4 shows the analysis results of the correlation between PIR expression level and prognosis in the TCGA clinical database. The relationship between PIR expression level in the TCGA clinical database and the prognosis of patients with glioblastoma (A), liver cancer (B), breast cancer (C) and kidney cancer (D) was analyzed. GBM: glioblastoma multiforme; LIHC: hepatocellular carcinoma; BRCA: breast cancer; KIRC: renal clear cell carcinoma.
图5为多种肿瘤细胞在感染可表达shPIR#1的病毒60h后用PI染色，采用流式细胞术检测细胞存活率的统计图。数据代表均值±标准差，三个独立的实验(非配对t检验，***p<0.001),CTRL：Control。HCT 116：人类结肠癌细胞；HeLa：人类宫颈癌细胞；MCF7：人类乳腺癌细胞；LLC：小鼠Lewis肺癌细胞；sMPNST：人类散发性恶性周围神经鞘膜瘤细胞；MV3：人类黑色素瘤细胞；OVCAR3：人类卵巢癌细胞；A549：人类肺腺癌细胞；786-O：人类肾透明细胞腺癌细胞。CTRL：Control。Figure 5 is a statistical graph of various tumor cells stained with PI 60 hours after infection with a virus expressing shPIR#1, and cell survival rates were detected by flow cytometry. Data represent mean ± standard deviation, three independent experiments (unpaired t test, ***p<0.001), CTRL: Control. HCT 116: human colon cancer cells; HeLa: human cervical cancer cells; MCF7: human breast cancer cells; LLC: mouse Lewis lung cancer cells; sMPNST: human sporadic malignant peripheral nerve sheath tumor cells; MV3: human melanoma cells; OVCAR3: human ovarian cancer cells; A549: human lung adenocarcinoma cells; 786-O: human renal clear cell adenocarcinoma cells. CTRL: Control.
图6为敲低或回补PIR蛋白表达水平后细胞凋亡情况。HCT 116细胞先单独感染可表达rHA-PIR或CTRL的病毒24h，再继续分别感染可表达shPIR#1的病毒72h，进一步用Annexin V-PI染色后用流式细胞术分析细胞凋亡情况(左)，对应的PIR蛋白用免
疫印迹的方法检测(右)。rHA-PIR:rescuing HA-PIR,即带HA标签的PIR的补救表达载体。Figure 6 shows the cell apoptosis after knocking down or replenishing the expression level of PIR protein. HCT 116 cells were first infected with viruses expressing rHA-PIR or CTRL for 24 hours, and then infected with viruses expressing shPIR#1 for 72 hours. After further staining with Annexin V-PI, cell apoptosis was analyzed by flow cytometry (left). The corresponding PIR protein was expressed by immunofluorescence staining. Detection by immunoblotting (right). rHA-PIR:rescuing HA-PIR, i.e., rescue expression vector of PIR with HA tag.
图7为PIR蛋白敲低后的RNA-Seq结果富集分析。HCT 116细胞感染可表达shPIR的病毒60h后提取细胞的总RNA,执行RNA-Seq实验，并进一步进行基因富集分析。Figure 7 shows the enrichment analysis of RNA-Seq results after PIR protein knockdown. Total RNA was extracted from HCT 116 cells 60 hours after infection with a virus expressing shPIR, and RNA-Seq experiments were performed, and gene enrichment analysis was further performed.
图8为敲低PIR蛋白水平后FAS信号通路中关键调控蛋白的免疫印迹结果图。在HCT 116细胞中先感染可表达rHA-PIR或CTRL的病毒24h，再分别感染可表达shPIR#1或shPIR#2的病毒60h，通过免疫印迹实验检测FAS信号通路中关键调控蛋白的变化。Casp8：Caspase 8；Casp3：Caspase 3。Figure 8 shows the immunoblotting results of key regulatory proteins in the FAS signaling pathway after knocking down the PIR protein level. HCT 116 cells were first infected with a virus expressing rHA-PIR or CTRL for 24 hours, and then infected with a virus expressing shPIR#1 or shPIR#2 for 60 hours, and the changes in key regulatory proteins in the FAS signaling pathway were detected by immunoblotting experiments. Casp8: Caspase 8; Casp3: Caspase 3.
图9为敲低FAS蛋白后再敲低PIR蛋白的肿瘤细胞的存活率。在HCT 116细胞中先感染可表达shFAS或CTRL的病毒24h后再分别感染可表达shPIR#1的病毒48h。细胞用PI染色，然后用流式细胞术进行生存分析(上)。PIR和FAS蛋白免疫印迹结果(下)。数据代表均值±标准差，五个独立的实验(非配对t检验，***p<0.001)。Figure 9 shows the survival rate of tumor cells after knocking down FAS protein and then knocking down PIR protein. HCT 116 cells were first infected with a virus expressing shFAS or CTRL for 24 hours and then infected with a virus expressing shPIR#1 for 48 hours. Cells were stained with PI and survival analysis was performed by flow cytometry (top). PIR and FAS protein immunoblotting results (bottom). Data represent mean ± SD, five independent experiments (unpaired t test, ***p < 0.001).
图10为FAS单抗处理不同PIR蛋白水平的细胞的存活率。在HCT 116细胞中梯度体积感染可表达shPIR的病毒24h，用2.5g mL-1FAS激活单抗(FAS mAb)处理细胞24h后用PI进行染色，进一步采用流式细胞术对上述细胞进行生存分析(上)，对应的PIR蛋白水平用免疫印迹实验分析(下)。数据代表均值±标准差，三个独立的实验(非配对t检验，**p<0.01)。Figure 10 shows the survival rate of cells with different PIR protein levels treated with FAS mAb. HCT 116 cells were infected with viruses expressing shPIR in gradient volume for 24 hours, and the cells were treated with 2.5 g mL -1 FAS activating mAb (FAS mAb) for 24 hours and then stained with PI. The above cells were further analyzed for survival by flow cytometry (upper), and the corresponding PIR protein levels were analyzed by immunoblotting (lower). Data represent mean ± standard deviation, three independent experiments (unpaired t test, **p<0.01).
图11为CD8+T细胞处理不同PIR蛋白水平和FAS蛋白水平敲低的细胞的存活率。在HCT 116细胞中梯度体积感染可表达shPIR的病毒24h，将HCT 116细胞与预激活的人效应CD8+T细胞孵育20h后用PI染色，进一步采用流式细胞术对上述细胞进行生存分析(上)，采用免疫印迹实验检测PIR和FAS蛋白水平(下)，数据代表均值±标准差，三个独立的实验(非配对t检验，***p<0.001，n.s.：无显著性差异)。Figure 11 shows the survival rate of cells with different PIR protein levels and FAS protein levels knocked down by CD8 + T cells. HCT 116 cells were infected with viruses expressing shPIR in gradient volumes for 24 hours, and HCT 116 cells were incubated with pre-activated human effector CD8 + T cells for 20 hours and then stained with PI. The above cells were further analyzed for survival by flow cytometry (top), and PIR and FAS protein levels were detected by immunoblotting (bottom). The data represent mean ± standard deviation, three independent experiments (unpaired t test, ***p<0.001, ns: no significant difference).
图12为PIR蛋白抑制剂TphA、CCG1423或CCG203971处理后细胞的存活情况。用DMSO(CTRL)或PIR蛋白抑制剂TphA(100μM)、CCG1423(10μM)、CCG203971(10μM)处理HCT 116、HeLa、sMPNST和HFF-1细胞，48小时后用显微镜拍摄照后用PI染色，进一步采用流式细胞术进行生存分析。数据代表均值±标准差，三个独立的实验(非配对t检验，***p<0.001，n.s.：无显著性差异)。HCT 116：人类结肠癌细胞系；HeLa：人类宫颈癌细胞系；sMPNST：人类散发性恶性周围神经鞘膜瘤细胞系；HFF-1：人类胚胎成纤维细胞系。
Figure 12 shows the survival of cells after treatment with PIR protein inhibitors TphA, CCG1423 or CCG203971. HCT 116, HeLa, sMPNST and HFF-1 cells were treated with DMSO (CTRL) or PIR protein inhibitors TphA (100 μM), CCG1423 (10 μM), CCG203971 (10 μM), and photographed with a microscope 48 hours later and stained with PI, and further survival analysis was performed by flow cytometry. Data represent mean ± standard deviation, three independent experiments (unpaired t test, ***p<0.001, ns: no significant difference). HCT 116: human colon cancer cell line; HeLa: human cervical cancer cell line; sMPNST: human sporadic malignant peripheral nerve sheath tumor cell line; HFF-1: human embryonic fibroblast cell line.
图13为不同浓度PIR蛋白抑制剂TphA处理后细胞的存活情况。用DMSO(CTRL)、50μM TphA或150μM TphA处理A549、MCF7、SN12-PM6、HeLa、U118、PANC-1、PC-3细胞，48小时后用显微镜拍摄照后用PI染色，进一步采用流式细胞术进行生存分析。数据代表均值±标准差，三个独立的实验(非配对t检验，***p<0.001)。A549：人类肺腺癌细胞系；MCF7：人类乳腺癌细胞系；SN12-PM6：人类肾癌细胞系；HeLa：人类宫颈癌细胞系；U118：人脑星形胶质母细胞瘤细胞系；PANC-1：人类胰腺癌细胞系；PC-3：人类前列腺癌细胞系。Figure 13 shows the survival of cells after treatment with different concentrations of PIR protein inhibitor TphA. A549, MCF7, SN12-PM6, HeLa, U118, PANC-1, and PC-3 cells were treated with DMSO (CTRL), 50 μM TphA, or 150 μM TphA. After 48 hours, the cells were photographed under a microscope and stained with PI. Flow cytometry was used for survival analysis. The data represent mean ± standard deviation, three independent experiments (unpaired t test, ***p<0.001). A549: human lung adenocarcinoma cell line; MCF7: human breast cancer cell line; SN12-PM6: human kidney cancer cell line; HeLa: human cervical cancer cell line; U118: human brain astrocytoblastoma cell line; PANC-1: human pancreatic cancer cell line; PC-3: human prostate cancer cell line.
图14为PIR蛋白抑制剂CCG1423处理裸小鼠移植瘤模型的肿瘤发展情况。将1×107HCT 116细胞接种到裸小鼠皮下，1周后对其注射PBS(对照组)或PIR蛋白抑制剂CCG1423(一周三次皮下注射10mg kg-1的CCG1423)，一个月后将裸小鼠安乐死，解剖取出肿瘤并进行称重。Figure 14 shows the tumor development of nude mouse transplanted tumor model treated with PIR protein inhibitor CCG1423. 1×10 7 HCT 116 cells were inoculated subcutaneously into nude mice, and PBS (control group) or PIR protein inhibitor CCG1423 (10 mg kg -1 CCG1423 was subcutaneously injected three times a week) was injected into the nude mice one week later. After one month, the nude mice were euthanized, and the tumors were removed and weighed by dissection.
图15为以PIR蛋白作为药物靶点的抗肿瘤药物的高通量筛选方法。该筛选方法以PIR的槲皮素2，3-双加氧酶活性进行药物效能评价，利用原核生物E.coli表达带6×His标签的PIR蛋白，经离心收集、超声裂解和Ni-NTA柱纯化后，用硼酸缓冲液(Boric acid buffer，80mM H3BO3，pH＝10.0)透析并将PIR蛋白稀释至5μM，以每孔50μL铺384孔透明平底板，使用Plate&Worker自动化筛选系统(美国PE公司)添加0.1μL待筛选化合物(DMSO溶解至10mM)，混匀后再添加0.1μL槲皮素(Quercetin，DMSO溶解至100mM)，转移至恒温培养箱37℃培养1h，分别在0min,20min,40min检测波长为400nm(槲皮素特征吸收峰)的紫外可见光谱吸光度，计算吸光度的降低速率，挑选速率显著低于阴性对照(仅不添加PIR蛋白)的待筛选化合物作为候选化合物(Candidates)进行ITC(等温滴定量热法)和MST(微量热泳动技术)等其他验证性实验。FIG15 is a high-throughput screening method for anti-tumor drugs using PIR protein as a drug target. This screening method uses the quercetin 2,3-dioxygenase activity of PIR to evaluate drug efficacy. The PIR protein with a 6×His tag is expressed in prokaryotic organism E. coli. After centrifugation, ultrasonic lysis and Ni-NTA column purification, the protein is purified by using a boric acid buffer (80 mM H 3 BO 3 , pH = 10.0) was dialyzed and the PIR protein was diluted to 5 μM, and 50 μL per well was used to cover 384-well transparent flat-bottom plates. 0.1 μL of the compound to be screened (dissolved in DMSO to 10 mM) was added using the Plate&Worker automated screening system (PE, USA), and 0.1 μL of quercetin (dissolved in DMSO to 100 mM) was added after mixing. The plates were transferred to a constant temperature incubator at 37°C and cultured for 1 h. The absorbance of the UV-visible spectrum at a wavelength of 400 nm (characteristic absorption peak of quercetin) was detected at 0 min, 20 min, and 40 min, respectively, and the rate of decrease of the absorbance was calculated. The compounds to be screened whose rate was significantly lower than that of the negative control (only without adding PIR protein) were selected as candidate compounds (Candidates) for other confirmation experiments such as ITC (isothermal titration calorimetry) and MST (microthermophoresis technology).
本发明通过在肿瘤组织和细胞中检测PIR蛋白水平(图1-2)并结合TCGA数据库分析(图3)发现PIR在多种肿瘤中高表达，并且PIR表达水平与预后负相关(图4)。通过RNA干扰技术敲低PIR后发现肿瘤细胞死亡明显增多(图5)，且通过RNA干扰技术敲低PIR并回补表达PIR蛋白后用流式细胞术分析发现PIR敲低引起的肿瘤细胞凋亡能被回补PIR所挽救(图6)，说明PIR是一个关键的抗凋亡蛋白。进一步RNA-Seq分析发现PIR敲低后FAS介导的凋亡通路被富集(图7)。敲低PIR能显著升高FAS蛋白的表达水平(图8)，提前将FAS敲低后再敲低PIR蛋白不再引起肿瘤细胞死亡(图9)，说明敲低PIR主要通过上调FAS引起细胞凋亡，PIR是通过抑制FAS表达维持细胞生存。由于FAS介导的凋亡通路是免疫细胞杀伤肿瘤的重要途径，为了确定PIR在其
中的功能，利用FAS单抗和激活的CD8+T细胞处理PIR不同程度敲低的HCT 116细胞(图10-11)，发现PIR敲低的细胞对FAS单抗和CD8+T细胞的敏感性显著增强，说明PIR通过抑制FAS表达对抗CD8+T细胞的杀伤作用。以上实验证明，PIR是一个极具潜力的肿瘤免疫增敏的靶点。为验证PIR作为肿瘤治疗靶点的可行性，利用现有的PIR抑制剂TphA、CCG1423或CCG203971处理HCT 116、HeLa、sMPNST和HFF-1细胞(图12)，发现PIR抑制剂处理会导致PIR蛋白表达水平高的细胞死亡，而对PIR蛋白表达水平低的细胞未产生有效的细胞毒性，且PIR抑制剂TphA处理浓度越高细胞死亡越明显(图13)，而在用CCG1423处理移植瘤小鼠(图14)时发现CCG1423对移植瘤的增殖有显著的抑制作用。此外，我们提供一种以PIR蛋白为药物靶点的抗肿瘤药物的高通量筛选方法(图15)。
The present invention detected the PIR protein level in tumor tissues and cells (Figures 1-2) and combined with the TCGA database analysis (Figure 3) to find that PIR is highly expressed in a variety of tumors, and the PIR expression level is negatively correlated with prognosis (Figure 4). After knocking down PIR by RNA interference technology, it was found that tumor cell death increased significantly (Figure 5), and after knocking down PIR by RNA interference technology and re-expressing PIR protein, flow cytometry analysis found that tumor cell apoptosis caused by PIR knockdown can be rescued by re-expressing PIR (Figure 6), indicating that PIR is a key anti-apoptotic protein. Further RNA-Seq analysis found that the FAS-mediated apoptosis pathway was enriched after PIR knockdown (Figure 7). Knocking down PIR can significantly increase the expression level of FAS protein (Figure 8). Knocking down PIR protein after knocking down FAS in advance no longer causes tumor cell death (Figure 9), indicating that knocking down PIR mainly causes cell apoptosis by up-regulating FAS, and PIR maintains cell survival by inhibiting FAS expression. Since the FAS-mediated apoptosis pathway is an important way for immune cells to kill tumors, in order to determine the role of PIR in its In order to investigate the function of PIR in tumor therapy, HCT 116 cells with different degrees of PIR knockdown were treated with FAS monoclonal antibody and activated CD8 + T cells (Figures 10-11). It was found that the sensitivity of PIR knockdown cells to FAS monoclonal antibody and CD8 + T cells was significantly enhanced, indicating that PIR inhibited the killing effect of CD8 + T cells by inhibiting FAS expression. The above experiments proved that PIR is a target with great potential for tumor immunosensitization. In order to verify the feasibility of PIR as a target for tumor therapy, HCT 116, HeLa, sMPNST and HFF-1 cells were treated with existing PIR inhibitors TphA, CCG1423 or CCG203971 (Figure 12). It was found that PIR inhibitor treatment would cause cell death with high PIR protein expression levels, but no effective cytotoxicity was produced for cells with low PIR protein expression levels. The higher the concentration of PIR inhibitor TphA treatment, the more obvious the cell death (Figure 13). When CCG1423 was used to treat mice with transplanted tumors (Figure 14), it was found that CCG1423 had a significant inhibitory effect on the proliferation of transplanted tumors. In addition, we provide a high-throughput screening method for anti-tumor drugs using PIR protein as drug target ( FIG. 15 ).
Claims (9)
- Pirin基因/蛋白作为抗肿瘤靶点的应用。Application of Pirin gene/protein as anti-tumor target.
- 根据权利要求1所述应用，其特征在于，所述Pirin基因即：Ensembl-ENSG00000087842，所述Pirin蛋白即：UniProtKB-O00625。The use according to claim 1 is characterized in that the Pirin gene is: Ensembl-ENSG00000087842, and the Pirin protein is: UniProtKB-O00625.
- 根据权利要求2所述应用，其特征在于，Ensembl-ENSG00000087842的核苷酸序列如SEQ NO 2；UniProtKB-O00625的氨基酸序列如SEQ NO 1。The use according to claim 2 is characterized in that the nucleotide sequence of Ensembl-ENSG00000087842 is such as SEQ NO 2; the amino acid sequence of UniProtKB-O00625 is such as SEQ NO 1.
- Pirin抑制剂在抗肿瘤中的应用，或Pirin抑制剂在制备抗肿瘤药物中的应用。Application of Pirin inhibitor in anti-tumor, or application of Pirin inhibitor in preparation of anti-tumor medicine.
- 检测Pirin含量在肿瘤诊断或肿瘤筛查或免疫治疗或预后判断的应用，检测Pirin含量的试剂在制备肿瘤诊断或肿瘤筛查或免疫治疗或预后判断检测试剂盒的应用。The application of detecting Pirin content in tumor diagnosis, tumor screening, immunotherapy or prognosis judgment, and the application of reagents for detecting Pirin content in the preparation of tumor diagnosis, tumor screening, immunotherapy or prognosis judgment detection kits.
- 根据权利要求1-5任一项权利要求所述应用，其特征在于，所述肿瘤为Pirin高表达的肿瘤，选自结直肠癌、肝癌、肺癌、乳腺癌、胰腺癌、前列腺癌、宫颈癌、肾癌、胃癌、胶质瘤、黑色素瘤、子宫内膜癌等。The use according to any one of claims 1 to 5 is characterized in that the tumor is a tumor with high expression of Pirin, selected from colorectal cancer, liver cancer, lung cancer, breast cancer, pancreatic cancer, prostate cancer, cervical cancer, kidney cancer, gastric cancer, glioma, melanoma, endometrial cancer, etc.
- 靶向Pirin基因或蛋白的药物作为肿瘤免疫增敏剂的应用。Application of drugs targeting Pirin gene or protein as tumor immunosensitizers.
- 根据权利要求7所述应用，其特征在于，所述肿瘤免疫增敏剂的应用包括将Pirin抑制剂作为增强免疫功能的成分，或将Pirin抑制剂与免疫治疗药物联合应用于抗肿瘤的应用。The use according to claim 7 is characterized in that the use of the tumor immunosensitizer includes using the Pirin inhibitor as a component for enhancing immune function, or combining the Pirin inhibitor with an immunotherapy drug for anti-tumor application.
- 一种筛选抗肿瘤药物的方法，其特征在于，通过使用Pirin蛋白作为药物筛选靶点进行筛选。 A method for screening anti-tumor drugs, characterized in that the screening is performed by using Pirin protein as a drug screening target.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7247426B2 (en) * | 2001-08-02 | 2007-07-24 | Agilent Technologies, Inc. | Classifying cancers |
| US20170326202A1 (en) * | 2014-12-23 | 2017-11-16 | 4D Pharma Research Limited | Pirin polypeptide and immune modulation |
| CN116942817A (en) * | 2022-12-14 | 2023-10-27 | 厦门大学 | Application of Pirin gene and protein in preparation of antitumor target medicaments |
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| Title |
|---|
| ARAI TAKAYUKI, KOJIMA SATOKO, YAMADA YASUTAKA, SUGAWARA SHO, KATO MAYUKO, YAMAZAKI KAZUTO, NAYA YUKIO, ICHIKAWA TOMOHIKO, SEKI NAO: "Pirin: a potential novel therapeutic target for castration‐resistant prostate cancer regulated by miR‐455‐5p", MOLECULAR ONCOLOGY, ELSEVIER, vol. 13, no. 2, 1 February 2019 (2019-02-01), pages 322 - 337, XP093180902, ISSN: 1574-7891, DOI: 10.1002/1878-0261.12405 * |
| HAI-LU JIANG, LIU FENG, DONG WEN-HUI, WU JIN, MAN JIANG-HONG: "Effect of Pirin on the proliferation and self-renew of glioma stem cell", JOURNAL OF INTERNATIONAL PHARMACEUTICAL RESEARCH, JUNSHI YIXUE KEXUEYUAN * DUWU YAOWU JIANJIUSUO, CN, vol. 44, no. 10, 30 October 2017 (2017-10-30), CN , pages 953 - 957, XP093180903, ISSN: 1674-0440, DOI: 10.13220/j.cnki.jipr.2017.10.009 * |
| JINRUI FAN, WANG LEI, ZOU JUNJIE: "Research Progress on PRNs Gene Family", CURRENT BIOTECHNOLOGY, vol. 12, no. 3, 25 May 2022 (2022-05-25), pages 325 - 331, XP093180909, DOI: 10.19586/j.2095•2341.2021.190 * |
| ZHANG YING, KNATKO ELENA V., HIGGINS MAUREEN, DAYALAN NAIDU SHARADHA, SMITH GILLIAN, HONDA TADASHI, DE LA VEGA LAUREANO, DINKOVA-K: "Pirin, an Nrf2-Regulated Protein, Is Overexpressed in Human Colorectal Tumors", ANTIOXIDANTS, MDPI AG, vol. 11, no. 2, 1 February 2022 (2022-02-01), pages 262, XP093180901, ISSN: 2076-3921, DOI: 10.3390/antiox11020262 * |
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