Diagnostic reagent and therapeutic reagent for human head and neck squamous cell carcinoma

Abstract

The invention discloses a clinical diagnosis reagent for human head and neck squamous cell carcinoma, which is a reagent for detecting the expression level of human NCAPH gene, the expression level of the human NCAPH gene is in negative correlation with clinical prognosis of the human head and neck squamous cell carcinoma, and experimental results show that the expression of the human NCAPH gene in a human head and neck squamous cell carcinoma cell line is higher than that of normal nasopharyngeal epithelial cells. By knocking down the expression of the human NCAPH gene, the proliferation of head and neck squamous carcinoma cells and the clone sphere forming ability can be remarkably inhibited; the invention further provides a medicine for inhibiting human NCAPH gene expression or an NPIDP small peptide medicine for inhibiting human PD-L1 gene expression, the NPIDP small peptide medicine acts on human head and neck squamous carcinoma cells, combination of NCAPH and HIP1R can be remarkably and competitively blocked, degradation of PD-L1 protein is promoted, and therefore the T cell killing effect is enhanced, and the NPIDP small peptide medicine can be used for treating human head and neck squamous carcinoma cells. The invention provides a novel diagnosis and treatment medicine for treatment of human head and neck squamous cell carcinoma.

Description

Technical Field

[0001] The present invention relates to the field of tumor treatment, and in particular to a diagnostic reagent and a therapeutic reagent for human head and neck squamous cell carcinoma, belonging to the field of biomedical technology. Background Art

[0002] Head and neck squamous cell carcinoma (HNSCC) originates from the mucosal epithelial cells of the oral cavity, pharynx, larynx and sinus tract, and is the most common malignant tumor of the head and neck. Epidemiological studies have shown that the main risk factors for HNSCC include smoking, drinking, exposure to environmental pollutants and infection with viral factors (i.e. HPV and EBV). Among them, the increase in HPV infection rate is one of the important reasons for the high incidence of HNSCC in recent years. Currently, HNSCC is the sixth most common cancer in the world, with a total of 880,000 new cases and 450,000 deaths in 2020. Its incidence rate continues to rise and is expected to increase by 30% in 2030 (i.e. 1.08 million new cases per year). The number of new cases and deaths of HNSCC in China is second only to India. In the past nearly two decades, surgical resection combined with chemoradiotherapy has improved the survival rate of HNSCC patients to a certain extent, and platinum, 5-fluorouracil, docetaxel and cetuximab have also become first-line drugs for systemic treatment of HNSCC. The most common factors affecting the development of HNSCC are high-frequency mutations in some tumor suppressor genes, including TP53, CDKN2A, and FAT1, but it is still very difficult to target these inactivated tumor suppressor genes for treatment. Recent studies suggest that the PI3K / AKT / mTOR signaling pathway and the cell cycle signaling pathway can be used as new therapeutic targets for HNSCC, but clinical studies of drugs targeting these signaling pathways have proven to be ineffective.

[0003] Tumor immunotherapy has become an emerging and popular tumor treatment option because of its good specificity and few side effects. The tumor immune microenvironment is mainly composed of tumor-infiltrating lymphocytes (T cells, B cells, and NK cells) and myeloid cells (macrophages, neutrophils, dendritic cells, and myeloid-derived suppressor cells). Under physiological conditions, the activity of T cells is intricately regulated, selectively eliminating pathogens and abnormal cells, but avoiding attacking normal cells to maintain immune homeostasis. Although HNSCC tumors are highly infiltrated by immune cells compared to other solid tumors, multiple studies have confirmed that the tumor microenvironment (TME) in most HNSCCs plays a highly immunosuppressive role. PD-1 (encoded by PDCD1) / PD-L1 (encoded by CD274) are important proteins for maintaining immune homeostasis. In the TME, the PD-1 / PD-L1 axis is hijacked by tumor cells. In advanced HNSCC, the expression of PD-L1 in tumor cells is upregulated, and the MDSC and Treg cells recruited to the TME upregulate the expression of PD-L1 and CTLA4. By phosphorylating SHP-2, the TCR signal cascade reaction is offset to escape immune detection, which greatly weakens the lytic activity of T cells against tumor cells. Therefore, targeted blockade of the PD-1 / PD-L1 axis is currently one of the mainstream solutions to avoid tumor cell immune escape and thus improve the effect of immunotherapy. Although some immunotherapy drugs targeting the PD-1 / PD-L1 signal axis, such as Keytruda (pembrolizumab), have entered clinical trials for HNSCC, the response rate is about 15%. Therefore, it is urgent to further analyze the expression and regulation mechanism of PD-L1 in HNSCC and find new targeted drugs.

[0004] So far, there are no reports on the correlation between NCAPH gene expression and human head and neck squamous cell carcinoma, or the function of NCAPH in regulating PD-L1 in tumor cells. Summary of the invention

[0005] The present invention provides a clinical diagnostic reagent for human head and neck squamous cell carcinoma. The clinical diagnostic reagent for human head and neck squamous cell carcinoma is prepared from a reagent for detecting the expression of human NCAPH gene. The reagent for detecting the expression of human NCAPH gene is a reagent for detecting the high expression of human NCAPH gene.

[0006] The invention designs RNA primer sequences of human NCAPH by using human NCAPH gene sequences, and detects the RNA level of human NCAPH by real-time quantitative PCR; the primer sequences for detecting the RNA level of human NCAPH are AAACACGCAGATTACGGAACA, GTTGGTTGGTTCGGTGTCTTT.

[0007] The diagnostic reagents of the present invention also include conventional commercially available reagents used in real-time quantitative PCR.

[0008] Another object of the present invention is to provide a drug for treating human head and neck squamous cell carcinoma, wherein the drug is obtained by screening for the purpose of inhibiting the expression of human NCAPH gene.

[0009] The present invention conducted single-cell omics analysis on 6 HNSCC patients (3 of whom responded to PD-1 monoclonal antibody immunotherapy and 3 did not respond) and found that the T cell subpopulations infiltrating the tumor of patients who did not respond to immunotherapy were significantly reduced, and their tumor cell subpopulations also had activation of cancer-promoting signaling pathways such as proliferation and metastasis; further through evolutionary biology multi-omics comparison and tumor cell subpopulation differential gene expression analysis, it was found that NCAPH (Non-SMCcondensin I complex subunit H) was significantly upregulated in patients who did not respond to immunotherapy. In-depth research found that NCAPH mainly affects T cell killing in HNSCC and is closely related to the activity of T cell receptor-related signaling pathways; further screening found that NCAPH promotes tumor immune escape by upregulating the expression of PD-L1 protein in tumor cells. In-depth research found that NCAPH can directly interact with PD-L1, and through truncation analysis, it was found that amino acids 271-360 of the NCAPH molecule can bind to PD-L1.

[0010] Another object of the present invention is to provide a drug for treating human head and neck squamous cell carcinoma, which is a NPIDP small peptide drug for the purpose of inhibiting the expression of human PD-L1 gene.

[0011] The NPIDP small peptide drug is a small peptide composed of the cell-penetrating peptide iRGD (CRGDKGPDC) and the amino acid sequence at positions 271-360 of the NCAPH molecule, and its amino acid sequence is shown in SEQ ID NO:1.

[0012] The present invention finds that the NPIDP small peptide drug simulates the binding of NCAPH to PD-L1 by binding to PD-L1 on human head and neck squamous cell carcinoma cells, thereby effectively blocking the binding of NCAPH to PD-L1, promoting PD-L1 to enter the lysosomal degradation pathway, thereby promoting immune activation, inhibiting the development of human head and neck squamous cell carcinoma, and achieving the purpose of treating human head and neck squamous cell carcinoma.

[0013] The drug for treating human head and neck squamous cell carcinoma of the present invention uses a drug for inhibiting human NCAPH gene expression or a NPIDP small peptide as an active ingredient, and may be added with one or more excipients acceptable to pharmaceutical preparations, or compounded with other active ingredients to exert a synergistic inhibitory effect on human head and neck squamous cell carcinoma; and may be prepared into a pharmaceutically suitable dosage form.

[0014] Advantages and technical effects of the present invention: The present invention provides a new approach for clinical diagnosis and clinical treatment of human head and neck squamous cell carcinoma. The present invention performs clinical diagnosis by detecting high expression of human NCAPH gene; it is found that NCAPH gene is highly expressed in human head and neck squamous cell carcinoma, and the expression of NCAPH gene is inhibited by targeting NCAPH gene to achieve the purpose of treating human head and neck squamous cell carcinoma; the present invention also finds through experiments that NPIDP small peptide drugs can significantly and specifically degrade PD-L1, thereby achieving immune activation and inhibiting the progression of human head and neck squamous cell carcinoma; and after treatment with NPIDP small peptide drugs, the protein level of PD-L1 in human head and neck squamous cell carcinoma cell lines is significantly reduced, the protein level of PD-L1 on the membrane surface is also reduced accordingly, and the number of PD-L1 positive cells is significantly reduced. When NPIDP small peptide drugs are co-cultured with T cells, the killing effect of T cells is significantly higher than that of T cells not treated with NPIDP small peptide drugs. Animal in vivo experiments have found that PD-1 monoclonal antibody and NPIDP small peptide drugs can effectively inhibit the growth of nude mouse transplanted tumors when treated alone, and the combination of the two can more effectively inhibit tumor growth. The NPIDP peptide drug treatment has no obvious toxic side effects on individual mice. The present invention provides a new approach for diagnosing and treating human head and neck squamous cell carcinoma, and has clinical application value and market promotion application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Figure 1 shows the expression of human NCAPH in head and neck squamous cell carcinoma and its relationship with prognosis; Figure A shows the expression of NCAPH in normal tissues and tumor tissues; Figure B shows the relationship between high and low NCAPH expression and prognosis; Figure 2 The expression levels of human NCAPH gene in normal human nasopharyngeal epithelial cells (NP69) and head and neck squamous cell carcinoma cell lines (CNE1, CNE2, SUNE1, 5-8F, SCC25, CAL27 and FADU). The upper figure is the qPCR test result, and the lower figure is the immunoblot test result; Figure 3 The efficiency test of knocking down the NCAPH gene in human head and neck squamous cell carcinoma cell lines CAL27 (left) and CNE2 (right); the upper figure is the RNA expression test result, and the lower figure is the protein test result; Figure 4 Immunoblotting experiments of EMT-related N-cadherin (N-cad) and Vimentin proteins after stable knockdown of NCAPH in head and neck squamous cell carcinoma cell lines CAL27 and CNE2; the upper figure is the immunoblotting results of N-cad and Vimentin proteins in various stable cell lines of CAL27, and the lower figure is the immunoblotting results of N-cad and Vimentin proteins in various stable cell lines of CNE2; Figure 5The growth curve results of head and neck squamous cell carcinoma CAL27 and CNE2 knockdown stable cells are shown in the figure on the left. The growth curve of each stable cell line of CAL27 is shown in the figure on the right. The growth curve of each stable cell line of CNE2 is shown in the figure on the right. Figure 6 The results of the clone sphere formation experiment after stable knockdown of the NCAPH gene in head and neck squamous cell carcinoma cell lines CAL27 and CNE2 are shown in the left figure. The clone sphere growth of each stable cell line of CAL27 and CNE2 is shown in the right figure. The statistical results of the clone sphere growth of each stable cell line of CAL27 and CNE2 are shown in the right figure. Figure 7 The results of transwell chamber migration experiments after stable knockdown of NCAPH gene in head and neck squamous cell carcinoma cell lines CAL27 and CNE2. Figure A is the chamber migration of each stable cell line of CAL27 and CNE2, and Figure B is the statistical results of chamber migration of each stable cell line of CAL27 and CNE2; Figure 8 The results of simultaneously overexpressing NCAPH-Myc and PD-L1-Flag or the N-terminal (PD-L1-N-Flag) or C-terminal (PD-L1-C-Flag) truncated proteins of PD-L1-Flag in 293T cells and detecting the interaction region between NCAPH protein and PD-L1 by co-immunoprecipitation method; Fig. 9 The results of overexpressing PD-L1-Flag and different truncated proteins of NCAPH-Myc in 293T cells and detecting the interaction region between PD-L1 protein and NCAPH by immunoprecipitation method; Fig.10 In order to design a small peptide for the screened NCAPH minimum segment N3 that interacts with PD-L1, the small peptide was purified and detected; Figure A is a schematic diagram of the design of the small peptide and its control; Figure B is the Coomassie brilliant blue staining result of the purified small peptide, and Figure C is the Ni column protein pull down experiment result; Fig.11 Results of the effects of PBS, Sumo, and NPIDP peptide on the growth of head and neck squamous cell carcinoma cell line CAL27: Fig.12 The results show the effects of PBS, Sumo and NPIDP peptide on the sphere formation of head and neck squamous cell carcinoma cell line CAL27. Fig.13The results of the effect of NPIDP small peptide on the expression levels of NCAPH, HIP1R, and PD-L1 in head and neck squamous cell carcinoma cells CAL27 and CNE2 cells. The left figure shows the expression levels of NCAPH, HIP1R, and PD-L1 in CAL27 cell lines after being treated with PBS, Sumo, and NPIDP under the stimulation of IFNγ; the right figure shows the expression levels of NCAPH, HIP1R, and PD-L1 in CNE2 cell lines after being treated with PBS, Sumo, and NPIDP under the stimulation of IFNγ. Fig.14 For competitive binding experiments, IP experiments were performed with PD-L1 antibodies to detect the effects of PBS, Sumo, and NPIDP treatment on the competitive binding of NCAPH and HIP1R to PD-L1; Fig.15 The cell killing experiment of CAL27 or CNE2 cells with or without PBMC co-culture after adding PBS, Sumo, and NPIDP treatment; the upper part shows the cell killing experiment results of each stable cell line of CAL27; the lower part shows the cell killing experiment results of each stable cell line of CNE2; Fig.16 This is an in vivo drug experiment to detect the inhibitory effect of different drugs (negative control vehicle, PD-1 antibody, NPIDP, PD-1 antibody and NPIDP at the same time) on in vivo tumors; Figure A is a schematic diagram of drug administration in mice; Figure B is the growth status of the tumor after drug administration; Figure C is a curve statistical result of tumor growth; Figure D is a statistical result of tumor weight; Fig.17 The expression and statistical results of immune-related molecules PD-L1, CD8, and GZMB were detected by immunohistochemistry in tumors under different drug treatments (negative control vehicle, PD-1 antibody, NPIDP, PD-1 antibody and NPIDP at the same time). The left side is a schematic diagram of immunohistochemical staining results under different drug treatments, and the right side is the statistical results of PD-L1+, CD8+, and GZMB+ cells. Fig.18 This is a test of whether the small peptide NPIDP has toxic side effects on mice; Figure A is the result of the mouse weight test; Figure B is the immunohistochemical staining of different internal organs after drug addition. DETAILED DESCRIPTION

[0016] The present invention is further described in detail below through examples, but the protection scope of the present invention is not limited to the contents described above. The methods in the examples are all conventional methods unless otherwise specified, and the reagents used are all conventional commercially available reagents or reagents prepared by conventional methods unless otherwise specified.

[0017] Example 1: Bioinformatics analysis RNAseq data and clinical data of the TCGA-HNSCC (head and neck squamous cell carcinoma) project were downloaded and organized from the TCGA database (https: / / portal.gdc.cancer.gov) as analysis materials. The software used was R (4.2.1) version. The ggplot2 package was used to visualize the data for expression difference analysis, and the stats package and car package were used for Wilcoxon rank sumtest statistical analysis. The survival package was used for proportional hazard hypothesis testing and fitted survival regression in survival analysis. The analysis results were visualized using the survminer package and ggplot2 package, and Logrank test statistical analysis was performed.

[0018] The results are as follows Figure 1 As shown in the figure, it can be seen from the results that the expression of human NCAPH gene in head and neck squamous cell carcinoma tissue is higher than that in normal tissue, and NCAPH expression is negatively correlated with patient survival. Patients with high expression have a short survival time, while patients with low expression have a longer survival time.

[0019] Example 2: Fluorescence quantitative PCR experiment When the target cells are cultured to the logarithmic growth phase, discard the culture medium, add PBS to rinse the cells and discard, add 1mL of Trizol lysis buffer, use a pipette to gently blow the cells in the dish, and transfer the Trizol lysis buffer sample to a 1.5mL collection tube after complete lysis; centrifuge the Trizol lysis buffer sample at 12000g and 4℃ for 5 minutes, transfer the supernatant to a new 1.5mL collection tube, and the precipitate below is the incompletely lysed cells and can be discarded. Add 200μL of chloroform to the Trizol lysis buffer, vortex to mix, let stand at room temperature for 5 minutes, and then centrifuge at 12000g and 4℃ for 15 minutes. Carefully transfer the top layer of liquid after centrifugation to a new RNase-free 1.5mL collection tube, add 750μL of isopropanol, gently invert up and down to mix, let stand at room temperature for 10 minutes, and then centrifuge at 12000g and 4℃ for 10 minutes. The white precipitate at the bottom of the tube after centrifugation is RNA. The isopropanol supernatant is discarded, and 1 mL of 75% ethanol prepared with DEPC water is added to wash the precipitate. Subsequently, the precipitate is centrifuged at 7500g and 4°C for 5 minutes. The 75% ethanol supernatant is discarded, and the collection tube is inverted. When the RNA precipitate is dried and translucent, an appropriate amount of DEPC water is added to dissolve the RNA, and the RNA concentration is determined using Nano-Drop. 1 μg of RNA is reversed into cDNA using a commercial reverse transcription kit and analyzed with commercial SYBR and NCAPH primers (Human NCAPH_F: AAACACGCAGATTACGGAACA; Human NCAPH_R:GTTGGTTGGTTCGGTGTCTTT) according to the corresponding reaction system (SYBR 20 μL, 2.5 μM Primer 2.4 μL, cDNA 1 μL, ddH2O 6.6 μL).

[0020] The results are as follows Figure 2 As shown, the expression of human NCAPH gene in head and neck squamous cell carcinoma (CNE1, CNE2, SUNE1, 5-8F, SCC25, CAL27, FADU) cell lines was significantly higher than that in normal immortalized nasopharyngeal epithelial cells NP69.

[0021] Example 3: Immunoblotting experiment 1. When the target cells grow to the logarithmic growth phase, place them on ice, discard the culture medium, add PBS to rinse the cells and discard, add an appropriate amount of RIPA lysis buffer, react on ice for 5 minutes, and use a cell scraper to transfer the lysate to a 1.5mL collection tube. After repeated freezing and thawing of liquid nitrogen 3 times, centrifuge at 15000g and 4℃ for 25 minutes (generally the highest speed of a refrigerated centrifuge), and transfer the supernatant to a clean 1.5mL collection tube for subsequent concentration determination. Add ddH2O and 5×SDS protein buffer to the protein supernatant to prepare a protein sample of a certain concentration. After flicking the bottom of the tube to mix, heat it in a 95℃ metal bath for 2~5 minutes, and then briefly centrifuge it for subsequent electrophoresis experiments. Assemble the protein SDS-PAGE gel into the electrophoresis tank and add the electrophoresis buffer. The electrophoresis conditions are constant voltage 150 V for 1 hour. When transferring, first soak the cut PVDF membrane in methanol for 5 minutes, then assemble the gel in a "sandwich" manner (sponge mesh-filter paper-gel-PVDF membrane-filter paper-sponge mesh) in the transfer buffer, and transfer the membrane at a constant current of 200~500 mA for 1~2 hours. After the transfer, cut the transferred PVDF membrane according to the desired experimental purpose, soak the cut PVDF membrane in 5% skim milk (prepared with 1×TBST membrane washing buffer) and block it on a shaker at room temperature for 1 hour, and use TBST membrane washing buffer to wash off the excess milk. Add the corresponding primary antibody (prepared with New Cyme Antibody Diluent), incubate on a shaker at 4°C overnight; wash three times with TBST membrane washing buffer, 10 minutes each time, add the secondary antibody of the species corresponding to the primary antibody (prepared with 5% skim milk), incubate on a shaker at room temperature for 2 hours, wash three times with TBST membrane washing buffer, 10 minutes each time, mix the HRP developer according to the ratio (A solution: B solution = 1:1), and soak the PVDF membrane that has been dried with special hand towels, and place the membrane in the developer for development after about 1 minute of reaction.

[0022] The results are as follows Figure 2 As shown, the expression level of NCAPH gene in head and neck squamous cell carcinoma (CNE1, CNE2, SUNE1, 5-8F, SCC25, CAL27, FADU) cell lines was significantly higher than that in normal human immortalized nasopharyngeal epithelial cells NP69.

[0023] 2. Head and neck squamous cell carcinoma cells CAL27 and CNE2 were first treated with 10 ng / mL IFNγ for 12 hours, and then PBS, Sumo (10 μg / mL, solubilizing tag), and NPIDP peptide (10 μg / mL, the sequence of NPIDP peptide is as follows: CRGDKGPDCGGGGSGGGGSGGGGSHHHHHHRSELLFPSDVQTLSTGEPLELPELGCVEMTDLKAPLQQCAEDRQICPSLAGFQFTQWDSETHNESVSALVDKFKKNDQVFDINAEVDESD) were added and treated for 12 hours. Then, the cells were lysed with IP buffer containing 0.5% NP-40. The protein samples were repeatedly frozen and thawed by liquid nitrogen for 3 times, and the BCA protein concentration was quantified. After that, an appropriate amount of 5×SDS protein buffer was added to mix with the magnetic beads, and the mixture was heated in a 95°C metal bath for 10 minutes and then briefly centrifuged for subsequent electrophoresis experiments.

[0024] The results are as follows Fig.13 As shown, the protein levels of NCAPH and HIP1R did not change significantly after drug treatment, and the protein level of PD-L1 did not change significantly under the treatment of PBS and 10μg / mL Sumo, but was significantly reduced after treatment with NPIDP peptide.

[0025] Example 4: Construction of a cell line with knockdown of NCAPH expression 1. Use the pLKO.1-shRNA lentiviral expression vector to knock down the target gene NCAPH, obtain the mRNA sequence of the NCAPH gene from the NCBI database, design and synthesize the shRNA target sequence (Human NCAPH sh#1:TCAGAGATTCTTAAACAGAAA; Human NCAPH sh#2: TCTCCTAAATTGATCTGTTAT), and use Nhe Ⅰ and EcoRⅠ restriction endonuclease cloned it into the pLKO.1-puro vector, and identified by enzyme digestion and sequencing. After the correct NCAPH knockdown plasmid was identified, lentivirus was prepared by lentivirus packaging system: first, the plasmid with shRNA target sequence and packaging plasmid (PMD2.G, PSPAX2) were co-transfected into HEK-293T cells by calcium phosphate transfection method, and normal culture medium was replaced after 8 hours, and then the supernatant culture medium with lentivirus was collected for 48 hours and 72 hours, Polybrene (10 μg / mL) was added and infected with different HNSCC tumor cell lines for 12 hours and normal culture medium was replaced. After 48 hours of infection, normal culture medium containing Puromycin (2 μg / mL) was replaced to screen the cells infected with lentivirus for stable cell lines. After the cells were passaged 3 times, the expression of RNA and protein in the cells after NCAPH knockdown was detected by qRT-PCR and immunoblotting techniques (same as in Examples 2 and 3); The results are as follows Figure 3 As shown, in head and neck squamous cell carcinoma cell lines CAL27 and CNE2 knockdown stable cell lines, the expression levels of RNA and protein of NCAPH gene were significantly reduced; Moreover, the expression levels of EMT-related N-cad and Vimentin proteins in head and neck squamous cell carcinoma CAL27 and CNE2 knockdown stable cell lines were significantly reduced compared with control cells ( Figure 4 ).

[0026] Example 5: Growth curve experiment 1. After the knockdown stable cell lines of head and neck squamous cell carcinoma cells CAL27 and CNE2 grew to the logarithmic growth phase, they were digested into single cells with 0.25% trypsin and then cultured at a rate of 1×10 4 The density of each well was set, and two replicate wells were inoculated into a 12-well cell culture plate. The cells were then digested and counted using 0.25% trypsin every day. The cell counting in each well was repeated 3 times, and the cell growth curve was drawn after 6 days.

[0027] The results are as follows Figure 5 As shown, the growth rate of stable cell lines with NCAPH knockdown in CAL27 (left) and CNE2 (right) was significantly lower than that of the control cell lines.

[0028] 2. Experiment on the effect of NPIDP peptide on the growth of head and neck squamous cell carcinoma CAL27 cell line The experiment set up a negative control (PBS buffer), a Sumo treatment group (added with 10 μg / mL Sumo, a solubilizing tag), and an NPIDP peptide treatment group (added with 10 μg / mL NPIDP peptide, the sequence of the NPIDP peptide is as follows: CRGDKGPDCGGGGSGGGGSGGGGSHHHHHHRSELLFPSDVQTLSTGEPLELPELGCVEMTDLKAPLQQCAEDRQICPSLAGFQFTQWDSETHNESVSALVDKFKKNDQVFDINAEVDESD); the operation procedure was the same as above.

[0029] The results are as follows Fig.11 As shown in the figure, it can be seen that there is no significant difference in the growth of CAL27 cells under NPIDP peptide treatment compared with the negative control group PBS group and Sumo group.

[0030] Example 6: Clone formation experiment NCAPH knockdown stable cell line clonal sphere formation experiment When the target cells grow to the logarithmic growth phase, they are digested into single cells with 0.25% trypsin and 600 target cells are inoculated into a 6-well plate containing 2 mL of normal culture medium. Fresh culture medium is replaced every other day. After culturing for about 2 weeks, the plates are washed twice with PBS, fixed with 4% paraformaldehyde for 20 minutes, washed twice with PBS, stained with 0.5% crystal violet stain, rinsed with distilled water to remove excess stain and dried, and the number of clones formed in each well was counted for analysis.

[0031] The results are as follows Figure 6 As shown in the figure, the number of clone spheres formed by the knockdown stable cell lines of head and neck squamous cell carcinoma CAL27 and CNE2 was significantly lower than that of the control cell line (SC group of head and neck squamous cell carcinoma CAL27 and CNE2).

[0032] 2. Experiment on the effect of NPIDP peptide on the clonal sphere formation of head and neck squamous cell carcinoma CAL27 cells The experiment set up a negative control (PBS buffer), a Sumo treatment group (added with 10 μg / mL Sumo, a solubilizing tag), and an NPIDP peptide treatment group (added with 10 μg / mL NPIDP peptide, the sequence of the NPIDP peptide is as follows: CRGDKGPDCGGGGSGGGGSGGGGSHHHHHHRSELLFPSDVQTLSTGEPLELPELGCVEMTDLKAPLQQCAEDRQICPSLAGFQFTQWDSETHNESVSALVDKFKKNDQVFDINAEVDESD); the operation procedure was the same as above.

[0033] The results are as follows Fig.12As shown in the figure, it can be seen that there is no significant difference in the clonal sphere formation ability of CAL27 cells added with NPIDP peptide and the Sumo treatment group and the control (PBS buffer) group.

[0034] Example 7: Transwell chamber migration assay When the target cells grew to the logarithmic growth phase, they were digested into single cells with 0.25% trypsin and 3×10 5 The target cells were centrifuged and the supernatant was removed, and the target cells were resuspended in 1 mL of serum-free medium. 100 μL of cell suspension was inoculated into the upper chamber of the Transwell chamber, and 600 μL of complete medium was added to the lower chamber. After culturing for 24 hours, the medium above the chamber was discarded, and a cotton swab moistened with PBS was used to gently wipe off the cells that did not pass through the upper chamber. 600 μL of 4% paraformaldehyde was added to the lower chamber to fix the cells for 20 minutes, and 600 μL of 0.5% crystal violet stain was added to the lower chamber for staining for 1 hour. The excess stain was rinsed with distilled water and dried, and the number of transferred cells was recorded under a microscope.

[0035] The results are as follows Figure 7 As shown, the number of migrating cells in the stable cell lines with NCAPH knockdown in CAL27 and CNE2 was significantly lower than that in the control cell lines.

[0036] Example 8: Immunoprecipitation reaction experiment 1. The full-length (NFL and PD-L1-Flag) and truncated (N1, N2, N3, N4, N5, N6 and PD-L1-N-Flag, PD-L1-C-Flag) cDNAs encoding human NCAPH, HIP1R and PD-L1 in this article were synthesized by Shanghai Jierui Biotechnology Co., Ltd. and cloned into the pCDNA3.1 vector through NheI and KpnI (NCAPH), Nhe I and EcoR I (PD-L1) and NheI and NotI (HIP1R); the constructed target plasmids were transfected into HEK-293T, and the target cells were collected 48h and 72h after transfection, and lysed using IP buffer containing 0.5% NP-40. The protein sample was repeatedly frozen and thawed in liquid nitrogen for 3 times, and then the BCA protein concentration was quantified. After the concentration was determined, the protein-antibody mixture was prepared according to the ratio of 1mg protein to 1μg antibody. The above ratio is a conventional ratio and can be adjusted appropriately according to the experimental results. Place the protein-antibody mixture on a vertical flip shaker at 4°C overnight. Place the Protein A / G magnetic beads on a magnetic rack and wash three times with IP buffer containing 0.2% NP-40 to remove residual magnetic bead storage solution components. Add 50μL of magnetic beads to each protein-antibody mixture and react on a vertical flip shaker at 4°C for 2 hours. After the reaction, place the magnetic bead-protein antibody mixture on a magnetic rack, discard the supernatant, and wash three times with IP buffer containing 0.2% NP-40. The last time, the supernatant should be aspirated as cleanly as possible. Then add 50μL of 1.5×SDS protein buffer and mix with the magnetic beads. Heat in a 95°C metal bath for 10 minutes and then briefly centrifuge for subsequent electrophoresis experiments; The amino acid sequences of the full-length NCAPH-Myc protein, N1, N2, N3, N4, N5, and N6 are shown in SEQ ID NOs: 2-8; The results are as follows Figure 8 As shown, immunoprecipitation experiments using Myc antibodies revealed that both the full-length PD-L1-Flag protein and the C-terminal truncated protein of PD-L1-C-Flag could interact with the full-length NCAPH-Myc protein, while the N-terminal truncated protein of PD-L1-N-Flag could not interact with the full-length NCAPH-Myc protein.

[0037] The results are as follows Fig. 9 As shown, immunoprecipitation experiments using Flag antibodies revealed that the full-length PD-L1-Flag protein could bind to the full-length NCAPH-Myc protein, N1, N3, N4, and N5, but could not bind to N2 and N6, thereby narrowing the intervention functional small peptide to the N3 peptide region.

[0038] 2. Treat CAL27 cells with 10 ng / mL IFNγ for 12 hours, then add 100 μM chloroquine and 10 μg / mL Sumo or NPIDP peptide for another 12 hours, and lyse with IP buffer containing 0.5% NP-40. The protein sample is frozen and thawed three times in liquid nitrogen and then the BCA protein concentration is quantified. After the concentration is determined, a protein-antibody mixture is prepared according to the ratio of 1 mg protein to 1 μg anti-PD-L1 antibody. The above ratio is a conventional ratio and can be adjusted appropriately according to the experimental results. Place the protein-antibody mixture on a vertical flip shaker at 4°C overnight. Place the Protein A / G magnetic beads on a magnetic stand and wash three times with IP buffer containing 0.2% NP-40 to remove residual components of the magnetic bead storage solution. Add 50 μL of magnetic beads to each protein-antibody mixture, react on a vertical flip shaker at 4°C for 2 hours. After the reaction, place the magnetic bead-protein-antibody mixture on a magnetic rack, discard the supernatant, and wash three times with IP buffer containing 0.2% NP-40. The last time, the supernatant should be as clean as possible. Then add 50 μL of 1.5×SDS protein buffer and mix with the magnetic beads. Heat in a 95°C metal bath for 10 minutes and then centrifuge briefly for subsequent electrophoresis experiments.

[0039] The results are as follows Fig.14 As shown, under the inhibition of autophagy by chloroquine and inflammatory stimulation, there was interaction between PD-L1 and NCAPH and HIP1R under PBS and Sumo treatment. After the addition of NPIDP small peptide, the overall expression level of the protein did not change, but the binding of PD-L1 to HIP1R was enhanced, while the binding to NCAPH was significantly inhibited.

[0040] Example 9: In vitro purification experiment of small peptides The DNA sequences of the cell-penetrating peptide iRGD (CRGDKGPDC) and the 271-360aa region of NCAPH were commercially synthesized and cloned into the prokaryotic expression plasmid pET-30a, and coupled with the fusion tag Sumo; 2 μL of the plasmid was transformed into BL21 (DE3) competent cells, coated on Kana-resistant LB plates, and inverted cultured at 37°C for 16-18 h. Pick a single clone into a test tube containing 4 mL LB medium with corresponding resistance, and culture at 37°C and 220 rpm until the bacterial OD600 is 0.6-0.8; add IPTG with a final concentration of 0.3 mM to the test tube, and induce expression at 16°C and 220 rpm; centrifuge at 8000 rpm for 5 min, discard the supernatant, and collect the bacterial sludge; add 25 mL PBS buffer to every 500 mL of bacterial liquid to resuspend the bacterial sludge until there is no obvious block precipitation, and crush it in an ice bath in an ultrasonic disruptor for 30 min; centrifuge at 10000 rpm for 35 min, take the supernatant, and filter it with a 0.45 μm filter; take 4 mL Ni-Beads purification filler to load the column, and rinse the filler with ultrapure water for 5-10 column volumes; then use resuspension buffer (containing 20 mM PB, 300 mM NaCl, 5% glycerol, 20 mM imidazole, pH 8.0) to balance for 5-10 column volumes; the filtered sample is purified on a chromatography column, and the loading flow rate is 2 mL / min; after loading, wash with resuspension / equilibrium buffer for 5-10 column volumes; wash with resuspension buffer containing 50mM imidazole for 5-10 column volumes; then use resuspension buffer containing 250mM imidazole for protein elution; dialyze the sample purified by Ni column into storage buffer (containing 20mM PB, 300mM NaCl, 5% glycerol, pH8.0) to determine the protein concentration; add 10μL SUMO enzyme according to 1mg protein, and cut in a 4℃ water bath for 16h; purify the cut sample using Ni-Beads; the loading flow rate is 2mL / min; after loading, wash with equilibration buffer for 5-10 column volumes; use resuspension buffer containing 250mM imidazole for protein elution; dialyze the eluted protein sample into storage buffer, with a dialysis ratio of >1000 times; filter with a 0.22μm filter head and store in aliquots.

[0041] The results are as follows Fig.10 As shown, Fig.10 A shows the schematic diagram of the structure of the small peptide purified in vitro. Fig.10 B shows the in vitro purification results of the small peptide.

[0042] Example 10: Ni pull down experiment CAL27 cells treated with 10 ng / mL IFNγ for 24 h were lysed on ice for 10 min using RIPA lysis buffer, and the lysate mixture was collected in a 1.5 mL centrifuge tube and centrifuged at 4°C and 15,000 rpm for 25 min. The supernatant was taken as the total cell protein. 50 μL Ni-Beads were washed three times with PBS, resuspended in 500 μL PBS, added with 100 μg of the target protein, incubated at 4°C for 30 min by inversion, centrifuged at 4°C and 3,000 rpm for 1 min, the supernatant was removed, 1 mg of total cell protein (the lysis buffer was fixed to 500 μL), incubated at 4°C for 1 h by inversion, washed three times with 500 μL PBS, resuspended in 50 μL Loading Buffer, and heated in a 100°C metal bath for 10 min for subsequent immunoblot detection.

[0043] The results are as follows Fig.10 As shown in C, the results of the nickel column pull-down experiment showed that the PD-L1 protein could be pulled down only on the NPIDP-bound nickel column, while Sumo could not pull down the PD-L1 protein.

[0044] Example 11: T cell killing experiment Extraction of human PBMC cells (peripheral blood mononuclear cells): Take about 10 mL of blood from a healthy person, add Fico separation solution restored to room temperature to a centrifuge tube, slowly add the blood to the centrifuge tube (do not break the boundary between the separation solution and the blood), centrifuge at 1500 rpm for 30 minutes at room temperature, and the cells in the middle white layer after centrifugation are PBMC cells. Slowly aspirate the PBMC cells and add them to PBS, centrifuge at 1500 rpm for 10 minutes at room temperature, repeat twice, then resuspend the cell pellet with RPMI-1640 complete medium, add it to a cell culture dish, place it in a cell culture incubator and precipitate for 2 hours, aspirate the non-adherent cells in the culture medium and transfer them to a new culture dish, then add CD3 (100 ng / mL) and IL-2 (10 ng / mL) to activate for 48 hours. Count the target cells and calculate the number of cells according to 1×10 5 The cells were seeded at a density of 10 μg / mL per well in a 24-well plate. After the cells adhered to the wall, PBS control group, Sumo group and NPIDP peptide group (10 μg / mL each) were added for pretreatment for 12 hours. Then, 5×10 5 The activated PBMC cells were co-cultured with the target tumor cells (CAL27 and CNE2). After 48 hours of culture, the supernatant was discarded, the cells were washed once with PBS, fixed with 4% paraformaldehyde for 20 minutes, washed twice with PBS, and 300 μL of 0.5% crystal violet was added to each well for staining for 5 minutes. The excess crystal violet was washed with distilled water, dried, and photographed.

[0045] The results are as follows Fig.15 As shown, after adding PBS, Sumo, and NPIDP peptide, after CAL27, CNE-2 and PBMC cells were co-cultured, the killing effect of PBMC added with NPIDP peptide on tumor cells was significantly higher than that of PBMC treated with PBS and Sumo.

[0046] Example 12: Animal model experiment SCCVII head and neck squamous cell carcinoma cells were subcutaneously transplanted into the inguinal region of 5-6 week-old C57BL / 6 mice. The tumor volume and mouse weight were monitored every other day. 3 The mice were given intraperitoneal injection of drugs (PD-1 monoclonal antibody: 100 μg; NPIDP small peptide: 200 μg; a total of 5 times) at different ages. They were grouped as I: Vehicle (PBS), II: PD-1 monoclonal antibody, III: NPIDP small peptide, and IV: PD-1 monoclonal antibody combined with NPIDP small peptide. After the drug treatment, the mice were killed by cervical dislocation, the tumor tissues were dissected out and weighed, and the tumor volume curve was drawn.

[0047] The results are as follows Fig.16 As shown, Figure A is a schematic diagram of mouse dosing; Figure B is a schematic diagram of mouse tumors under different dosing conditions. It can be seen that the administration of PD-1 monoclonal antibody or NPIDP peptide alone can inhibit tumor growth to a certain extent, and the combination of PD-1 monoclonal antibody and NPIDP can more effectively inhibit tumor growth; Figure C is the statistical result of tumor volume under different drug treatment conditions; Figure D is the statistical result of tumor weight under different drug treatment conditions.

[0048] Example 13: IHC Experiment Formalin-fixed paraffin-embedded (FFPE) tumor tissue sections were dewaxed in a 65°C oven for 1 hour, dewaxed in xylene I for 10 minutes, dewaxed in xylene II for 10 minutes, dewaxed in xylene III for 10 minutes, and rehydrated in graded ethanol: 100% ethanol I for 5 minutes, 100% ethanol II for 5 minutes, 100% ethanol III for 5 minutes, 90% ethanol for 5 minutes, 80% ethanol for 5 minutes, and 70% ethanol for 5 minutes. The slides were immersed in pH 9.0 EDTA antigen retrieval solution or pH 6.0 citric acid tissue antigen buffer and boiled at high pressure for 8 minutes for antigen retrieval. After rinsing in PBS, PBST (0.3% TritonX-100) was used for membrane permeabilization for 15 minutes, and hydrogen peroxide blocking agent was used for 10 minutes. The tissue slides were incubated with primary antibodies at 4°C overnight in the environment. Then, biotinylated universal secondary antibodies were added and incubated for 30 minutes at room temperature. After rinsing in PBS for 5 minutes, the slides were developed using a 3,5-diaminobenzidine (DAB) substrate kit (DAB staining solution), counterstained with hematoxylin, dehydrated with graded ethanol, transparentized with xylene, and mounted with a neutral resin.

[0049] The results are as follows Fig.17 As shown in the figure, it can be seen that compared with the Vehicle (PBS) group, the administration of PD-1 monoclonal antibody or NPIDP peptide alone can inhibit the expression of PD-L1 to a certain extent and promote the infiltration of CD8+T cells and GZMB positive cells. The combination of PD-1 monoclonal antibody and NPIDP peptide can further inhibit the expression of PD-L1 and promote the infiltration of CD8+T cells and GZMB positive cells.

[0050] Example 14: Animal model drug toxicity experiment The mice treated with different drugs (vehicle, PD-1 antibody, NPIDP peptide, PD-1 antibody and NPIDP peptide co-treated) were weighed to detect the changes in the weight of the mice under the action of drugs. Different tissues of the mice were fixed with formalin, and the paraffin-embedded (FFPE) tissue sections were dewaxed in a 65°C oven for 1 hour, dewaxed in xylene I for 10 minutes, dewaxed in xylene II for 10 minutes, and dewaxed in xylene III for 10 minutes. They were rehydrated with gradient ethanol, 100% ethanol I for 5 minutes, 100% ethanol II for 5 minutes, 100% ethanol III for 5 minutes, 90% ethanol for 5 minutes, 80% ethanol for 5 minutes, and 70% ethanol for 5 minutes, rehydrated, stained with hematoxylin for 5 minutes, differentiated with differentiation solution, rinsed with water to return to blue, stained with eosin for 20 seconds, dehydrated with gradient ethanol, transparentized with xylene, and sealed with neutral resin.

[0051] Fig.18 Results A showed that different drug treatments (vehicle, PD-1 antibody, NPIDP peptide, and PD-1 antibody and NPIDP peptide co-treatment) had no effect on the body weight of mice; Fig.18 B The results showed that the small peptide NPIDP did not cause obvious abnormalities in the structure of the mouse's visceral tissues (heart, liver, spleen, lungs, and kidneys).

Claims

1. A clinical diagnostic reagent for human head and neck squamous cell carcinoma, which is a reagent for detecting the expression level of human NCAPH gene.

2. The clinical diagnostic reagent for human head and neck squamous cell carcinoma according to claim 1, characterized in that: The reagent for detecting the expression level of human NCAPH gene is a reagent for detecting the high expression level of human NCAPH gene.

3. The clinical diagnostic reagent for human head and neck squamous cell carcinoma according to claim 2, characterized in that: The invention comprises primers for detecting the RNA level of human NCAPH, and the primer sequences are AAACACGCAGATTACGGAACA, GTTGGTTGGTTCGGTGTCTTT.

4. The clinical diagnostic reagent for human head and neck squamous cell carcinoma according to claim 2, characterized in that: When the diagnostic reagent detects a high expression level of the human NCAPH gene, the patient is diagnosed as a patient with poor response to immunotherapy.

5. A clinical therapeutic drug for human head and neck squamous cell carcinoma, which is a drug screened for the purpose of inhibiting the expression of human NCAPH gene.

6. A clinical therapeutic drug for human head and neck squamous cell carcinoma, which is a NPIDP small peptide drug aimed at inhibiting the expression of human PD-L1 gene.

7. The clinical therapeutic drug for human head and neck squamous cell carcinoma according to claim 6, characterized in that: The amino acid sequence of the NPIDP small peptide drug is shown in SEQ ID NO:

1.

8. A clinical therapeutic drug for human head and neck squamous cell carcinoma, characterized in that: NPIDP small peptide drugs are used in combination with immunotherapy drugs.

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