Application of NCCRP1 as lung cancer immunotherapy biomarker

Abstract

The invention discloses application of NCCRP1 as a lung adenocarcinoma immunotherapy biomarker, and belongs to the field of biological medicine. Bioinformatics analysis finds that the NCCRP1 is remarkably and highly expressed in lung adenocarcinoma tissues, and the expression level of the NCCRP1 is in negative correlation with prognosis of a patient. Secondly, the expression level of lung adenocarcinoma cells NCCRP1 is in negative correlation with infiltration of anti-tumor immune cells such as CD8 + T cells. Experiments show that the knockout of the NCCRP1 can significantly inhibit the growth of mouse lung tumors by increasing the infiltration quantity of CD8 + T cells and NK cells in a lung adenocarcinoma microenvironment. The invention provides a new target spot for immunotherapy of lung adenocarcinoma, including NCCRP1 gene editing (knockout / knockdown) and application of an inhibitor in preparation of antitumor drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of NCCRP1 as a biomarker for immunotherapy of lung adenocarcinoma. Background Art

[0002] Targeted therapies and immunotherapies have been widely used in the clinical practice of lung adenocarcinoma, significantly improving the prognosis of some patients. However, a considerable proportion of patients still do not benefit from these therapies. The tumor immune microenvironment plays a critical role in the progression and treatment resistance of lung adenocarcinoma and is a key factor in the low response rate to targeted therapies and immunotherapies. The tumor immune microenvironment is a complex and dynamic integrated system, comprising cellular components such as tumor cells and immune cells, as well as non-cellular components such as cytokines and the extracellular matrix. The dynamic changes and interactions among these components not only promote the growth, metastasis, and immune escape of lung adenocarcinoma but also significantly inhibit targeted therapies and immunotherapies for lung adenocarcinoma. Therefore, targeting the tumor immune microenvironment has become a new direction in the development of therapeutics for lung adenocarcinoma. However, targets that effectively reshape the immune microenvironment of lung adenocarcinoma are currently scarce. Actively identifying new targets and elucidating their biological functions and molecular mechanisms are crucial for improving the survival and prognosis of lung adenocarcinoma patients. Summary of the Invention

[0003] In response to the problems of the existing technology, the present invention aims to provide the application of NCCRP1 as a biomarker for lung cancer immunotherapy. Targeted intervention of the immune microenvironment of lung adenocarcinoma using NCCRP1 as a target is expected to provide new ideas for the treatment of lung adenocarcinoma.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0005] The present invention discloses the application of NCCRP1 as a target in the preparation of immunotherapy drugs for lung adenocarcinoma.

[0006] Furthermore, the application includes increasing the expression or activity of CD8 in the tumor microenvironment by inhibiting NCCRP1. + Infiltration of T cells and NK cells.

[0007] The present invention also discloses the use of an NCCRP1 inhibitor in the preparation of a lung adenocarcinoma immunotherapy drug.

[0008] Furthermore, the inhibitor includes siRNA, shRNA, sgRNA or small molecule inhibitor targeting NCCRP1.

[0009] Furthermore, the nucleotide sequence of the sgRNA is shown in any one of SEQ ID NO.1 to SEQ ID NO.2.

[0010] The present invention also discloses a gene-edited lung adenocarcinoma cell, characterized in that the NCCRP1 gene in the cell is knocked out or knocked down, preferably an LLC cell with NCCRP1 gene knockout constructed by CRISPR / Cas9 technology.

[0011] Furthermore, the gene-edited cells described above are used to prepare an experimental model for studying lung tumor growth inhibition.

[0012] The present invention also discloses the use of NCCRP1 as a biomarker in the prognosis assessment of lung adenocarcinoma, wherein high expression of NCCRP1 indicates a poor prognosis.

[0013] The present invention also discloses a method for detecting the level of immune cell infiltration in lung adenocarcinoma, characterized in that the expression level of NCCRP1 in the sample is detected, wherein low expression of NCCRP1 indicates CD8 + High infiltration of T cells and / or NK cells.

[0014] The present invention also discloses a method for regulating the immune microenvironment of lung adenocarcinoma, comprising using an NCCRP1 inhibitor or a gene editing tool to knock down NCCRP1 expression to increase CD8 + T cell and NK cell infiltration.

[0015] The present invention also provides a modified lung adenocarcinoma cell LLC, characterized in that the cell is a lung adenocarcinoma cell with NCCRP1 gene knocked out.

[0016] The present invention also provides an application of NCCRP1 gene knockout lung adenocarcinoma cells, characterized in that the application includes research on inhibiting lung tumor growth.

[0017] Compared with the prior art, the present invention has the following beneficial effects.

[0018] The present invention uses the TCGA database to analyze and confirm that NCCRP1 is significantly overexpressed in lung adenocarcinoma tissues, and its expression is negatively correlated with the patient's prognosis. The TISCH2 database is used to further reveal that NCCRP1 is significantly more expressed in lung adenocarcinoma cells than in other cell types. The CancerSCEM database found that the high expression of NCCRP1 in lung adenocarcinoma cells is associated with anti-tumor immune cells such as CD8 + T cell infiltration was negatively correlated.

[0019] This study reveals for the first time the key role of NCCRP1 in the immune microenvironment of lung adenocarcinoma, filling the gap in the lack of effective targets in existing technologies. +The infiltration of NCCRP1 by T cells and NK cells promotes tumor growth, providing a direct basis for targeted intervention. By targeting NCCRP1 to reshape the immune microenvironment, the response rate to immunotherapy and survival of patients with lung adenocarcinoma can be improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Comparative graphs showing the expression and clinical relevance of NCCRP1 in lung adenocarcinoma tissues provided by the present invention. (A) shows the differential expression of NCCRP1 between lung adenocarcinoma and normal tissues; (B) shows the expression of NCCRP1 in various cell types; (C) shows the relationship between NCCRP1 expression and the prognosis of lung adenocarcinoma patients; and (D) shows the percentage of various cell types in lung adenocarcinoma cells with high and low NCCRP1 expression.

[0021] Figure 2 Comparative graphs showing the effects of NCCRP1 knockout on lung adenocarcinoma growth, as provided by the present invention. (A) shows NCCRP1 gene expression in LLC cells detected by DNA sequencing; (B) shows NCCRP1 protein expression in LLC cells detected by Western blot; (C) shows lung tumor volume curves in NSG mice; (D) shows survival curves in NSG mice; (E) shows lung tumor volume curves in C57BL / 6 mice; and (F) shows survival curves in C57BL / 6 mice.

[0022] Figure 3 Comparative graphs showing the effects of NCCRP1 knockout on the immune microenvironment of lung adenocarcinoma. (A) shows the single-cell transcriptome sequencing cell annotation map (UMAP) of subcutaneous lung xenografts; (B) shows the relative expression levels of marker genes in all cells from single-cell transcriptome sequencing; (C) shows the single-cell transcriptome sequencing cell annotation map (UMAP) of subcutaneous lung xenografts from the two groups; and (D) shows the percentage analysis of various cell types between the two groups.

[0023] Figure 4 The present invention provides a method for knocking out NCCRP1 by recruiting CD8 + The results of T cells and NK cells inhibiting lung adenocarcinoma were verified. Among them, (A) is the flow cytometry detection of CD8 + The number of T cells; (B) is the number of NK cells in subcutaneous lung transplant tumors in the two groups detected by flow cytometry; (C) is the number of CD8 + Volume curve of subcutaneous lung transplanted tumor in mice after treatment with T cell / NK cell neutralizing antibody; (D) CD8 + Survival curve of mice after treatment with T cell / NK cell neutralizing antibodies. DETAILED DESCRIPTION

[0024] The present invention is further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the present invention to the following embodiments, as all technologies implemented based on the present invention fall within the scope of the present invention.

[0025] Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

[0026] Example 1.

[0027] 1. A lung adenocarcinoma dataset was selected from the TCGA database (https: / / www.cancer.gov / ccg / research / genome-sequencing / tcga), which included 515 lung adenocarcinoma tissues and 59 normal lung tissues. The mRNA expression of NCCRP1 was then analyzed and violin plots were prepared. The statistical method used was the unpaired t-test (e.g. Figure 1 A).

[0028] 2. Select the human non-small cell lung cancer single cell transcriptome dataset (NSCLC_GSE148071) from the TISCH2 database (http: / / tisch.compbio.cn), analyze the mRNA expression of NCCRP1 in various cell types online, and download the result graph (such as Figure 1 B).

[0029] 3. Select a lung adenocarcinoma dataset from the GEPIA database (http: / / gepia.cancer-pku.cn / ), which contains 477 lung adenocarcinoma tissues. Then analyze the relationship between NCCRP1 mRNA expression and overall survival of patients online and download the result graph (e.g. Figure 1 C).

[0030] 4. A human lung adenocarcinoma single-cell transcriptome dataset (ID 1055415-BioProject-NCBI: PRJNA1055415) was selected from the CancerSCEM database (https: / / ngdc.cncb.ac.cn / cancerscem / ), which includes 7 patients (28 lung adenocarcinoma samples). The mRNA expression of NCCRP1 in lung adenocarcinoma cells and the number of various cell types infiltrating in each sample were analyzed online. The median of the average mRNA expression value of NCCRP1 in the 7 patients was then taken and the samples were divided into two groups with high and low NCCRP1 expression, and the proportion of each type of cell was analyzed (e.g. Figure 1 D).

[0031] Example 2.

[0032] 1. Use CRISPR / Cas9 to construct a cell line with Nccrp1 gene knockout.

[0033] The LLC cell line with stable knockout of Nccrp1 gene was constructed by CRISPR / Cas9 technology. GV392-sgNccrp1 plasmid and control plasmid GV392-sgCtrl were purchased from Shanghai GeneGene, and lentiviral packaging plasmids psPAX2 and PMD2.G were purchased from Addgene.

[0034] The sgNccrp1 sequence information is as follows: Forward sequence: (5'-TTCAACCTCAGTCGGTGCAT-3'); Reverse sequence: (5'-ATGCACCGACTGAGGTTGAA-3').

[0035] Negative control, denoted as sgCtrl, has the following sequence information: Forward sequence: (5'-CGCTTCCGCGGCCCGTTCAA-3'); Reverse sequence: (5'-TTGAACGGGCCGCGGAAGCG-3').

[0036] The specific operations are as follows.

[0037] (1) Lentiviral packaging.

[0038] HEK293 cells (purchased from the National Cell Bank of the Chinese Academy of Sciences) were seeded in 10 cm culture dishes 24 hours in advance to a density of 50% for lentiviral packaging. 1 mL of DMEM basal medium (Servicebio, G4515) was pipetted into a 1.5 mL Eppendorf tube. The target plasmids GV392-sgNccrp1 or GV392-sgCtrl (5 μg) and the lentiviral packaging plasmids psPAX2 (1.25 μg) and pMD2.G (3.75 μg) were added. After mixing, 30 μL of liposomal transfection reagent GTR (Shanghai GeneCare, REVG007) was added and incubated at room temperature for 10 minutes. The transfection mixture was gently added dropwise to the cell culture medium, and the HEK293 cells were incubated at 37°C. The medium was changed 12 hours after transfection. After 48 hours of fluid replacement, the viral supernatant was collected, centrifuged at 3000 rpm for 30 min, and then filtered through a 0.45 μm filter to obtain the lentivirus.

[0039] (2) Infect LLC cells with lentivirus.

[0040] LLC cells (purchased from the National Cell Bank of the Chinese Academy of Sciences) were seeded in six-well plates 24 hours in advance to a density of 50% in preparation for lentiviral infection. All medium was aspirated, and 1 mL of RPMI 1640 complete medium (Servicebio, G4535) was added, followed by 1 mL of supernatant containing GV392-sgNccrp1 or GV392-sgCtrl virus. The cells were incubated in an incubator for 24 hours. The medium was then replaced with RPMI 1640 complete medium and cultured for an additional 24 hours. Selection was then continued for two weeks using RPMI 1640 complete medium supplemented with 5 μg / mL puromycin (Beyotime, ST551) to obtain puromycin-resistant stably transfected cell lines.

[0041] (3) Monoclonal screening of NCCRP1-KO cells and verification of knockout effect.

[0042] The LLC cells selected by puromycin were diluted in 96-well plates and cultured. The wells containing single cells were marked and cultured to obtain monoclonal cell lines. After expansion, DNA from some cells was extracted using a high-performance monoclonal genotyping kit (Yuanjing Bio, YK-MVH-250) and sent to BGI for Sanger sequencing to identify whether DNA recombination occurred in the Nccrp1 target sequence (e.g. Figure 2 A). NCCRP1 DNA sequencing primers: (Forward sequence: 5'-ACGCCCTCTCTTAAAAGCCC-3'); (Reverse sequence: 5'-ATTAAGGAGGTTGGTGGGCG-3'). Expand the remaining cells, extract cell proteins, and detect NCCRP1 expression by Western Blot to confirm that the cell line has completely knocked out NCCRP1 protein (e.g. Figure 2 B). The primary antibody used was NCCRP1 antibody (Prosci, 56-470), the internal control was α-Tubulin (Abmart, MC33039M), and the secondary antibody was Goat Anti-Rabbit IgG, Peroxidase Conjugated, H+L (biosharp, BL003A) or Goat Anti-Mouse IgG, Peroxidase Conjugated, H+L (biosharp, BL001A).

[0043] 2. Construction of mouse lung transplant tumor model.

[0044] In this study, 8-week-old female C57BL / 6 mice and female NSG immunodeficient mice of the same weight were used for the experiment. 6The cell suspension was used as a carrier in 100 μL PBS (phosphate buffered saline) and accurately inoculated into the subcutaneous area on the right back of the mouse to establish a stable and reliable subcutaneous transplant tumor model.

[0045] The steps are as follows.

[0046] (1) Cell culture before inoculation: Expand the culture of the cells to be inoculated, namely LLC cells (sgCtrl or sgNccrp1). Digest the cells in the logarithmic growth phase, count them, and resuspend them in PBS to adjust the final concentration to 1×10 7 / mL, place the cells in an ice box and prepare for inoculation.

[0047] (2) Subcutaneous inoculation: 3% tribromoethanol solution was injected intraperitoneally at a dose of 200 μL per mouse for anesthesia. After the mouse was fully anesthetized, the cells were fully resuspended and mixed, and 1×10 6 LLC cells (sgCtrl or sgNccrp1) were placed in the subcutaneous area on the right side of the back of mice.

[0048] (3) Observe the tumor formation in mice: Observe and record the date of tumor formation in each mouse. Once a tumor is found, use a vernier caliper to measure the long diameter (a mm) and short diameter (b mm) of the subcutaneous tumor every 3 days. The tumor volume is calculated as volume = 1 / 2 × a × b 2 The formula for calculation (such as Figure 2 C, 2E). The statistical method of tumor growth curve was two-way ANOVA. In addition, when the mice died of natural causes or the tumor volume exceeded 1500mm 3 When the tumor diameter exceeds 15 mm or the weight loss reaches 20%, the mouse is considered dead. The survival status of each mouse is recorded during the experiment until 40 days after tumor formation, and a survival curve is drawn (e.g. Figure 2 D, 2F). Survival curves were analyzed using the log-rank test.

[0049] Example 3.

[0050] Single-cell transcriptome sequencing and data processing.

[0051] Six female, 8-week-old C57BL / 6 mice of equal weight were selected for the experiment. The mice were randomly divided into two groups, 3 mice each: sgCtrl and sgNccrp1. 1×10 6The corresponding LLC cells were used to establish a xenograft tumor model. Once the tumors reached the appropriate size, the mice were euthanized and the xenografts were removed. The xenografts were infiltrated in preservation solution and sent to Beijing SeekOne Biotechnology Co., Ltd. for single-cell transcriptome sequencing. The SeekOne® DD Single-Cell 3' Transcriptome Kit includes: a chip (SeekOne® DD Chip S3), a gasket, carrier oil, barcoded beads, amplification reagents, and library construction reagents. Based on microfluidics, the kit achieves single-cell isolation and capture through an oil-in-water process. It then uses nucleic acid-modified barcoded beads to molecularly label RNA from different cell sources, ultimately constructing a high-throughput single-cell transcriptome library. The SeekOne® DD Single-Cell 3' Transcriptome Kit User Manual was strictly followed to construct a single-cell transcriptome library compatible with high-throughput sequencers. Once the library passed the test, it was sequenced. Qualified libraries had a main peak fragment size between 350 and 750 bp, no small fragments, and a library concentration of at least 1 ng / μL (as measured by Qubit 4.0). Sequencing was performed using the Illumina high-throughput sequencing platform, with ≥50,000 reads per cell to ensure the accuracy of single-cell sequencing data analysis. The raw sequencing data (sequenced reads) were first processed using fastp to remove low-quality reads. The resulting matrix was then analyzed using SeekSoul Tools. Cell clustering, cell annotation, and cell proportion analysis were performed on the SeekSoul Online cloud platform.

[0052] Single-cell data from sgCtrl and sgNccrp1 subcutaneous xenografts were integrated for comprehensive analysis using the R package Seurat (v4.2.0). Cells failing quality control (>20,000 or <500 transcripts, >4,000 or <500 genes, or mitochondrial gene expression greater than 5%) were filtered out. After quality control, 27,341 cells from the sgCtrl group and 23,393 cells from the sgNccrp1 group were retained. Gene expression data were normalized using the default parameters of the NormalizeData function, and highly variable genes were identified using the FindVariableFeatures function. The top 2,000 highly variable genes were scaled using the "ScaleData" function. Dimensionality reduction was performed using principal component analysis (PCA), and the top 50 principal components were selected for cluster analysis. To correct for batch effects, the RunHarmony function was applied. Unbiased clustering was performed at a resolution of 3, and two-dimensional visualization was performed using Unified Mapping (UMAP). The FindAllMarkers function was used to identify the signature genes of each cluster and compared with known lineage-specific markers for cell type annotation, including tumor cells (expressing Myc), CD8 + T cells (expressing Cd8a), CD4 + Eleven cell types were identified, including T cells (expressing Cd4), NK cells (expressing Ncr1), macrophages (expressing C1qa, Chil3), neutrophils (expressing S100a9), dendritic cells (expressing Cd209a), B cells (expressing Ms4a1), endothelial cells (expressing Cdh5), fibroblasts (expressing Col1a1), and pericytes (expressing Rgs5), and UMAP maps were drawn. Subsequently, the SeekSoul Online cloud platform was used to select the drawing tool to analyze the differences in the number of various cells in the sgCtrl and sgNccrp1 groups, and a stacked bar graph was drawn (e.g. Figure 3 A-3D).

[0053] Example 4.

[0054] 1. Flow cytometry detection of CD8 + The number of T cells and NK cells.

[0055] Fresh tumor tissue was washed with Hanks solution (biosharp, BL561A), placed in HBSS-FBS (Hanks solution + 5% FBS), and minced into 1-2 mm pieces. 1 mg / mL Collagenase I (GLPBIO, GC19589) and 1 mg / mL DNase I (MedChemExpress, HY-108882) were then added and digested in a 37°C water bath for 1 hour. The tissue solution was filtered through a 70 μm filter, the suspension was washed with HBSS-FBS, and then centrifuged at 4°C, 600 g for 5 minutes. The cell pellet was resuspended in 10 mL of red blood cell lysis buffer (biosharp, BL503B) and lysed at room temperature for 10 minutes. The cells were then washed twice with HBSS-FBS and centrifuged at 4°C. The cell pellet was resuspended in Stain Buffer (BD, 554656), and the cells were counted and diluted to 6 × 10 6 Flow cytometry was performed to detect CD8 + For the number of T cells, the single cell suspension of each tumor was divided into 5 tubes, each with 500 μL. They were labeled as blank control, APC-IgG, APC-CD3, APC-CD3+FITC-IgG, and APC-CD3+FITC-CD8a. For the number of NK cells, the single cell suspension of each tumor was divided into 3 tubes, each with 500 μL. They were labeled as blank control, BrilliantViolet 421 TM -IgG and Brilliant Violet 421 TM -NK1.1. Except for the blank tube, 15 μL of the corresponding antibody (1×10 6 Each cell corresponds to 5 μL of antibody), incubate at 4°C in the dark for 1 hour. Then add 700 μL of Stain Buffer, centrifuge at 4°C, 600g for 5 minutes, and discard the supernatant. Wash the cells with 1 mL of Stain Buffer and centrifuge. Finally, resuspend the cells with 500 μL of Stain Buffer (FBS), filter and detect on the machine, and analyze with Flowjo software. The primary antibodies used are APC-IgG (Elabscience, AN00338E), APC-CD3 (Elabscience, E-AB-F1013E), FITC-IgG (Elabscience, AN00338C), FITC-CD8a (Elabscience, E-AB-F1104C), Brilliant Violet 421 TM -IgG (BioLegend, 400259) and Brilliant Violet 421 TM-NK1.1 (BioLegend, 108731).

[0056] 2. CD8 + In vivo T cell and NK cell depletion experiments.

[0057] Twenty female, 8-week-old C57BL / 6 mice of uniform weight were selected for the experiment. The mice were randomly divided into four groups, with 5 mice in each group: sgCtrl (IgG), sgNccrp1 (IgG), sgNccrp1 (anti-CD8), and sgNccrp1 (anti-NK1.1). 1×10 6 Each LLC (sgCtrl or sgNccrp1) cell line was injected intraperitoneally with the corresponding antibody on days 0 and 6 of subcutaneous inoculation: the sgNccrp1 (anti-CD8) group received intraperitoneal injections of InVivoMAb anti-mouse CD8α (BioXCell, BE0004-1); the sgNccrp1 (anti-NK1.1) group received intraperitoneal injections of InVivoMAb anti-mouse NK1.1 (Bio XCell, BE0036-1); and the sgCtrl (IgG) and sgNccrp1 (IgG) groups received intraperitoneal injections of InVivoMAb rat IgG2a isotype control (Bio X Cell, BE0089). The antibody injection dose was 200 μg / mouse. Mice were observed for tumor formation. Once tumor formation was detected, the long diameter (a mm) and short diameter (b mm) of the subcutaneous tumor were measured every 3 days with a vernier caliper. Tumor volume was calculated as volume = 1 / 2 × a × b. 2 Calculate the growth curve (such as Figure 4 C). The statistical method of growth curve was two-way ANOVA. At the same time, the survival of each mouse was recorded during the experiment, and the records were kept until 40 days after tumor formation, and the survival curve was drawn (such as Figure 4 D). The log-rank test was used as the statistical method for survival curve.

[0058] The C57BL / 6 mice and NSG immunodeficient mice used in this experiment were purchased from Saiye (Suzhou) Biotechnology Co., Ltd. All animal experiments in this study were approved by the ethics review of Bengbu Medical University.

[0059] The experimental results are as follows.

[0060] Figure 1The expression and clinical relevance of NCCRP1 in lung adenocarcinoma tissues were shown. Bioinformatics analysis was performed using the TCGA database, GEPIA database, TISCH2 database, and CancerSCEM database. The results showed that NCCRP1 expression was significantly upregulated in lung adenocarcinoma tissues compared with normal tissues ( Figure 1 A); NCCRP1 expression in lung adenocarcinoma cells is significantly higher than that in other cell types ( Figure 1 B); Patients with high NCCRP1 expression have shorter overall survival and worse prognosis ( Figure 1 C); The lower the NCCRP1 expression in lung adenocarcinoma cells, the more anti-tumor immune cells such as CD8 + T cells and CD4 + The greater the number of T infiltrations ( Figure 1 D). In summary, NCCRP1 may play an important role in the progression of lung adenocarcinoma and also emphasizes that it may play a key role in regulating immune cell infiltration.

[0061] Figure 2 The effect of NCCRP1 on the growth of lung adenocarcinoma was shown. First, monoclonal cells with NCCRP1 knockout were constructed and screened using CRISPR / Cas9, and then identified using DNA sequencing and Western blot. Subsequently, immunodeficient NSG mice and immune-normal C57BL / 6 mice were selected to construct a mouse subcutaneous lung transplant tumor model, and the tumor size and mouse survival were recorded. The results showed that monoclonal cells with NCCRP1 knockout were successfully constructed and screened, and identified by DNA sequencing and Western blot ( Figure 2 A, 2B); In the NSG mouse model, there was no significant difference in tumor size and mouse survival between the two groups ( Figure 2 C, 2D); In the C57BL / 6 mouse model, the tumor size of the NCCRP1 knockout group was significantly reduced and the survival of the mice was significantly prolonged ( Figure 2 E, 2F). In summary, NCCRP1 significantly promotes the growth of subcutaneous lung xenografts in C57BL / 6 mice.

[0062] Figure 3 The study showed that NCCRP1 regulates the immune microenvironment of lung adenocarcinoma. First, single-cell transcriptome sequencing was performed on two groups of subcutaneous lung transplant tumors, and cell-specific genes were used to mark various cell populations. Then, the differences in the number of various cell types in the two groups were compared. The results showed that by using cell-specific genes to mark various cell populations, 11 cell types were identified, including tumor cells (expressing Myc), CD8 + T cells (expressing Cd8a), CD4 +T cells (expressing Cd4), NK cells (expressing Ncr1), macrophages (expressing C1qa, Chil3), neutrophils (expressing S100a9), dendritic cells (expressing Cd209a), B cells (expressing Ms4a1), endothelial cells (expressing Cdh5), fibroblasts (expressing Col1a1), and pericytes (expressing Rgs5) ( Figure 3 A, 3B); By comparing the differences in the number of various cells in the two groups, it was found that knocking out NCCRP1 significantly increased the number of CD8 + The number of infiltrating T cells and NK cells ( Figure 3 C, 3D). In summary, knockout of NCCRP1 increased CD8 + The number of infiltrating T cells and NK cells.

[0063] Figure 4 It was shown that knockout of NCCRP1 recruits CD8 + T cells and NK cells inhibit lung adenocarcinoma. First, flow cytometry was used to detect the CD8 + The number of T cells and NK cells was then depleted using neutralizing antibodies in a subcutaneous lung xenograft model. + The results showed that CD8 T cells and NK cells were detected by flow cytometry after NCCRP1 knockout. + The number of T cells and NK cells increased significantly ( Figure 4 A, 4B); CD8 + Depletion of T cells or NK cells significantly weakened the inhibitory effect of NCCRP1 knockout on lung adenocarcinoma ( Figure 4 C, 4D). In summary, knockout of NCCRP1 recruits CD8 + T cells and NK cells to inhibit the growth of lung adenocarcinoma.

[0064] In summary, this study reveals for the first time the core mechanism by which NCCRP1 promotes lung adenocarcinoma progression by inhibiting CD8⁺T cell and NK cell infiltration, filling a gap in immune microenvironment-targeted therapy. Based on bioinformatics analysis and in vitro and in vivo experimental validation, it was confirmed that targeting NCCRP1 (gene editing or inhibitors) can reshape the immune microenvironment, significantly inhibit tumor growth, and prolong survival, providing new targets and biomarkers for immunotherapy of lung adenocarcinoma.

[0065] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. Application of NCCRP1 as a target in the preparation of immunotherapy drugs for lung adenocarcinoma.

2. The use according to claim 1, characterized in that The application includes increasing CD8 in the tumor microenvironment by inhibiting NCCRP1 expression or activity. + Infiltration of T cells and NK cells.

3. Application of an NCCRP1 inhibitor in the preparation of immunotherapy drugs for lung adenocarcinoma.

4. The use according to claim 3, characterized in that The inhibitors include siRNA, shRNA, sgRNA or small molecule inhibitors targeting NCCRP1.

5. The use according to claim 4, characterized in that The nucleotide sequence of the sgRNA is shown in any one of SEQ ID NO.1 to SEQ ID NO.

2.

6. A gene-edited lung adenocarcinoma cell, characterized in that: The NCCRP1 gene in the cells is knocked out or knocked down, preferably LLC cells with NCCRP1 gene knockout constructed by CRISPR / Cas9 technology.

7. Use of the gene-edited cells as described in claim 6 in preparing an experimental model for studying lung tumor growth inhibition.

8. Application of NCCRP1 as a biomarker in the prognosis assessment of lung adenocarcinoma, where high expression of NCCRP1 indicates poor prognosis.

9. A method for detecting the level of immune cell infiltration in lung adenocarcinoma, characterized in that: Detect the expression level of NCCRP1 in the sample, where low expression of NCCRP1 indicates CD8 + High infiltration of T cells and / or NK cells.

10. A method for regulating the immune microenvironment of lung adenocarcinoma, comprising knocking down NCCRP1 expression using an NCCRP1 inhibitor or gene editing tool to increase CD8 + T cell and NK cell infiltration.

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