Application of short hairpin RNA and NRBP1 in preparation of medicine for preventing or treating lung adenocarcinoma

Abstract

The invention provides application of short hairpin RNA and NRBP1 in preparation of a medicine for preventing or treating lung adenocarcinoma, and belongs to the technical field of biological medicine. The research proves that the NRBP1 has huge potential as an LUAD prognostic marker and an effective immunotherapy target; the high expression thereof in tumor tissue is significantly related to the unfavorable overall lifetime and higher pathological grading of the patient. Further correlation analysis shows that the polypeptide is strongly correlated with tumor immune infiltration degree, TMB (tetramethylbenzidine), tumor microenvironment-related pathways and the like; more importantly, in in-vitro and in-vivo experiments, NRBP1 expression regulates and controls proliferation, migration and invasion capabilities of lung adenocarcinoma.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and specifically relates to the application of short hairpin RNA and NRBP1 in the preparation of drugs for preventing or treating lung adenocarcinoma. Background Art

[0002] Lung cancer is the second most common cancer and the leading cause of cancer death worldwide. In China, the incidence and mortality of lung cancer also rank first. Lung cancer is a malignant tumor that occurs in the bronchial mucosal epithelium and mucous glands, and its pathogenic factors are diverse, including smoking, environmental pollution, occupational exposure, genetic factors, and certain chronic pulmonary diseases. In addition, recent studies have shown that epigenetic factors, chronic inflammation, and microbial infections may also be related to the occurrence and development of lung cancer. Small cell lung cancer (SCLC, 15%) and non-small cell lung cancer (NSCLC, 75%) are the two main types of lung cancer. The two main subtypes of NSCLC are lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD). Lung adenocarcinoma (LUAD) is one of the most common subtypes of lung cancer, and most patients have reached the advanced stage at the time of diagnosis. Its treatment methods include surgery, targeted therapy, radiotherapy, immunotherapy, and chemotherapy. The 5-year survival rate of advanced LUAD patients is still only 50%. In addition, this comprehensive treatment plan is associated with an increase in treatment-related toxicity and an increase in surgical risk. Advanced LUAD patients still have a high risk of poor clinical outcomes, and invasive or advanced LUAD patients still face the problem of poor clinical prognosis. Therefore, there is an urgent need to develop methods that can identify patient groups who may benefit from active treatment. Correspondingly, for LUAD patients, it is particularly necessary to develop innovative diagnostic biomarkers for predicting prognosis and treatment response.

[0003] Nuclear Receptor Binding Protein 1 (NRBP1) is a highly conserved and widely expressed protein. This gene is located in the region of human chromosome 2p23. Due to the lack of key catalytic residues in the kinase core structure, NRBP1 is regarded as a pseudokinase. NRBP1 also includes a binding region of Src homology 2 (SH2) domain, a bipartite nuclear localization signal, and three regions rich in proline, glutamate, serine, and threonine residues (PEST sequence). Although NRBP1 does not have the ability to phosphorylate substrates, it still has a typical eukaryotic protein kinase structure and may retain other non-catalytic functions. NRBP1 has been confirmed to be mainly localized in the cytoplasm, but it has also been observed to be expressed in the nucleus. In vitro studies have found that NRBP1 shuttles between the nucleus and the cytoplasm, regulating protein localization and the activity of transcription factors. NRBP1 can bind to MLF, JAB1, and activated RAC3, inhibiting cell differentiation, JAB1-mediated AP1 activation, and the redistribution of the Golgi marker protein p58. In addition, NRBP1 also interacts with key ubiquitination molecules such as Elongin BC and CUL5, affecting the intracellular concentration of proteins such as TSC22d2 and SALL4, suggesting that it may be involved in the metabolic regulation of these proteins.

[11] The role of NRBP1 in tumors has only been proposed recently, and the exact regulatory mechanism remains unknown. However, due to the high conservation of NRBP1 and the characteristics of multiple protein domains, it plays an important role in cell growth regulation. In addition, since NRBP1 has putative nuclear localization signal (NLS) and nuclear export signal (NES) sequences, NRBP1 may also have the function of binding specific ligands for its intracellular transport, which may also be closely related to cell proliferation. NRBP1 may have different regulatory mechanisms in different types of tumors. Nrbp1 is considered to act as an intestinal tumor suppressor gene in a mouse conditional knockout model. However, NRBP1 inhibits the development of breast cancer and certain types of lymphoma, but shows the opposite effect in prostate cancer. Notably, using the analysis data of human tumor cell lines, NRBP1 is downregulated in lung and colorectal adenocarcinomas, and the low expression level of NRBP1 in lung tumors is associated with reduced survival rate, suggesting the potential role of NRBP1 in the progression of lung and colon tumors. However, the specific mechanism of action of NRBP1 in different tumor subtypes is not fully understood. The role of NRBP1 in the progression of lung adenocarcinoma has not been fully elucidated.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The present invention provides the application of short hairpin RNA and NRBP1 in the preparation of drugs for preventing or treating lung adenocarcinoma. Research has found that NRBP1 is upregulated in a variety of tumors, especially in lung adenocarcinoma (LUAD). The high expression of NRBP1 is associated with poor prognosis in LUAD patients, including shorter overall survival and higher pathological grades. The expression of NRBP1 is significantly associated with the infiltration levels of multiple immune cells in the tumor immune microenvironment and is positively correlated with tumor mutational burden (TMB). In vitro experiments show that NRBP1 knockout inhibits the proliferation and migration abilities of lung cancer cells. In vivo experiments indicate that NRBP1 knockout significantly slows down tumor growth and affects the expression of immune checkpoint-related genes.

[0006] The technical solution of the present invention is as follows:

[0007] The first object of the present invention is to provide a short hairpin RNA that knocks down NRBP1, and its coding sequence is as shown in SEQ ID NO.1 in the sequence listing.

[0008] The second object of the present invention is to provide the application of the above short hairpin RNA that knocks down NRBP1 in the preparation of drugs for preventing or treating lung adenocarcinoma.

[0009] The third object of the present invention is to provide the application of an expression inhibitor or knockout reagent or silencing reagent of the NRBP1 gene in the preparation of drugs for preventing or treating lung adenocarcinoma.

[0010] In a specific embodiment of the present invention, the expression inhibitor includes a short hairpin RNA capable of inhibiting the expression of the NRBP1 gene; or a construct capable of expressing or forming the short hairpin RNA.

[0011] In a specific embodiment of the present invention, the coding sequence of the short hairpin RNA is as shown in SEQ ID NO.1 in the sequence listing.

[0012] In a specific embodiment of the present invention, the construct is a lentiviral plasmid.

[0013] In a specific embodiment of the present invention, the drug inhibits the proliferation of lung adenocarcinoma cells, or inhibits the migration of lung adenocarcinoma cells, or inhibits the invasion of lung adenocarcinoma cells, or inhibits the growth of lung adenocarcinoma tumors, or reduces the expression level of immune checkpoint-related genes, and the immune checkpoint-related genes include CD274, PDCD1, TIGIT, LAG3, CTLA4.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] 1. NRBP1 is a potential biomarker for the prognosis and immunotherapy response of LUAD. Changes in its expression level are not only related to tumor progression but may also affect the tumor immune microenvironment and the efficacy of immunotherapy. This study provides new insights into the potential application of NRBP1 in the treatment of lung adenocarcinoma.

[0016] 2. High expression of NRBP1 in tumor tissues is significantly correlated with poor overall survival (OS) and higher pathological grades of patients. Further correlation analysis shows that it has a strong association with the degree of tumor immune infiltration, TMB, tumor microenvironment-related pathways, etc. More importantly, in vitro and in vivo experiments, NRBP1 expression regulates the proliferation, migration, and invasion abilities of lung adenocarcinoma. Figure Legends

[0017] Figure 1 For the exploration of the expression of the NRBP1 gene in pan-cancer and LUAD, A. Expression differences and distributions of NRBP1 between pan-cancer tumor tissues and adjacent tissues; B, C. Correlation studies of the expression of NRBP1 with fibroblasts and phagocytes in different tumors; D. Significant differences in the expression of NRBP1 between LUAD tumor tissue and adjacent tissue samples; E. Significant difference studies of NRBP1 between different tumor grade samples; F. Survival curve of the best CutOff (P<0.05); G. Co-expression study of the NRBP1 gene; H. GO and KEGG enrichment analysis.

[0018] Figure 2 For the study of NRBP1 gene related to immunity, A. GSEA analysis of single genes in GO, KEGG, and Reactome; B, C, D. CIBERSORT, TIMER, and ESTIMATE analyses reveal the correlation between high and low expression of single genes and the degree of immune infiltration.

[0019] Figure 3 For the study of NRBP1 gene related to immunity: A. Correlation between NRBP1 expression and TMB (tumor pan-genome); B. Significant correlation studies of NRBP1 gene expression with immune checkpoint, M6a, MCH, immune activation, immune suppression, chemokine, chemokine receptor, ferroptosis and other related genes in pan-cancer.

[0020] Figure 4 For the drug sensitivity and prognostic characteristics of the NRBP1 gene in pan-cancer: A. GSVA analysis of NRBP1 expression related to pathways; B. Correlation of copy number variation of NRBP1 expression in pan-cancer; C. Relationship between differential expression of NRBP1 and drug sensitivity; D. Influence of single gene expression of NRBP1 on the prognosis of pan-cancer.

[0021] Figure 5Expression of NRBP1 in different cells: A, B. The expression of NRBP1 in BEAS-2B, H1650, H1975, A549, H1299, MLE-12 and LLC cells was detected by qRT-PCR. The results of three independent experiments are presented as mean ± standard deviation.

[0022] Figure 6 Effect of NRBP1 knockdown on the proliferation and migration of H1975 and LLC cells: H1975 cells and LLC cells were treated with sh-NC or sh-NRBP1 respectively. After 7 days of culture, colony formation assay was performed. After 48 hours of culture, scratch assay was carried out. (A, C) The proliferation ability of H1975 cells in each group was detected by colony formation assay; (B, D) The horizontal migration ability of H1975 cells was detected by scratch assay, Bar = 200 μm. (E, G) The proliferation ability of LLC cells in each group was detected by colony formation assay. (F, H) The horizontal migration ability of LLC cells was detected by scratch assay, Bar = 200 μm. The results of three independent experiments are presented as mean ± standard deviation. Compared with sh-NC, *P < 0.05, **P < 0.01.

[0023] Figure 7 Effect of NRBP1 gene knockdown in vivo: LLC cells (1×107 cells) treated with sh-NC or sh-NRBP1 were subcutaneously injected into C57BL / 6 mice. The mice died 21 days after inoculation. A. Tumor volume; B. Body weight of mice; D. Ex vivo tumor image; E. Tumor weight; The expression of NRBP1, CD274, PDCD1, TIGIT, LAG3, CTLA4 in tumors was detected by qRT-PCR, as shown in C, F, G, H, I, J respectively. The results of six independent experiments are presented as mean ± standard deviation. Compared with sh-NC, *P < 0.05, **P < 0.01.

[0024] Figure 8 Role of NRBP1 in the immunomodulation of lung adenocarcinoma: LLC cells (1×107 cells) treated with sh-NC or sh-NRBP1 were subcutaneously injected into C57BL / 6 mice. The mice died 21 days after inoculation. Flow cytometry was used to detect the proportions of CD3+CD8+ cells (A, B), CD4+CD25+FoxP3+Helios+ cells (B, C), CD4+CD25+FoxP3+Helios- cells (B, D) and F4 / 80+CD11b+ cells (B, E) in tumor tissues of each group. The results of six independent experiments are presented as mean ± standard deviation. Compared with sh-NC, *P < 0.05, **P < 0.01. Detailed implementation

[0025] In this study, by analyzing the data of gene expression, mutation, and clinical information from The Cancer Genome Atlas (TCGA) database, we deeply explored the role of NRBP1 in LUAD. First, in the pan-cancer study, the gene NRBP1 showed differential expression in multiple tumor types. NRBP1 was highly expressed in pan-cancer, especially in tumors such as LUAD, liver hepatocellular carcinoma (LIHC), breast cancer (BRCA), and esophageal carcinoma (ESCA), where its expression increase was more significant. This was significantly different from the previous study by Wilson et al. In Wilson's study, lung tissue and fetal lung tissue were used as controls. NRBP1 played an important role in embryonic development, participated in the structural development of the intestine by regulating Wnt-responsive genes, and thus might also play an important role in lung development, and its expression might be different from that adjacent to cancer. In addition, factors such as the sample size and ethnic differences would also have a certain impact on the results. Moreover, to explore the relationship between the gene expression of NRBP1 and the sensitivity of LUAD to tumor drugs, we explored the expression differences using the CI50 of 192 drugs. Entinostat, Entospletinib, Crizotinib, and Buparlisib showed relatively significant differences. Univariate COX regression analysis was performed to analyze the predictive ability of the single-gene expression of NRBP1 on the prognosis of tumors.

[0026] NRBP1 is also significantly associated with the infiltration levels of multiple immune cells in the tumor immune microenvironment. Further bioinformatics analysis indicates that the expression of NRBP1 is closely related to biological processes such as the cell cycle, mitosis, and RNA transport. In addition, the tumor mutational burden (TMB) of samples with high NRBP1 expression levels is relatively high across pan-cancers, suggesting that it may serve as a predictive indicator of immunotherapy responsiveness. These findings suggest that NRBP1 not only plays a key role in tumor development but also acts as a regulator of immune responses in the tumor microenvironment and may become an important biomarker for cancer treatment and prognosis assessment. Immune cells can recognize and eliminate tumor cells, and tumors avoid immune-mediated attacks through elimination, balancing, and escape mechanisms. Tumor development often progresses rapidly after immune escape. Immunotherapy relies on the use of antibodies or immune vaccines to better stimulate the host's anti-tumor immune response and promote the clearance of tumor cells. In this study, methods such as CIBERSORT, TIMER, and ESTIMATE were used to explore the potential association between NRBP1 expression and the immune status, and significant correlations with macrophages, M0 macrophages, lymphocytes, plasma cells, etc. were found. Macrophage M0 is an undifferentiated cell type that can potentially be induced to differentiate into M1 or M2 polarized cell types according to corresponding signals and the microenvironment. The regulatory role of macrophage M0 in LUAD has not been fully confirmed. The NRBP1 expression level is also related to immune cells such as CD8 T, nTreg, iTreg, MAIT, and macrophages. Evidence for the tumor mutational burden (Tumor Mutational Burden, TMB) of LUAD patients as an NSCLC immune-related biomarker mainly comes from subgroup analyses of clinical trials. In the CheckMate 227 study, regardless of PD-L1 expression, in NSCLC patients with high TMB (≥10 mutations per megabase), the progression-free survival (PFS) of first-line nivolumab combined with ipilimumab was significantly better than that of chemotherapy. However, long-term follow-up data showed that the TMB level was not related to overall survival (OS). This study found that the NRBP1 expression level was significantly and positively correlated with the TMB status of LUAD patients, and the expression of NRBP1 was significantly different from OS survival and was closely related to pathological grade. Further analyzing the mechanism by which NRBP1 affects immunotherapy, we found that genes related to immune checkpoints, M6a, MCH, immune activation, immune suppression, chemokines, chemokine receptors, ferroptosis, etc. were significantly correlated with the expression of NRBP1. Although there were no significant differences in tumor microenvironment-related pathways and NRBP1 expression, multiple related indicators still showed significant differences. These findings provide a promising basis for the immunotherapy of LUAD and suggest that the differential expression of NRBP1 may have potential relevance in the immune response status of LUAD patients.

[0027] In this study, through in-depth analysis of in vitro and in vivo models of LUAD, the upregulated expression of NRBP1 in lung cancer cell lines was further independently verified. Among cell lines such as BEAS-2B, H1650, H1975, A549, H1299, MLE-12, and LLC, the H1975 cell line with high expression of NRBP1 and the LLC cell line with low expression of NRBP1 were screened out. By knocking out NRBP1 in the H1975 and LLC cell lines, the importance of NRBP1 for the proliferation of lung cancer cells was further evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) technology. In subsequent colony formation and cell scratch assays, it was observed that the knockout of NRBP1 could inhibit the proliferation of lung cancer cells. We also supplemented cell migration assays to demonstrate the regulatory effect of NRBP1 expression on the migratory ability of lung cancer cells. In in vivo experiments, we found that subcutaneous injection of lung cancer cells with knocked-out NRBP1 effectively inhibited tumor growth, and at the same time confirmed the regulatory effect of NRBP1 on immune checkpoint-related genes such as CD274, PDCD1, TIGIT, LAG3, and CTLA4. The combination of CTLA4 antibody and PD1 / PDL1 antibody is currently a clinically proven effective immunotherapy strategy. CD274 is expressed on activated T cells, B cells, and NKT cells, binds to PD-1 (programmed death receptor 1) encoded by PDCD1 on effector T cells, conducts immunosuppressive signals, and inhibits the activation of immune effector T cells. LAG-3 is the third immune checkpoint applied to the clinic after PD-1 / PD-L1 and CTLA-4. It is a cell surface inhibitory receptor with multiple biological activities in terms of T cell activation and effector functions. Studies have shown that LAG-3 and PD-1 are co-expressed in tumor-infiltrating lymphocytes (TILs) of progressive tumors, and their combined blockade has a synergistic effect on immune escape, increasing anti-tumor responses, enhancing T cell proliferation, and cytokine production. TIGIT is expressed by activated CD8+ T and CD4+ T cells, natural killer (NK) cells, regulatory T cells (Tregs), and follicular helper T cells, and is one of the most promising and potential targets. Multiple lines of evidence support a key role for TIGIT in restricting adaptive and innate immunity against tumors. And it was shown that the knockout intervention of NRBP1 could effectively promote the activities of CD8+ T cells, iTregs, and macrophages, promote immune infiltration, and has the potential to evaluate the efficacy of immunotherapy. Comprehensive bioinformatics data analysis and related experimental results fully demonstrated that NRBP1 expression could promote the proliferation, migration, and invasion abilities of lung cancer.

[0028] This study used bioinformatics methods to provide profound biological insights into the potential mechanism of NRBP1 in regulating the development of anti-lung cancer and tumor immune responses. In vitro experiments also demonstrated the role of NRBP1 in promoting the development of lung cancer. Further in vivo experiments effectively verified the function of NRBP1 and showed its potential to enhance the effectiveness of tumor immunotherapy.

[0029] In order to make the objectives, technical solutions and advantages of the present invention more clear and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0030] Example 1

[0031] I. Materials and Methods

[0032] 1. Data acquisition and screening

[0033] Using TCGA (The Cancer Genome Atlas, TCGA, https: / / portal.gdc.cancer.gov / ), gene expression data and mutation data of 560 LUAD (lung adenocarcinoma) patients were downloaded. The samples included 502 tumor samples (01A) and 58 adjacent cancer samples (11A), and clinical data including age, gender, stage, and tumor, node, and metastasis (TNM staging) were collected. The data normalized by the "normalizeBetweenArrays" function of the limma 3.9.19 package in R language was used. After excluding samples with a gene deletion rate of more than 20%, a total of 502 tumor samples and 19,603 genes were included.

[13] Samples with a gene deletion rate of more than 20% were excluded, and a total of 502 tumor samples and 19,603 genes were included.

[0034] 2. Expression analysis of NRBP1 in pan-cancer tissues

[0035] The online website GEPIA (http: / / gepia.cancer-pku.cn / detail.php?gene = CUL3) was used to analyze the expression differences of the NRBP1 gene in cancer patients of different tissues and the correlation study of the degree of immune infiltration, which mainly included fibroblasts and macrophages.

[0036] 3. Identification of the expression difference of NRBP1 in cancer tissues and adjacent cancer tissues in lung adenocarcinoma

[0037] The t-test method provided by the limma 3.9.19 package (http: / / www.bioconductor.org / packages / release / bioc / html / limma.html) was used to test the difference in the mean expression of the NRBP1 gene between two groups of samples (Tumor VS Normal) (P Value < 0.05). At the same time, the paired-sample t-test was performed to explore the individual differential expression characteristics of the samples.

[0038] 4. Relationship between NRBP1 expression difference and clinicopathological grading of lung adenocarcinoma

[0039] The Whitney U test method was used to calculate the expression difference of the NRBP1 gene in the pathological grades pairwise. The ggplot2 package in R language was used to draw the box plot of significant differences to explore the difference in NRBP1 expression among different pathological grades (Stage I, Stage II, Stage III, Stage IV).

[0040] 5. Establishment of the KM survival curve of NRBP1 and lung adenocarcinoma prognosis

[0041] The population was classified into high and low risks (High&Low) using the median value of NRBP1 gene expression. Then, the Log-rank Test method provided in the package was used to estimate the survival probabilities of the two groups (P value < 0.05). The population was classified by the best cut-off value to explore the best survival estimation model (P value < 0.05). The AUC of the prognostic model of the 1-year, 3-year, and 5-year prognoses of the LUAD population and the NRBP1 single gene was evaluated.

[0042] 6. Co-expression analysis of the NRBP1 gene in lung adenocarcinoma

[0043] Pearson correlation research was used to study the correlation between the expression profile of NRBP1 and all genes (P<0.05). The R language Heatmap package was used to draw the heatmap (Top50 genes). The Gene ID was converted through the H.sapiens Entrez gene IDs (2021-02-01) of the R language clusterProfiler package. The following database sources were used for pathway and process enrichment analysis of the gene list to explore the biological functions of the pathways where genes with significant correlation characteristics with NRBP1 are located (P value<0.05). The analysis databases included: GO (BP\MF\CC), KEGG. The pathway threshold was set to find significantly changed enrichment entries (p-value<0.05, enrichment factor>1.5).

[0044] 7. GSEA analysis

[0045] For the Msigdb data [https: / / www.gsea-msigdb.org / gsea / msigdb / index.jsp], GO and KEGG functional gene sets were provided: "KEGG subset of CP" and "GO:Gene Ontology gene sets". The GSEA algorithm was used to calculate the contribution degree and enrichment degree of the NRBP1 gene in each pathway (p.adjust<0.05). The R language ggplot2 was used to draw the contribution percentage wave graph.

[0046] 8. Immune infiltration and immune cell pathway correlation analysis

[0047] Through CIBERSORT (R 4.0.2), the infiltration fractions of 22 immune cells were determined. The "LM22 leukocyte gene matrix set" was used in the package, and the method of Deconvolution was used to analyze multiple immune cell types in all TCGA-LUAD samples (LM22 consists of 547 genes, with a very wide coverage, and can distinguish 22 immune cells, including different subtypes of B cell types, T cell types, natural killer cells (NKs), plasma cells and myeloid cell types). After calculating the proportion of immune infiltrating cells, the Mann Whithey U test was used to compare the differences in the immune infiltration results between the high-risk (High Risk) and low-risk (Low Risk) group patients (P value<0.05). The Pearson algorithm was used to calculate the correlation between the two (P value<0.05). The R language ggplot2 package was used for drawing pictures.

[0048] Using the expression profiles of genes included in the TME-related and immune cell-related pathways provided by the msigdb database (https: / / www.gsea-msigdb.org / gsea / msigdb / index.jsp), where immune cells include: CD8_naive, Tc, Tex, Tr1, nTreg, iTreg, Th1, Th2, Th17, Tfh, Tcm, Tem, NKT, MAIT, DC, B_cell, Monocyte, Macrophage, NK, Neutrophil, Tgd, CD4_T, CD8_T, InfiltrationScore; The TME-related pathways and genes include: CD_8_T_effector, Immune_Checkpoint, Antigen_processing_machinery, Mismatch_Repair, Nucleotide_excision_repair, DNA_damage_response, DNA_replication, Base_excision_repair, Pan_F_TBRs, EMT1, EMT2, EMT3. The GSVA enrichment scores of the above gene sets were calculated using the GSVA algorithm, and the GSVA scoring was performed using the R language GSVA package (v 1.36.3). And based on the GSVA scores, the Wilcox rank sum test was used to perform a differential comparison analysis of the TME score results between the high-expression (High) and low-expression (Low) groups of patients with the NRBP1 gene (P value < 0.05).

[0049] 9. Analysis of the correlation between NRBP1 expression and TMB (tumor mutation burden)

[0050] The GDCquery_Maf function provided by TCGAbiolinks was used to download the mutation data of LUAD. The TMB score was calculated for the mutation processes of muse and somaticsniper. The R language toolkits involved include: maftools, TCGAbiolinks, tidyverse.

[0051] 10. Analysis of the correlation between NRBP1 expression and immunity

[0052] Pearson test was used to perform a significant correlation analysis of gene expression and immune checkpoint gene expression (P value < 0.05). Secondly, Pearson correlation exploration was performed on the gene sets of M6a, MCH, immune activation, immune suppression, chemokines, chemokine receptors, and ferroptosis genes respectively to screen for significantly different immune-related genes.

[0053] 11. GSVA analysis of NRBP1 expression related to pathways

[0054] The GSVA enrichment scores of the pathways in the high-expression (High) and low-expression (Low) grouped populations were calculated using the GSVA algorithm. The gene sets included in the study were: "hallmark gene sets" (https: / / www.gsea - msigdb.org / gsea / msigdb / human / collections.jsp). The GSVA scores were performed using the GSVA package (v 1.36.3) in R language to obtain the expression enrichment scores of the pathways for individual samples. Then, the Whitney U test was used to compare the enrichment differences between the High and Low gene expression groups (P < 0.05).

[0055] 12. Correlation analysis of NRBP1 expression and gene copy number variation

[0056] Using the differential expression method provided by the GEPIA website (http: / / gepia.cancer - pku.cn / index.html), the mutation spectrum based on TCGA - LUAD patient samples was studied. Pearson correlation was used to explore the linear correlation between gene expression and gene copy number variation, and its correlation (Pearson) was also explored in pan - cancer and lollipop plots were drawn.

[0057] 13. Drug sensitivity analysis of NRBP1 expression

[0058] Using the pRRophetic package in R language, 192 predicted drugs included in the possibleDrugs2016 reference file were used to evaluate the sensitivity of each patient to each chemical drug, and the sensitivity was represented by the IC50 value. Then, the Wilcox rank sum test was used to perform differential comparison analysis on the IC50 results of each drug for patients in the high - expression (High) and low - expression (Low) groups of the gene (P value < 0.05). The method provided by limma 3.9.19 in R language was used to test the expression between the two groups to find differentially expressed genes and determine the drugs sensitive to the difference with P value < 0.05. Pearson's method was used for correlation research (P < 0.05).

[0059] 14. Univariate COX regression analysis of the effect of NRBP1 expression on prognosis

[0060] Based on the expression of the NRBP1 gene, univariate COX regression was used to explore the predictive ability of single-gene expression for the prognosis of LUAD (P value < 0.05). Overall survival (OS), disease-specific survival (DSS), progression-free interval (PFI), and disease-free interval (DFI) were used as prognostic factors for four models of exploration, and forest plots were drawn using R language to display the hazard ratio (HR) and P values.

[0061] 15. Differential analysis of NRBP1 expression and somatic mutations

[0062] To explore the differences in somatic mutation rates between patients with high and low expression of the NRBP1 gene in LUAD, the differential expression method provided by the GEPIA website (http: / / gepia.cancer-pku.cn / index.html) was used in this study. The mutation spectrum based on TCGA-LUAD patient samples was studied. The mutation data were classified using High and Low, and the R package maftools (http: / / www.bioconductor.org / packages / release / bioc / html / maftools.html) was used to calculate the mutation rates of the two groups, and waterfall plots were drawn to display the specific mutation information of the genes.

[0063] 16. Construction of lentiviral plasmids

[0064] Short hairpin RNA (shRNA-NRBP1#1: 5’-CCGGGGCCATGGATACAGAGGAACTCGAGTTCCTCTGTATCCATGGCCTTTTTG-3’) was designed for the sh-NRBP1 sequence, and a lentiviral plasmid for knocking down the NRBP1 gene was constructed. As a negative control, scrambled shRNA against NRBP1 was also constructed.

[0065] 17. Cell culture and transfection

[0066] BEAS-2B, H1650, H1975, A549, H1299, MLE-12, and LLC cells were purchased from the American Tissue Culture Collection (ATCC, Rockville, MD, USA). BEAS-2B cells were cultured in airway epithelial cell basal medium, and H1650, H1975, and H1299 cells were cultured in RPMI-1640 medium (containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin). MLE-12 and LLC cells were cultured in DMEM medium (containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin), and A549 cells were cultured in Ham’s F-12K medium (containing 10% fetal bovine serum, 1×105 U / L penicillin, and 100 mg / L streptomycin). All cells were cultured in a constant temperature incubator at 37°C and 5% CO2.

[0067] BEAS-2B, H1650, H1975, A549, H1299, MLE-12, and LLC cells were cultured normally. sh-NC and sh-NRBP1 plasmids were transfected into H1975 and LLC cells, and mRNA was extracted from the cells to analyze the efficiency of NRBP1 silencing.

[0068] 18. Establishment and grouping of mouse models

[0069] Twelve specific pathogen-free (SPF) grade C57BL / 6 male mice, 6 - 8 weeks old, with a body weight of 20 ± 2 g, were provided by Changzhou Cavens Experimental Animal Co., Ltd. After 1 week of adaptive feeding, the animals were randomly divided into the sh-NC and sh-NRBP1 groups (N = 6). Mice in the sh-NC group were subcutaneously injected with 0.2 mL (1×107 / mL) of LLC cells transfected with the sh-NC plasmid to establish a transplanted tumor model; mice in the sh-NRBP1 group were subcutaneously injected with 0.2 mL (1×107 / mL) of LLC cells transfected with the sh-NRBP1 plasmid to establish a transplanted tumor model.

[0070] The tumor volume and body weight were measured every 3 days. After 21 days of modeling, the mice were sacrificed, and the tumor tissues were photographed macroscopically and weighed. The expression of NRBP1 and the levels of immune checkpoint-related genes CD274, PDCD1, TIGIT, LAG3, and CTLA4 in the tumor tissues were detected by qPCR; the infiltration of immune cells CD8+T, nTreg, iTreg, and Macrophage in the tumor tissues was detected by flow cytometry.

[0071] 19. mRNA extraction and qPCR detection

[0072] After 48 h of cell transfection, the cells were collected and RNA was extracted. For tissue samples, 100 mg was taken, ground with a homogenizer, and then RNA was extracted. After RNA extraction was completed, the mRNA concentration and purity were measured using a Nanodrop 2000.

[0073] Reverse transcription was carried out into cDNA according to the instructions of the iScript cDNA Synthesis Kit (1708891EDU) (Bio-Rad, Hercules, CA, USA). Subsequently, quantitative real-time PCR (qRT-PCR) was performed using TB Green Premix Ex Taq (Tli RNase H Plus) (RR420A) (Takara, Japan). GAPDH was used as the internal reference gene, and 3 replicate wells were set for different samples. The primer sequences are as follows:

[0074]

[0075]

[0076] 20. Colony formation assay

[0077] Take the cells in the logarithmic growth phase of each group. After digestion, adjust the cell density to 1×10 3 cells / well, inoculate into a 6-well plate, mix well, and then place in an incubator for incubation. After 7 days of culture, fix with 4% paraformaldehyde, stain with crystal violet, take pictures, and count and analyze. 3 replicates were set for each group.

[0078] 21. Cell scratch assay

[0079] After 48 h of transfection of H1975 and LLC with sh-NC and sh-NRBP1 plasmids, the cells were resuspended and the cell density was adjusted. 2 mL of cells with a density of 6×10 5 cells / mL were seeded into the scratched 6-well plate. After incubation in a 37 °C, 5% CO2 cell culture incubator for 24 h, use a sterile 200 μL pipette tip to scratch perpendicular to the marked horizontal line on the back in a laminar flow hood. Remove the scratched cells, add serum-free medium, continue to culture in the incubator, sample according to time points, and take pictures and record under a 100× light microscope. Detect the colony formation of cells in each group.

[0080] 22. Flow cytometry detection of immune cell infiltration

[0081] Take the tumor tissues of mice in each group, grind them, and pass them through a 400-mesh cell sieve. After centrifugation, adjust to a cell suspension with a concentration of 1×10 6 cells / mL with PBS. Subsequently, detect CD8 + T, nTreg (CD4 + CD25 +FoxP3 + Helios + )、iTreg(CD4 + CD25 + FoxP3 + Helios - ) and macrophages.

[0082] 23. Statistical analysis

[0083] Analysis and graphing were performed using Graphpad Prism 9 (Version 9.4.0), and the figures were collated using Adobe Illustrator (Version 26.3.1). All data were expressed as mean ± SD. Statistical differences between groups were tested using t-test, one-way ANOVA, and two-way ANOVA. A P-value less than 0.05 was considered statistically significant.

[0084] II. Experimental results

[0085] 1. Expression differences of NRBP1 affect LUAD immune infiltration and prognosis

[0086] By analyzing the expression differences of the NRBP1 gene in tumor patients of different tissues through the online website GEPIA, we found 15 tumor types with significant differences ( Figure 1 A), mainly including LUAD, liver hepatocellular carcinoma (LIHC), breast cancer (BRCA), esophageal carcinoma (ESCA), etc. Notably, the NRBP1 gene we focused on showed a relatively high expression level in TCGA ( Figure 1 B). On the other hand, we also conducted a correlation study on the degree of immune infiltration. Among them, fibroblasts were significantly correlated with more tumor tissues, especially in LUAD, where Cancer associated fibroblast_MCPCOUNTER, Cancer associated fibroblast_TIDE, and Cancer associated fibroblast_EPIC were all significantly correlated (P < 0.05, Figure 1 B). In addition, macrophages also showed significant correlation in LUAD (P < 0.05, Figure 1 C), indicating that LUAD has strong immune infiltration characteristics.

[0087] Based on the special immune infiltration characteristics of LUAD and the expression differences of the NRBP1 gene in tumor and adjacent tissues of LUAD, we conducted a paired exploration of the expression differences of the NRBP1 gene in LUAD, and the results showed significant expression changes (P < 0.01,Figure 1 D). Then, the expression difference of NRBP1 gene was studied in different pathological grades of LUAD (Stage I, Stage II, Stage III, Stage IV). There were significant expression differences in moderate and mild LUAD, namely Stage I, Stage II, and Stage III (P<0.05, Figure 1 E).

[0088] In order to explore the prognostic ability of NRBP1 gene in LUAD, we used the optimal cut-off value (Cut-Off) to divide the population into two groups: NRBP1 High and Low. The significance test of OS was performed. The results showed that there was a significant difference between the expression of NRBP1 gene and OS (P<0.05, Figure 1 F), but no significant difference was found in PFS. We further explored whether there were co-expressed genes of NRBP1 gene in LUAD patients, and the results showed that 9515 genes had significant positive correlation ( Figure 1 G), 51 significantly negatively correlated genes ( Figure 1 G). Then, the biological functions of the pathways of genes with significant correlation characteristics with NRBP1 were analyzed (P value < 0.05), and the results showed that 206 BP, 101 CC, 49 MF and 14 KEGG significant pathways were enriched ( Figure 1 H). Taken together, these data suggest that NRBP1 is upregulated in LUAD and is associated with poor prognostic outcomes.

[0089] In order to further explore the contribution of NRBP1 single gene to GO and KEGG pathway scores, we calculated GSEA scores (p.adjust<0.05) based on the GO, KEGG, and Reactome functional gene sets provided by Msigdb data [https: / / www.gsea-msigdb.org / gsea / msigdb / index.jsp]. The results showed that in GO, NRBP1 single gene has a high contribution in many pathways, including cell cycle checkpoint, nuclear chromosome, G2 / M transition of mitotic cell cycle, mitotic cell cycle, mitotic sister chromatid segregation, etc. ( Figure 2Secondly, KEGG indicated that the NRBP1 single gene had a high contribution in the following pathways, such as Mitophagy–animal, RNA transport, Cell cycle, Ribosome biogenesis in eukaryotes, mRNAsurveillance pathway, etc. Finally, Reactome indicated pathways such as Programmed Cell Death, MembraneTrafficking, Apoptosis, Regulation of TP53 Activity, Mitotic G1-G1 / S phases, SPhase, G1 / S Transition, Cell Cycle Figure 2 A(c)).

[0090] 2. NRBP1 expression affects immune cells and the tumor microenvironment

[0091] We conducted an immune infiltration study based on three algorithms: CIBERSORT, TIMER, and ESTIMATE. The results showed a significant association between NRBP1 expression and 21 immune cells (P<1E-6, |Coef|>0.2), mainly including four immune cells: macrophages, M0 macrophages, lymphocytes, and plasma cells( Figure 2 B, C), and there were significant differences in the infiltration degree of immune cells between the High group and the Low group (P<0.05, Figure 2 D). Secondly, the study also used the GSVA algorithm to calculate the GSVA enrichment scores of the pathways in the high-expression (High) and low-expression (Low) grouped populations, and used the R language GSVA package (v 1.36.3) for GSVA scoring to obtain the expression enrichment scores of the pathways of individual samples. Then, the Whitney U test was used to compare the enrichment differences between the High and Low gene expression groups (P<0.05). The algorithm scored the pathways of 26 common immune cells respectively, and then conducted a correlation study with NRBP1 gene expression. The results showed a significant correlation between 8 cells and gene expression Figure 2D, E), including CD8_T, nTreg, iTreg, MAIT, Macrophage, etc. Finally, the study also explored the significant correlation between the scores of tumor microenvironment-related pathways in pan-cancer patients and the NRBP1 gene based on the GSVA algorithm. The results showed that there was no significant difference in TMEscore, but significant differences were found in indicators such as CD_8_T_effector, Immune_Checkpoint, Antigen_processing_machinery, TMEscoreA, Mismatch_Repair, Nucleotide_excision_repair, DNA_damage_response, DNA_replication, Base_excision_repair, Pan_F_TBRs, EMT1, EMT2, EMT3, TMEscoreB, etc.( Figure 2 F, G).

[0092] 3. Immunotherapy benefit analysis

[0093] The GDCquery_Maf function provided by TCGAbiolinks was used to download the mutation data of pan-cancer (a total of 503 LUAD tumor samples were included). The TMB score was calculated for the mutation processes of muse and somaticsniper. The results showed that the overall TMB score of LUAD was relatively high among pan-cancers( Figure 3 A(a)). Secondly, the TMB scores of READ and LUAD had the most significant correlation characteristics with the expression of the NRBP1 gene( Figure 3 A(b)). Finally, the study conducted a correlation study between the TMB score and the gene expression of NRBP1. The results showed that there was a significant positive correlation between the gene expression of NRBP1 and the TMB score in LUAD (P<0.001, Coeff = 0.23 (pearson), Figure 3 A(c)).

[0094] We further conducted a correlation study between the expression of NRBP1 in LUAD and immune checkpoints. The results showed that 5 genes including CD274, PDCD1, TIGIT, LAG3, CTLA4 (P<0.05) had significant differences with immune checkpoints( Figure 3 B(a)).

[0095] Pearson correlation exploration was performed on immune checkpoints, M6a, MCH, immune activation, immune suppression, chemokines, chemokine receptors, and ferroptosis gene sets. It was found that TAP2, CD276, CCR1, CXCL16, and ferroptosis genes were significantly correlated with NRBP1 in most tumors, except that IL10RB had no significant correlation in ESCA( Figure 3 B). In the further analysis of LUAD, 13 M6A genes were significantly correlated with the single-gene expression of NRBP1 in LUAD( Figure 3 B(b)), 13 MCH genes( Figure 3 B(c)), 30 immune activation genes( Figure 3 B(d)), 18 immune suppression genes( Figure 3 B(d)), 8 chemokines( Figure 3 B(f)), 29 chemokine receptors, and 53 ferroptosis-related genes( Figure 3 B(h)).

[0096] 4. Drug Sensitivity and Prognostic Characteristics of NRBP1 Gene in Pan-Cancer

[0097] To identify the significantly different pathways between the high-expression and low-expression groups of NRBP1. Based on the "hallmark gene sets" (https: / / www.gsea-msigdb.org / gsea / msigdb / human / collections.jsp), extensive pathway enrichment scores were performed. First, significant difference studies were conducted on the High and Low expression groups, and the results showed 1102 down-regulated pathways and 5469 up-regulated pathways( Figure 4 A(a), P<0.05, |Log FC|>0.2). Then, Pearson correlation studies were used to explore the correlation between single-gene expression and pathway enrichment scores, and the results showed 3033 pathways significantly positively correlated with gene expression and 378 significantly negatively correlated pathways( Figure 4 A(a), P<0.05, |Coef|>0.4). Secondly, we also analyzed the differences in somatic mutation rates between high-expression and low-expression LUAD patients, and LUAD showed a strong correlation( Figure 4 B(a), P<0.05). Therefore, the expression of NRBP1 may be affected by the somatic mutation rate and is involved in the regulation of multiple pathways. We further explored whether the expression of NRBP1 affects the sensitivity of anti-tumor drugs in LUAD. The study used the CI50 of 192 drugs to explore the expression differences, and the results showed that 29 drugs were significantly correlated with the high and low expression of NRBP1Figure 4 C(a), P < 0.05), where Entinostat, Entospletinib, Crizotinib, and Buparlisib showed significant differences (P < 0.0001, Figure 4 C(b)(c)). Finally, the predictive ability of NRBP1 single-gene expression for the prognosis of pan-cancer was analyzed using univariate COX regression (P value < 0.05). Four models were explored using OS, DSS, PFI, and DFI as prognostic factors respectively. The results showed that under different prognostic factors, NRBP1 had prognostic evaluation potential for different tumor types. For example, when DFI or PFI was used as the prognostic factor, NRBP1 had significant prognostic evaluation potential for ACC( Figure 4 D(a, d), P value < 0.05). When DSS or OS was used as the prognostic factor, NRBP1 had significant prognostic evaluation potential for ACC, BLCA, and BRCA( Figure 4 D(b, c), P value < 0.05). Thus, it can be seen that the expression of NRBP1 affects the sensitivity of anti-tumor drugs and may serve as a specific marker for anti-tumor drugs. At the same time, the expression of NRBP1 also has the potential to be a prognostic marker.

[0098] 5. Knockout of NRBP1 inhibits the proliferation, migration, and invasion of lung cancer cell lines

[0099] The mRNA expression levels of NRBP1 in lung cancer cell lines BEAS-2B, H1650, H1975, A549, H1299, MLE-12, and LLC were quantitatively analyzed. The results showed that the expression of NRBP1 was most significant in the H1975 cell line, while the expression level was relatively low in the LLC cell line( Figure 5 ). To further explore the biological function of NRBP1 in LUAD, this study silenced the NRBP1 gene in H1975 and LLC cell lines. By analyzing the mRNA level, we screened out the shRNA with the best knockout efficiency to effectively knockdown NRBP1 mRNA. In addition, colony formation assays also showed that knockout of the NRBP1 gene inhibited the proliferation ability of LUAD cells( Figure 6 A, C, E, G). Wound healing assays further demonstrated that knockout of the NRBP1 gene significantly inhibited the migration ability of LUAD cells( Figure 6 B, D, F, H). In summary, knockout of the NRBP1 gene can inhibit the proliferation and migration of lung adenocarcinoma cells.

[0100] 6. Knockout of NRBP1 inhibits LUAD proliferation, migration, and invasion

[0101] In the in-vivo experiment with mice, in C57BL / 6 mice injected with LLC cells with sh-NRBP1 gene knockout, the tumor growth rate was significantly slower than that of the control group ( Figure 7 D). In addition, the subcutaneous tumor volume and weight of NRBP1 knockout mice were significantly smaller than those of the negative control group ( Figure 7 A-C). In addition, quantitative real-time PCR analysis of genes such as NRBP1, PDCD1, TIGIT, LAG3, and CTLA4 showed that the expression levels of these immune checkpoint-related genes decreased when the NRBP1 gene was knocked out ( Figure 7 E-J).

[0102] 7. Functional study of NRBP1 in the immunomodulation of lung adenocarcinoma

[0103] Immunotherapy is one of the most crucial methods in the current treatment of lung cancer. We injected LLC cells transfected with sh-NC or sh-NRBP1 into C57BL / 6 mice. Analysis of the effect of NRBP1 knockout on immune cells by flow cytometry showed that the nTreg cells increased significantly, while the contents of CD8+ T cells, iTreg, and macrophages decreased significantly ( Figure 8 ). These results indicate that NRBP1 inhibition intervention can effectively promote the activities of CD8+ T cells, iTreg, and macrophages, promote immune infiltration, and has the potential to evaluate the efficacy of immunotherapy.

[0104] This invention confirms the great potential of NRBP1 as a prognostic marker and an effective immunotherapy target for LUAD. Its high expression in tumor tissues is significantly correlated with the unfavorable overall survival (OS) of patients and a higher pathological grade. Further correlation analysis shows that it has a strong association with the degree of tumor immune infiltration, TMB, tumor microenvironment-related pathways, etc. More importantly, in in-vitro and in-vivo experiments, the expression of NRBP1 regulates the proliferation, migration, and invasion abilities of lung adenocarcinoma.

[0105] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A short hairpin RNA for knocking down NRBP1, characterized in that: Its coding sequence is shown in the sequence table SEQ ID NO.

1.

2. Use of the short hairpin RNA for knocking down NRBP1 according to claim 1 in the preparation of a drug for preventing or treating lung adenocarcinoma.

3. Use of an expression inhibitor, knockout agent or silencing agent of the NRBP1 gene in the preparation of drugs for preventing or treating lung adenocarcinoma.

4. The use according to claim 3, characterized in that: The expression inhibitor includes a short hairpin RNA capable of inhibiting the expression of the NRBP1 gene; or a construct capable of expressing or forming the short hairpin RNA.

5. The use according to claim 4, characterized in that: The coding sequence of the short hairpin RNA is shown in the sequence table SEQ ID NO.

1.

6. The use according to claim 4, characterized in that: The construct is a lentiviral plasmid.

7. The use according to claim 3, characterized in that: The drug inhibits the proliferation of lung adenocarcinoma cells, or inhibits the migration of lung adenocarcinoma cells, or inhibits the invasion of lung adenocarcinoma cells, or inhibits the growth of lung adenocarcinoma tumors, or reduces the expression level of immune checkpoint-related genes, and the immune checkpoint-related genes include CD274, PDCD1, TIGIT, LAG3, and CTLA4.

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