Application of C1RL-AS1 / hsa-miR-424-5p / ADRB2 axis in preparation of immunoregulatory drugs for lung adenocarcinoma

Abstract

The application belongs to the technical field of biological medicine, and relates to application of a C1RL-AS1 / hsa-miR-424-5p / ADRB2 axis in preparation of an immunoregulatory drug for lung adenocarcinoma. The application discloses that the regulation axis can be used for preparing an immunoregulatory treatment drug for lung adenocarcinoma, and simultaneously provides a therapeutic preparation and a drug based on the axis. The therapeutic preparation comprises a C1RL-AS1 overexpression related preparation, a hsa-miR-424-5p inhibiting reagent, an ADRB2 overexpression vector and an ADRB2 agonist, and can be used alone or in combination with an immune checkpoint inhibitor. The drug contains at least one active ingredient and a pharmaceutical carrier, and the dosage form is an injection, a freeze-dried powder injection or a sustained-release preparation. The application further discloses that the ADRB2 can be used for preparing a prognosis evaluation, an individualized immunotherapy guidance reagent and a screening tool for an immunoregulatory drug for lung adenocarcinoma. The application clearly regulates the mechanism and the immune function, provides a new target, a marker and a screening tool, and has important clinical conversion value.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and in particular relates to the application of the C1RL-AS1 / hsa-miR-424-5p / ADRB2 axis in the preparation of immunomodulatory drugs for lung adenocarcinoma. Background Technology

[0002] Lung cancer is one of the most common and deadliest types of cancer worldwide, drawing significant attention due to its high incidence and mortality rates. Of all lung cancer cases, approximately 85% are non-small cell lung cancer (NSCLC), while only about 15% are small cell lung cancer. NSCLC is primarily divided into lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD), with LUAD being the most common subtype. Despite extensive research into the treatment of LUAD, patient survival rates remain unsatisfactory, highlighting the urgent need to identify effective therapeutic targets and reliable prognostic biomarkers for LUAD.

[0003] β2-adrenergic receptor 2 (ADRB2) is dysregulated in various cancers, including lung adenocarcinoma, and is closely related to inflammatory responses. Increasing research indicates that ADRB2 plays a crucial role in the development and progression of various cancers, with its expression varying across different cancer types, and it is a potential gene associated with lung adenocarcinoma. Previous studies have revealed the expression pattern of ADRB2 in lung cancer and its interaction with the tumor microenvironment, while also confirming its close association with the development and progression of various human diseases such as gastric cancer, breast cancer, and hepatocellular carcinoma. ADRB2 antagonists can inhibit tumor cell proliferation, metastasis, and invasion by suppressing transcription factors and proteins associated with the ERK1 / 2-JNK-MAPK pathway (such as activator protein 1, STAT3, cyclic adenosine monophosphate response element-binding protein, and NF-κB). Furthermore, ADRB2 participates in immune signaling; systemic activation of ADRB2 in vivo can enhance the expansion and anti-tumor activity of γδT cell receptor T cells. However, the specific expression characteristics of ADRB2 in lung adenocarcinoma, its clear association with patient prognosis, and its core regulatory mechanisms remain unclear. In particular, the direct association between ADRB2 and immune infiltration of lung adenocarcinoma tumors has not been reported, which greatly limits its clinical translational application as a therapeutic target and prognostic biomarker for lung adenocarcinoma.

[0004] Non-coding RNAs (ncRNAs), including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), have been identified as key regulatory molecules in the occurrence, development, and treatment of lung cancer. Among them, miRNAs participate in the pathological process of lung cancer by targeting and regulating the expression of target genes, possessing both tumor-suppressing and tumor-promoting functions, and have potential value in early diagnosis and prognostic assessment of lung cancer. Long non-coding RNAs can affect lung cancer cell proliferation by regulating gene expression or chromatin state. Circular RNAs can act as miRNA sponges to regulate downstream gene expression, showing good potential in early diagnosis of lung cancer. Furthermore, the application value of non-coding RNAs in lung cancer treatment is gradually becoming apparent, providing new research directions for precision treatment of lung cancer. However, currently, the specific molecular pathways by which non-coding RNAs regulate ADRB2 expression in lung adenocarcinoma, and the association between this regulatory network and immune invasion in lung adenocarcinoma, remain unclear. These research gaps have not yet been filled, and cannot provide new effective targets and strategies to support precision treatment of lung adenocarcinoma. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings and deficiencies of existing technologies, such as the unclear regulatory mechanism of ADRB2 in lung adenocarcinoma, the lack of elucidation of its association with immune infiltration, and the lack of targeted immunomodulatory therapeutic targets and accurate prognostic biomarkers. This invention provides the application of the C1RL-AS1 / hsa-miR-424-5p / ADRB2 axis in the preparation of immunomodulatory drugs for lung adenocarcinoma. The aim is to overcome the limitations of existing lung adenocarcinoma treatments, such as single therapeutic targets, inaccurate prognostic assessments, and limited immunotherapeutic effects. The goal is to clarify the core regulatory pathway of ADRB2, discover novel therapeutic targets, provide accurate prognostic assessment evidence, and offer personalized medication guidance.

[0006] This invention provides the application of the C1RL-AS1 / hsa-miR-424-5p / ADRB2 axis in the preparation of immunomodulatory therapeutic drugs for lung adenocarcinoma.

[0007] This invention also provides the application of a C1RL-AS1 overexpression-related formulation in the preparation of an immunomodulatory therapeutic drug for lung adenocarcinoma.

[0008] The present invention also provides the application of an hsa-miR-424-5p inhibitor in the preparation of an immunomodulatory therapeutic agent for lung adenocarcinoma, wherein the inhibitor is selected from hsa-miR-424-5p antagonists, antisense oligonucleotides, or lentiviral knockdown vectors.

[0009] This invention also provides the application of an ADRB2 overexpression vector in the preparation of an immunomodulatory therapeutic drug for lung adenocarcinoma, wherein the overexpression vector contains an ADRB2 coding sequence; the overexpression vector is pLV17-EF1α-ADRB2-Luciferase17-Puro.

[0010] This invention also provides the application of ADRB2 agonists in the preparation of immunomodulatory therapeutic drugs for lung adenocarcinoma.

[0011] Furthermore, the ADRB2 agonist is used in combination with a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

[0012] The present invention also provides an immunomodulatory therapeutic drug for lung adenocarcinoma, comprising at least one active ingredient with immunomodulatory function and a pharmaceutically acceptable carrier, wherein the active ingredient is selected from: (1) a C1RL-AS1 overexpression vector; (2) an hsa-miR-424-5p inhibitor; (3) an ADRB2 overexpression vector or an ADRB2 agonist, and the drug dosage form is an injection, a lyophilized powder injection or a sustained-release preparation.

[0013] The present invention also provides the application of ADRB2 in the preparation of a prognostic assessment reagent for lung adenocarcinoma, wherein the reagent is used to detect the expression level of ADRB2 in tumor tissue or peripheral blood, and the detection includes nucleic acid level detection or protein level detection.

[0014] The present invention also provides a personalized immunotherapy guidance reagent for lung adenocarcinoma, which is used to detect the expression level of ADRB2 in tumor tissue or peripheral blood mononuclear cells to determine the sensitivity of patients to immunotherapy-related drugs for lung adenocarcinoma.

[0015] This invention also provides the application of ADRB2 in screening immunomodulatory therapeutic drugs for lung adenocarcinoma, using ADRB2 expression levels, protein activity, or CD4+ as indicators. + Indicators related to the infiltration of T cells and dendritic cells are used as detection indicators.

[0016] Compared with the prior art, the beneficial effects of the present invention are:

[0017] 1. The C1RL-AS1 / hsa-miR-424-5p / ADRB2 regulatory axis was first clearly identified, revealing the core regulatory mechanism of ADRB2 in lung adenocarcinoma and filling the research gap on non-coding RNA regulation of ADRB2 and its association with related immune infiltration.

[0018] 2. This study confirmed that ADRB2 possesses both anti-tumor and immunomodulatory functions; its overexpression can significantly inhibit the growth of lung adenocarcinoma tumors and promote CD4+ expression. + T cell and dendritic cell infiltration provide new targets for immunomodulatory therapy of lung adenocarcinoma.

[0019] 3. It was found that ADRB2 is significantly positively correlated with immune checkpoints such as PD-1, PD-L1, and CTLA-4, providing experimental evidence for the combined treatment of ADRB2 agonists and immune checkpoint inhibitors, thereby improving treatment efficacy.

[0020] 4. The study found that high expression of ADRB2 is associated with sensitivity to classic chemotherapy drugs such as afatinib and docetaxel, which can guide individualized drug selection for lung adenocarcinoma and improve the precision of treatment.

[0021] 5. This study clarifies that ADRB2 is expressed at low levels in lung adenocarcinoma and is closely related to patient prognosis, providing a molecular marker for the development of prognostic assessment reagents for lung adenocarcinoma and aiding in disease prognostic stratification. Attached Figure Description

[0022] Figure 1 Analysis of ADRB2 expression in different cancer types; Figure 1 In Figure A, the expression of ADRB2 in TCGA cancer tissue is compared with that in the corresponding TCGA and GTEx normal tissues. Figure 1 In Figure B, ADRB2 expression is found in seven cancer types based on gene expression profile interaction analysis (GEPIA) data from cancer and normal samples.

[0023] Figure 2 To investigate and verify the correlation between LUAD and ADRB2; Figure 2 In the figure, A represents the association between overall survival and ADRB2 expression in LUAD; Figure 2 Image B shows the immunoblot image of ADRB2 in the LUAD and LUSC cell lines;

[0024] Figure 3 Enrichment analysis of genes co-expressed by ADRB2 using the Kyoto Gene and Gene Encyclopedia (KEGG);

[0025] Figure 4 The causes of ADRB2 dysregulation involve a multidimensional mechanism involving gene mutations, DNA methylation, and miRNAs; Figure 4 In this context, A represents the ratio of wild-type (wt), deletion type (del), and amplified type (amp) in LUAD. Figure 4 In this context, B represents the correlation between copy number variation and ADRB2 mRNA expression in the LUAD sample; Figure 4 In this context, C represents the correlation between amplified and ADRB2 mRNA expression in LUAD samples; Figure 4 In this context, D represents the correlation between the deletion type and ADRB2 mRNA expression in the LUAD sample; Figure 4 E in the figure represents the correlation between DNA methylation and ADRB2 mRNA in LUAD samples;

[0026] Figure 5 In this context, A represents the miRNA-ADRB2 regulatory network generated using Cytoscape software. Figure 5 B in the figure refers to the expression of miR-424-5p in LUAD and normal samples detected using the StarBase database; Figure 5 In this context, C represents the result of the dual-luciferase reporter gene experiment; Figure 5 In this figure, D represents the immunoblotting results of ADRB2 protein levels after overexpression of miR-424-5p;

[0027] Figure 6 Analysis of the long non-coding RNA upstream of miRNA-424-5p in LUAD; Figure 6 In this context, A stands for STAG3L5P-PVRIG2P-PILRB; Figure 6 In this context, B stands for C1RL-AS1; Figure 6 C in the text stands for GABPB1-AS1; Figure 6 In this context, D represents the results of the dual-luciferase reporter gene experiment; Figure 6 E in the figure represents the immunoblotting results of ADRB2 protein levels after C1RL-AS1 interference;

[0028] Figure 7 The levels of various immune cell infiltration in lung adenocarcinomas under different ADRB2 copy numbers;

[0029] Figure 8 The correlation between ADRB2 expression and the level of immune cell infiltration in lung adenocarcinoma;

[0030] Figure 9 The correlation between ADRB2 and tumor immune-related genes; Figure 9 In this context, A represents the correlation between ADRB2 and the chemokine receptor gene; Figure 9 In this context, B represents the correlation between ADRB2 and chemokine genes; Figure 9 C in the equation represents the correlation between ADRB2 and immune activation genes; Figure 9 In this context, D represents the correlation between ADRB2 and immunosuppressive genes;

[0031] Figure 10 To investigate the effects of ADRB2 overexpression on tumor size and changes in immune cells in C57BL / 6 mice; Figure 10 In the figure, A represents the construction and verification of a stable cell line overexpressing ADRB2; Figure 10 In the figure, B represents a comparison of tumor volume during growth between the control group and the ADRB2 overexpression group; Figure 10 In this context, C represents the size of the tumor observed through in vivo imaging of small animals; Figure 10 In this context, D represents a comparison of tumor size between the control group and the ADRB2 overexpression group in C57BL / 6 mice.

[0032] Figure 11 Analysis of immune cell infiltration in tumor tissues of C57BL / 6 mice after ADRB2 overexpression; Figure 11 A in the text represents CD4 in the tumor tissue of C57BL / 6 mice.+ Flow cytometry analysis of T cell infiltration (CD45) + CD11b - CD45R - CD3 + CD4 + ); Figure 11 B in the figure represents flow cytometry analysis of dendritic cell infiltration (CD45) in tumor tissue of C57BL / 6 mice. + CD11c + MHCII + );

[0033] Figure 12 In the figure, A represents the correlation between ARB2 and programmed death protein 1 (PD-1) expression in lung adenocarcinoma; Figure 12 In the figure, B represents the correlation between ADRB2 and programmed death-ligand 1 (PD-L1) expression in lung adenocarcinoma; Figure 12 C in the equation represents the correlation between ARB2 expression and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) expression in LUAD. Figure 12 D in the figure represents the validation of the correlation between ADRB2 and PD-1 expression in LUAD using the Gene Expression Interaction Analysis (GEPIA) database; Figure 12 E in the figure represents the correlation between ADRB2 and PD-L1 expression in LUAD, verified using the GEPIA database. Figure 12 F in the figure represents the correlation between ADRB2 and CTLA-4 expression in lung adenocarcinoma as verified by the GEPIA database;

[0034] Figure 13 Drug sensitivity analysis for ADRB2. Data from GDSC showed an association between ADRB2 expression and drug sensitivity;

[0035] Figure 14 This is a schematic diagram of the C1RL-AS1 / miR-424-5p / ADRB2 axis in lung adenocarcinoma. Detailed Implementation

[0036] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] 1. Materials and Methods

[0038] 1.1. ADRB2 Expression Analysis

[0039] Download data from the UCSCXena database (https: / / xenabrowser.net / datapages / ) containing the Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) datasets for 30 cancer types, including adrenocortical carcinoma, invasive breast cancer (BRCA), bladder urothelial carcinoma (BLCA), cervical squamous cell carcinoma and endometrial adenocarcinoma (CESC), colorectal cancer (COAD), cholangiocarcinoma (CHOL), diffuse large B-cell lymphoma, glioblastoma multiforme, esophageal cancer (ESCA), and head and neck squamous cell carcinoma (HNSC). Renal chromophobe carcinoma (KICH), renal papillary cell carcinoma (KIRP), renal clear cell carcinoma (KIRC), hepatocellular carcinoma (LIHC), acute myeloid leukemia, lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), pheochromocytoma and paraganglioma (PCPG), pancreatic cancer (PAAD), prostate cancer (PRAD), rectal cancer (READ), ovarian serous cystadenocarcinoma, sarcoma (SARC), gastric cancer (STAD), cutaneous melanoma (SKCM), testicular germ cell tumors, thymoma (THYM), thyroid cancer (THCA), endometrial cancer (UCEC), and uterine carcinosarcoma. The histological correspondences between the TCGA and GTEx datasets, the expression level of ADRB2 in pan-cancers, the level of long non-coding RNA in lung adenocarcinoma, and the correlation between ADRB2 and immune checkpoints were analyzed using the Gene Expression Interaction Analysis (GEPIA) database (http: / / gepia.cancer-pku.cn / help.html). The correlation between ADRB2 expression levels and the levels of immune cell infiltration and immune checkpoint expression in lung adenocarcinoma was analyzed using the tumor immunoassay resource (https: / / cistrome.shinyapps.io / timer / ). Data analysis was performed using R4.2 software (https: / / www.R-project.org / ), and the results were plotted using the ggplot2 package. P < 0.05 was considered statistically significant, and R > 0.1 was set as the screening criterion.

[0040] 1.2. ADRB2 Drug Sensitivity Analysis

[0041] To clarify the relationship between ADRB2 expression and drug sensitivity (IC50), ADRB2 expression data and drug sensitivity data were integrated using the Cancer Drug Sensitivity Genomics (GDSC) database (https: / / www.cancerrxgene.org / ). Pearson correlation analysis was used to determine the correlation between ADRB2 expression and drug sensitivity, and P < 0.05 was considered statistically significant.

[0042] 1.3. Copy Number Variation (CNV) and DNA Methylation Analysis

[0043] ADRB2 copy number data, DNA methylation data, and corresponding expression data were obtained from the UCSCXena database (https: / / xenabrowser.net / ). Copy number variation classification was performed based on TCGA gene copy data, using a genomic identification threshold of significant cancer targets: a copy number of -1 or -2 was defined as copy loss, 0 as normal copy number, and 1 or 2 as copy increase. P < 0.05 was considered statistically significant.

[0044] 1.4. Candidate miRNA Prediction and Analysis

[0045] The miRNAs that interact with the ADRB2 gene were predicted using the miRNA prediction tools RNA22 (https: / / cm.jefferson.edu / rna22v2 / ), PITA (http: / / genie.weizmann.ac.il / pubs / mir07 / mir07_data.html), miRmap (https: / / mirmap.ezlab.org / ), miRanda (www.microrna.org / ), microT (http: / / www.microrna.gr / microT-CDS), PicTar (https: / / pictar.mdc-berlin.de / ), and TargetScan (https: / / www.targetscan.org / vert_80 / ). The miRNAs predicted by two or more of these tools were considered as candidate miRNAs.

[0046] Information on the above candidate miRNAs was obtained from the StarBase database (http: / / starbase.sysu.edu.cn / ), including their expression levels in lung adenocarcinoma and normal tissues, correlation analysis with ADRB2 expression, and predicted candidate interacting long non-coding RNAs.

[0047] 1.5. Cell Culture and Transfection

[0048] LLC mouse Lewis lung cancer cells (BNCC338433, Onco Biomedical Technology Co., Ltd.), BEAS-2B normal human lung epithelial cells (ZQ0381, Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.), A549 human non-small cell lung cancer cells (ZQ0003, Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.), HCC827 human non-small cell lung cancer cells (ZQ0386, Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.), H520 human lung squamous cell carcinoma cells (ZQ0014, Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.), and H1703 human lung squamous cell carcinoma cells (ZQ0015, Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.) were cultured in DMEM medium (Gibco, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Gibco; Thermo Fisher Scientific) and 1% penicillin-streptomycin at 37°C and 5% CO2 humidification.

[0049] The lentiviral vector used for ADRB2 overexpression was pLV17-EF1α-ADRB2-Luciferase17-Puro (Germage Gene, Shanghai Germage Gene Co., Ltd.). The negative control group was transfected with the empty vector construct (pLV17-EF1α-Luciferase17-Puro, without ADRB2). When the cell confluence reached 70%-80%, lentiviral transfection was performed to establish a stable ADRB2 expression cell line: fresh DMEM medium was replaced, and lentivirus carrying the ADRB2 expression construct, pre-thawed on ice, was added to infect cells at an appropriate ratio. To improve transfection efficiency, 5 μg / ml polybrene (Germage Gene) was added, and the mixture was gently shaken to ensure even distribution of the virus. The cells were incubated at 37°C in a humidified incubator for 6 hours. After incubation, the lentiviral medium was removed, replaced with fresh complete DMEM medium, and cultured for another 48 hours to ensure adequate transgene expression. To establish a stable transfected cell line, 3 μg / ml puromycin was added for selection, and surviving cells were passaged continuously for at least three generations under selection pressure. Transfection efficiency was verified using Western blotting to ensure stable ADRB2 expression.

[0050] C1RL-AS1 expression was inhibited using siRNA-C1RL-AS1, while hsa-miR-424-5p overexpression (Germazon gene) was achieved using miRNA mimics. Cell transfection was performed using Lipofectamine 3000 transfection reagent. Forty-eight hours after transfection, cells were lysed using RIPA lysis buffer containing protease and phosphatase inhibitors, and total cellular protein was extracted for Western blotting analysis.

[0051] 1.6. Western blot analysis

[0052] Cells were lysed on ice using RIPA lysis buffer supplemented with 1% protease inhibitor and 1% phosphatase inhibitor to extract total protein. Protein quantification was performed using the BCA method, followed by protein separation via 10% SDS-PAGE gel electrophoresis and electrotransfer to a nitrocellulose membrane (Bio-Rad). ADRB2 protein was detected using primary antibody (anti-ADRB2, 29864-1-AP, Wuhan Proteintech Group Co., Ltd.) and secondary antibody (SA00001-2, Wuhan Proteintech Group Co., Ltd.). Protein bands were quantified using ImageJ software and normalized using HSP90 as an internal control.

[0053] 1.7. Dual-luciferase reporter gene assay

[0054] Dual-luciferase reporter gene assays were performed. A sequence approximately 200 bp near the binding sites of ADRB2 and C1RL-AS1 wild-type (WT) / mutant (MT) was synthesized and inserted into the pmirGLO vector containing the firefly luciferase and Renilla luciferase genes, with Renilla luciferase activity serving as a control. hsa-miR-424-5p mimics and negative control (NC) mimics were synthesized. The plasmids were transfected into 293T cells (SCC-120511, Beijing Solarbio Science & Technology Co., Ltd.) in the following combinations to detect the interaction between hsa-miR-424-5p and ADRB2 and C1RL-AS1: i) NC mimic + ADRB2-WT; ii) hsa-miR-424-5p mimic + ADRB2-WT; iii) NC mimic + ADRB2-MT; iv) hsa-miR-424-5p mimic + ADRB2-MT; v) NC mimic + C1RL-AS1-WT; vi) hsa-miR-424-5p mimic + C1RL-AS1-WT; vii) NC mimic + C1RL-AS1-MT; viiii) hsa-miR-424-5p mimic + C1RL-AS1-MT. Luciferase activity was measured using a dual-luciferase reporter assay kit (R41128).

[0055] 1.8. Animal Model Construction

[0056] Six-week-old male C57BL / 6 mice were randomly divided into two groups (n=5 per group) and housed under specific pathogen-free (SPF) conditions. ADRB2-overexpressing LLC cells with 80% confluence and control cells transfected with empty vector were collected and resuspended in ice-cold PBS (pH 7.4; Gibco; Thermo Fisher Scientific). 100 μl of PBS containing 4 × 10⁴ mg / L of the drug was injected using a 27-gauge insulin syringe (BD Biosciences, Charles River Laboratories, Inc.). 5A suspension of live cells was subcutaneously injected into the left back of mice. Mice were induced to be anesthetized by inhaling a mixture of 4% (v / v) isoflurane (Pfizer) and oxygen via a nasal cone, and then anesthesia was maintained with 1.5%-2% (v / v) isoflurane, with respiratory rate monitored throughout.

[0057] Starting from day 7 post-inoculation, tumor size was measured every two days using a digital caliper. The tumor volume was calculated using the formula: V = 0.5 × length × width² (mm³). When the tumor volume reached 2000 mm³ or the experimental endpoint (day 22), mice were deeply anesthetized with 5% isoflurane and euthanized by cervical dislocation. Mice were confirmed dead when spontaneous respiration ceased for more than 3 minutes, paw reflexes were lost, and pupils became fixed and dilated. A humane endpoint was reached if mice exhibited rapid weight loss, severe debilitating diarrhea, respiratory distress, bleeding from any body cavity, self-mutilation, or impaired mobility.

[0058] All animal experiments were approved by the Laboratory Animal Welfare and Ethics Committee of Hunan University of Traditional Chinese Medicine (Approval No.: A11051, Hunan, China) and were conducted in accordance with the American Veterinary Medical Association's guidelines for animal euthanasia.

[0059] 1.9. Flow cytometry

[0060] Immediately after mouse sacrifice, single-cell suspensions were prepared from fresh tumor tissue. Brief procedure: Tumor tissue was minced and enzymatically digested with collagenase IV (Millipore Sigma) at 37°C for 30 minutes. The resulting cell suspension was filtered through a 70 μm cell sieve, washed twice with PBS, and surface stained at 4°C for 30 minutes. CD4 cells were stained with fluorescently labeled antibodies. + T cells (CD45) + CD11b-CD45R-CD3 + CD4 + ) and dendritic cells (DCs; CD45) + CD11c + MHCII + Immunophenotypic analysis was performed using a Cytoflex LX flow cytometer (Beckman Coulter Ltd.), and immune cell population frequency data were analyzed using FlowJo X (10.0.7 R2 x64) software.

[0061] 1.10. Statistical Analysis

[0062] All quantitative data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using IBM SPSS Statistics 22.0 software, and graphs were generated using GraphPad Prism 8.0.1 software. Two-tailed unpaired t-tests were used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups, followed by a post-hoc LSD test. Statistical significance was defined as P < 0.05, where * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0063] 2. Results

[0064] 2.1. Expression analysis of ADRB2 in pan-cancer

[0065] Compared with normal tissue samples, ADRB2 was found in adrenocortical carcinoma (ACC), bladder cancer (BLCA), breast cancer (BRCA), cholangiocarcinoma (CHOL), colorectal cancer (COAD), and diffuse large B-cell carcinoma. Significantly downregulated in leukocytic lymphoma (DLBC), esophageal cancer (ESCA), head and neck squamous cell carcinoma (HNSC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), ovarian cancer (OV), rectal adenocarcinoma (READ), cutaneous melanoma (SKCM), gastric cancer (STAD), thyroid cancer (THCA), endometrial cancer (UCEC), and uterine sarcoma (UCS); significantly upregulated in glioblastoma (GBM), renal chromophobe carcinoma (KICH), renal papillary cell carcinoma (KIRP), acute myeloid leukemia (LAML), low-grade glioma (LGG), hepatocellular carcinoma (LIHC), pancreatic cancer (PAAD), pheochromocytoma and paraganglioma (PCPG), prostate cancer (PRAD), and testicular germ cell tumor (TGCT); but no significant difference was observed in cervical squamous cell carcinoma (CESC), renal clear cell carcinoma (KIRC), sarcoma (SARC), and thymoma (THYM). Figure 1 A). Further investigation of ADRB2 gene expression in cancer using the GEPIA database revealed decreased ADRB2 expression in BRCA, BLCA, CHOL, LUSC, LUAD, STAD, and UCEC. Figure 1 B), with no significant difference in expression among other cancer types.

[0066] In summary, ADRB2 was downregulated in BRCA, BLCA, CHOL, LUSC, LUAD, STAD, and UCEC, suggesting that it plays a tumor-suppressive role in these seven cancers.

[0067] Survival analysis of the ADRB2 gene in BRCA, BLCA, CHOL, LUSC, LUAD, STAD, and UCEC showed that lung adenocarcinoma patients with high ADRB2 expression had a better prognosis. Figure 2 A), while no prognostic value of ADRB2 was observed in patients with BLCA, BRCA, CHOL, LUSC, STAD, and UCEC. This result suggests that ADRB2 is a potential prognostic biomarker for patients with lung adenocarcinoma. Furthermore, experimental studies have shown that ADRB2 is significantly downregulated in lung adenocarcinoma cells, but not significantly changed in lung squamous cell carcinoma cells (LCCC). Figure 2 B).

[0068] 2.2. Biological functions of ADRB2

[0069] To clarify the biological function of ADRB2 in lung adenocarcinoma, the correlation between protein-coding genes and ADRB2 in lung adenocarcinoma was analyzed using the cor.test() function in R4.2 software. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) gene set enrichment analysis (GSEA) was performed on these genes. Figure 3 Enrichment results indicate that ADRB2 co-expressed genes are mainly involved in chemokine signaling pathways, calcium signaling pathways, actin cytoskeleton regulation, cell adhesion molecules, Jak-Stat signaling pathway, B cell receptor signaling pathway, and natural killer cell-mediated cytotoxicity.

[0070] 2.3. Multidimensional mechanisms of ADRB2 dysregulation and its upstream miRNA prediction

[0071] To elucidate the causes of ADRB2 dysregulation, a comprehensive analysis was conducted on factors related to ADRB2 expression, including genetic variations, DNA methylation, and related miRNAs. Copy number variations of ADRB2 were relatively common in lung adenocarcinoma patients, with a deletion rate of 34.81% and an amplification rate of 19.96%. Figure 4 A). Among them, copy number deletion is the genetic variant most closely associated with ADRB2 expression (R=0.29; P=2×10). -4 Copy number amplification was not significantly correlated with expression level. Figure 4 BD). ADRB2 lacks introns, and its various polymorphic forms, mutations, and downregulation are associated with a variety of diseases. Besides copy number, DNA methylation also affects gene expression. Figure 4 E), ADRB2 expression was negatively correlated with DNA methylation (R=-0.49; P=1.2×10), -6 In addition, miRNAs also play an important role in regulating mRNA expression.

[0072] Non-coding RNAs are known to regulate gene expression. To determine whether the ADRB2 gene is regulated by non-coding RNAs, we first predicted potential upstream miRNAs targeting ADRB2. A total of 15 potential miRNAs were identified (let-7a-5p, let-7b-5p, let-7c-5p, let-7i-5p, let-7e-5p, let-7g-5p, let-7f-5p, let-7d-5p, miR-15a-5p, miR-15b-5p, miR-16-5p, miR-98-5p, miR-195-5p, miR-424-5p, and miR-497-5p). A miRNA-ADRB2 regulatory network was constructed using Cytoscape software. Figure 5 A). Figure 5 B showed that miR-424-5p was significantly upregulated in lung adenocarcinoma. Based on the regulatory mechanism of miRNAs and target genes, miRNAs were negatively correlated with ADRB2. Subsequent expression correlation analysis revealed a negative correlation between ADRB2 and miR-424-5p (R = -0.108; P = 1.41 × 10⁻⁶). -2 ), and let-7a-5p (R=0.219; P=5.51×10), and let-7a-5p (R=0.219; P=5.51×10). -7 ), let-7b-5p (R=0.269; P=6.60×10 -10 ), let-7c-5p (R=0.392; P=2.66×10 -20 ), let-7f-5p (R=0.142; P=1.32×10 -3 ), let-7g-5p (R=0.149; P=7.03×10 -4 ), miR-195-5p (R=0.352; P=2.43×10 -16 ) and miR-497-5p (R=0.337; P=5.05×10) -15 It showed a positive correlation with miR-424-5p, but no statistically significant correlation with the other 7 predicted miRNAs. The expression of miR-424-5p in lung adenocarcinoma was detected. Figure 5 C). Dual-luciferase reporter gene assays showed that, compared with the NC mimic + ADRB2-WT transfection group, the relative luciferase activity of the miR-424-5p mimic + ADRB2-WT transfection group was significantly lower (P<0.01), while there was no significant difference among the ADRB2-MT groups. Finally, Western blotting experiments after miR-424-5p overexpression revealed a significant decrease in ADRB2 expression levels (P<0.01). Figure 5 D); These results indicate that miR-424-5p is the most likely regulatory miRNA of ADRB2 in lung adenocarcinoma.

[0073] 2.4. Prediction and Analysis of Long Non-coding RNA Upstream of miR-424-5p

[0074] Next, StarBase was used to predict upstream long non-coding RNAs of miR-424-5p, resulting in 73 potential long non-coding RNAs. Further analysis revealed that only STAG3L5P-PVRIG2P-PILRB (ENSG00000272752), C1RL-AS1 (ENSG00000205885), and GABPB1-AS1 (ENSG00000244879) were significantly upregulated in lung adenocarcinoma. Figure 6 A, B, and C). According to the competitive endogenous RNA (ceRNA) hypothesis, miRNAs should be negatively correlated with long non-coding RNAs, or mRNAs should be positively correlated with long non-coding RNAs. Using StarBase to detect the correlation between the expression of STAG3L5P-PVRIG2P-PILRB, C1RL-AS1, GABPB1-AS1, and miR-424-5p / ADRB2 in lung adenocarcinoma, we found that STAG3L5P-PVRIG2P-PILRB (R=-0.15; P=5.33×10⁻⁶) was significantly correlated with miR-424-5p / ADRB2. -4 ), C1RL-AS1 (R=-0.22; P=4.99×10 -7 ) and GABPB1-AS1 (R=-0.15; P=5.97×10) -4 The expression of all three was negatively correlated with miR-424-5p, while ADRB2 expression was positively correlated only with C1RL-AS1 (R=0.112; P=1.05×10). -2 The results of the dual-luciferase reporter gene assay also showed that, compared with the NC mimic + C1RL-AS1-WT transfection group, the relative luciferase activity of the miR-424-5p mimic + C1RL-AS1-WT transfection group was significantly reduced (P<0.01). Figure 6 (D) There were no significant differences among the ADRB2-MT groups. Interference with C1RL-AS1 revealed a significant decrease in ADRB2 expression levels. Figure 6 E); combined with expression and correlation analysis, it is suggested that C1RL-AS1 is an upstream long non-coding RNA of the miR-424-5p / ADRB2 axis in lung adenocarcinoma.

[0075] 2.5. Association between ADRB2 and immune cell infiltration in lung adenocarcinoma

[0076] ADRB2 encodes a β2-adrenergic receptor, belonging to the G protein-coupled receptor superfamily, and mediates the production of anti-inflammatory cytokines. We hypothesize that it may play an important role in the immune system. Analysis of ADRB2 expression levels in immune cell-infiltrated tumor tissues showed that the copy number status of ADRB2 significantly affected the level of immune cell infiltration in lung adenocarcinoma. Figure 7 and Figure 8 In lung adenocarcinoma, ADRB2 is present in B cells and CD8+ cells. + T cells, CD4 + Positive expression was observed in T cells, macrophages, neutrophils, and dendritic cells. Data from the GSM3516676 and GSE127465 datasets showed that ADRB2 is widely expressed in various immune cells, particularly in CD8 cells. + ADRB2 was significantly enriched in T cells and macrophages, consistent with the functional role identified by bulk RNA-seq analysis. Detailed comparisons revealed higher expression in activated macrophages, neutrophils, and dendritic cells, suggesting its involvement in innate and adaptive immune responses. Subsequently, the correlation between ADRB2 expression and immune-related genes (including chemokines, chemokine receptor proteins, and genes related to immune activation or inhibition) in lung adenocarcinoma was investigated. Analysis showed that ADRB2 was co-expressed with chemokine and chemokine receptor genes. Figure 9 (A and B), for example, CC motif chemokine receptor (CCR)4, CCR6, C-X3-C motif chemokine receptor 1, CC motif chemokine ligand (CCL)14, and CCL23 are strongly positively correlated with ADRB2 expression. ADRB2 expression is also significantly correlated with the expression of most immune activation and immunosuppression genes. Figure 9 (C and D). In the tumor immune microenvironment of lung adenocarcinoma, ADRB2 regulates immune cell infiltration and the function of immune-related genes.

[0077] 2.6. Association between ADRB2 expression and immune cell markers in lung adenocarcinoma

[0078] To further investigate the function of ADRB2 in tumor immunity, the TCGA dataset was used to determine the correlation between ADRB2 expression and some immune cell biomarkers in lung adenocarcinoma. The results showed that ADRB2 was associated with the B cell biomarker CD19 (R=0.23, P=5.5×10⁻⁶). -7 CD79A: R=0.19, P=2.4×10 -5 CD8 + T cell biomarkers (CD8A: R=0.14, P=1.4×10⁻⁶) -4 CD8B: R=0.14, P=2.4×10 -4 CD4 +T cell biomarkers (CD4: R=0.54, P=2.7×10⁻⁶) -38 M1 macrophage biomarker (NOS2: R=0.17, P=2.5×10⁻⁶) -5 IRF5: R=0.26, P=4.8×10 -9 PTGS2: R=0.15, P=9.4×10 -5 M2 macrophage biomarkers (CD163: R=0.35, P=8.7×10⁻⁶) -16 VSIG4: R=0.39, P=3.1×10 -19 ;MS4A4A: R=0.43, P=3.6×10 -23 ), neutrophil biomarkers (CEACAM8: R=0.42, P=4.5×10), -22 ITGAM: R=0.49, P=1.5×10 -30 CCR7: R=0.45, P=8.3×10 -26 ) and dendritic cell biomarkers (HLA-DPB1: R=0.56, P=7.6×10) -41 ;HLA-DQB1: R=0.36, P=1.6×10 -16 ;HLA-DRA: R=0.52, P=3.9×10 -34 ;HLA-DPA1: R=0.54, P=2.4×10 -37 CD1C: R=0.57, P=3×10 -42 NRP1: R=0.29, P=1.1×10 -10 ITGAX: R=0.39, P=1×10 -18 Both ADRB2 and dendritic cell biomarkers showed significant correlation in lung adenocarcinoma. The results of this study suggest that low ADRB2 expression may be associated with a stronger correlation with these biomarkers. These findings provide strong evidence that the ADRB2 gene is positively correlated with immune cell infiltration, and that low ADRB2 expression is associated with elevated levels of tumor-invasive neutrophils and dendritic cells in lung adenocarcinoma.

[0079] 2.7. ADRB2 overexpression inhibits tumor growth and regulates immune infiltration in lung adenocarcinoma mice.

[0080] To further investigate the role of ADRB2 in tumor growth and its effect on immune cell infiltration, a tumor model was established in C57BL / 6 mice, and lung adenocarcinoma cells (LLC) were stably transfected with ADRB2 overexpression. Figure 10A). Experimental results showed that, compared with control mice that received empty vector-transfected cells, mice transplanted with ADRB2-overexpressing LLC cells had significantly smaller tumor volumes (A). Figure 10 B, C, and D) suggest that ADRB2 overexpression significantly inhibits tumor growth (P<0.05).

[0081] To assess the impact of ADRB2 on the tumor immune microenvironment, CD4+ in mouse tumor tissue was analyzed. + Flow cytometry analysis was performed on T cells and dendritic cells. Results showed that CD4+ was present in tumor tissues of ADRB2-overexpressing mice. + The proportion of T cells increased significantly ( Figure 11 A), the proportion of dendritic cells also showed a similar increasing trend ( Figure 11 (B) This finding is consistent with results from public databases, suggesting that ADRB2 expression levels may regulate immune cell infiltration, thereby affecting the tumor microenvironment. These findings indicate that ADRB2 not only plays a crucial role in regulating tumor cell proliferation but may also alter the tumor microenvironment by influencing immune cell infiltration, thus positively impacting the prognosis of lung adenocarcinoma.

[0082] 2.8. Relationship between ADRB2 and immune checkpoints in lung adenocarcinoma

[0083] In immune checkpoint therapy, the main targets are programmed death protein 1 (PD-1), programmed death ligand 1 (PD-L1), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Therefore, this study used tumor immune assessment resources to analyze the relationship between ADRB2 and PD-1, PD-L1, and CTLA-4. Figure 12 (A, B, and C). After purity correction, PD-L1 and ADRB2 showed a significant positive correlation (R = 0.215; P = 1.45 × 10⁻⁶). -6 CTLA-4 and ADRB2 were also significantly positively correlated (R=0.152; P=7.21×10). -4 ) Validation using TCGA data revealed that ADRB2 and PD-1 (R=0.13; P=5.7×10) -3 ), PD-L1 (R=0.28; P=3.6×10 -10 ) and CTLA-4 (R=0.27; P=2.1×10) -9 All showed a significant positive correlation ( Figure 12 (D, E, and F). The results suggest that ADRB2 is involved in the immunotherapy of lung adenocarcinoma.

[0084] 2.9. Drug Sensitivity Analysis of ADRB2

[0085] To clarify the relationship between ADRB2 and drug sensitivity, the drug sensitivity of ADRB2 was analyzed using the GDSC drug database. The results showed that ADRB2 was associated with responses to multiple drugs. Patients with high ADRB2 expression may be more sensitive to afatinib, docetaxel, gefitinib, and bleomycin, but resistant to navitoclax, SB52334, NPK76-II-72-1, YM201636, and KIN001-102. Afatinib, docetaxel, gefitinib, WZ-1-84, and bleomycin are all components of classic chemotherapy regimens for lung adenocarcinoma. Among the top 30 drugs, patients with high ADRB2 expression may be sensitive to 21 drugs and resistant to 9 drugs. Figure 13 This finding suggests that the association between ADRB2 expression and drug sensitivity is closely related to poor prognosis in lung adenocarcinoma.

[0086] 3. Conclusion

[0087] This study systematically elucidates the expression characteristics, regulatory mechanisms, and functional value of β2-adrenergic receptor (ADRB2) in lung adenocarcinoma (LUAD). The core findings are as follows:

[0088] 1. ADRB2 Expression and Prognostic Significance: ADRB2 is significantly downexpressed in LUAD tissues and cell lines, and its low expression is closely related to shortened overall survival, making it a potential prognostic biomarker for LUAD. Pan-cancer analysis shows that ADRB2 is downregulated in seven cancers, including invasive breast cancer and urothelial carcinoma of the bladder, suggesting its broad-spectrum tumor-suppressing potential, but it only shows clear prognostic value in LUAD.

[0089] 2. Multidimensional Regulatory Mechanisms of ADRB2 Dysregulation: Low expression of ADRB2 in LUAD is regulated by multiple mechanisms: at the genetic level, the copy number loss rate of ADRB2 reaches 34.81%, and copy number loss is significantly correlated with downregulation of expression; at the epigenetic level, ADRB2 expression is strongly negatively correlated with DNA methylation (R=-0.49; P=1.2×10⁻⁶). -6 At the non-coding RNA level, a novel regulatory axis of C1RL-AS1 / hsa-miR-424-5p / ADRB2 was identified. hsa-miR-424-5p can directly target the 3' uncoding region of ADRB2 to inhibit its expression, while the long non-coding RNA C1RL-AS1 relieves the post-transcriptional inhibition of ADRB2 by competitively binding to hsa-miR-424-5p. This regulatory relationship was confirmed by dual-luciferase reporter gene assays.

[0090] 3. Dual Functions and Applications of ADRB2: Functional experiments confirmed that ADRB2 has both tumor-suppressive and immunomodulatory effects: In vivo experiments showed that ADRB2 overexpression significantly inhibited LUAD tumor growth (P<0.05); in terms of immunomodulation, ADRB2 overexpression promoted CD4+ expression. + T cells and dendritic cells infiltrate tumor tissue, and their expression is similar to that of B cells and CD8. + Biomarkers of immune cells such as T cells and macrophages showed a significant positive correlation, as did immune checkpoint markers such as PD-1, PD-L1, and CTLA-4 (P<0.05), providing a basis for the combined use of ADRB2-targeted therapy and immune checkpoint inhibitors. Drug sensitivity analysis showed that LUAD patients with high ADRB2 expression were more sensitive to classic chemotherapy drugs such as afatinib, docetaxel, and gefitinib, providing molecular clues for personalized drug selection.

[0091] 3. Innovation and Translational Potential of the Core Regulatory Axis: This invention is the first to clearly identify the C1RL-AS1 / hsa-miR-424-5p / ADRB2 axis as a key pathway for the regulation of ADRB2 expression in LUAD, filling a research gap in this field. Based on this pathway, novel targeted drugs such as C1RL-AS1 overexpression vectors, hsa-miR-424-5p inhibitors, and ADRB2 agonists can be developed. Simultaneously, by combining ADRB2 expression levels, copy number variations, and DNA methylation status, early diagnostic reagents and prognostic assessment kits for LUAD can be developed, providing multi-dimensional support for precise screening, prognostic stratification, and personalized treatment of the disease.

[0092] In summary, this study revealed the antitumor effect and immune regulatory function of ADRB2 in LUAD, and clarified the regulatory mechanism of the C1RL-AS1 / hsa-miR-424-5p / ADRB2 axis. Figure 14 This provides important theoretical basis and technical support for the diagnosis, prognostic assessment and development of novel immunomodulatory therapy strategies for LUAD.

[0093] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. Application of the C1RL-AS1 / hsa-miR-424-5p / ADRB2 axis in the preparation of immunomodulatory therapeutic drugs for lung adenocarcinoma.

2. Application of a C1RL-AS1 overexpression-related agent in the preparation of an immunomodulatory therapeutic drug for lung adenocarcinoma.

3. The application of an hsa-miR-424-5p inhibitor in the preparation of immunomodulatory therapeutic drugs for lung adenocarcinoma, characterized in that, The inhibitory agent is selected from hsa-miR-424-5p antagonists, antisense oligonucleotides, or lentiviral inhibitory vectors.

4. The application of ADRB2 overexpression vector in the preparation of immunomodulatory therapeutic drugs for lung adenocarcinoma, characterized in that, The overexpression vector contains an ADRB2 coding sequence; the overexpression vector is pLV17-EF1α-ADRB2-Luciferase17-Puro.

5. Application of ADRB2 agonists in the preparation of immunomodulatory therapeutic drugs for lung adenocarcinoma.

6. The application according to claim 5, characterized in that, The ADRB2 agonist is used in combination with a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

7. An immunomodulatory therapeutic drug for lung adenocarcinoma, characterized in that, The drug comprises at least one active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is selected from: (1) a C1RL-AS1 overexpression carrier; (2) an hsa-miR-424-5p inhibitor; (3) an ADRB2 overexpression carrier or an ADRB2 agonist, and the drug dosage form is an injection, a lyophilized powder for injection or a sustained-release formulation.

8. The application of ADRB2 in the preparation of a prognostic assessment reagent for lung adenocarcinoma, characterized in that, The reagent is used to detect the expression level of ADRB2 in tumor tissue or peripheral blood, and the detection includes nucleic acid level detection or protein level detection.

9. A personalized immunotherapy guidance reagent for lung adenocarcinoma, characterized in that, The reagent is used to detect the expression level of ADRB2 in tumor tissue or peripheral blood mononuclear cells to determine the patient's sensitivity to immunotherapy drugs for lung adenocarcinoma.

10. The application of ADRB2 in screening immunomodulatory therapeutic drugs for lung adenocarcinoma, characterized in that, Based on ADRB2 expression levels, protein activity, or CD4 + Indicators related to the infiltration of T cells and dendritic cells are used as detection indicators.

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