Application of ADAM32 gene in diagnosis and treatment of lung adenocarcinoma

Abstract

This invention relates to the application of the ADAM32 gene in the diagnosis and treatment of lung adenocarcinoma, and more particularly to the application of a biomarker detection reagent in the preparation of products for diagnosing lung adenocarcinoma, wherein the biomarker is the ADAM32 gene. This invention establishes for the first time the relationship between the ADAM32 gene and lung adenocarcinoma, demonstrating that inhibiting ADAM32 expression can delay the progression of lung adenocarcinoma and prolong patient lifespan. This invention provides an effective new approach for the treatment of lung adenocarcinoma.

Description

Technical Field

[0001] This invention relates to the field of medical technology, specifically to the application of the ADAM32 gene in the diagnosis and treatment of lung adenocarcinoma. Background Technology

[0002] Lung tumors originate from the bronchial mucosal epithelium and can be classified into small cell lung cancer and non-small cell lung cancer. Non-small cell lung cancer is further divided into squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. Small cell lung cancer accounts for approximately 15%-20% of bronchogenic lung cancers. At diagnosis, about 30% of small cell lung cancer patients have tumors in the limited stage, while the rest are in the extensive stage. Extensive stage refers to tumors that have spread beyond the supraclavicular region. Compared to small cell carcinoma, non-small cell lung cancer cells grow and divide more slowly, and metastasis occurs relatively later. Non-small cell lung cancer accounts for approximately 80% of all lung cancers, and about 75% of patients are diagnosed at an intermediate or advanced stage, with a very low 5-year survival rate. Lung adenocarcinoma is one of the most common malignant tumors. Unlike squamous cell lung cancer, lung adenocarcinoma is more likely to occur in women and non-smokers. It originates from the bronchial mucosal epithelium, and a few originate from the mucous glands of the large bronchi. Its incidence is lower than squamous cell carcinoma and undifferentiated carcinoma, with a younger age of onset, and it is relatively more common in women. Most adenocarcinomas originate from smaller bronchi and are peripheral lung cancers. In its early stages, it generally presents with no obvious clinical symptoms and is often discovered during chest X-ray examinations. It manifests as a round or oval mass, typically growing slowly, but sometimes hematogenous metastasis occurs early. Lymphatic metastasis occurs later. Traditional drugs targeting known targets are no longer sufficient to meet treatment needs, necessitating the exploration of new therapeutic targets.

[0003] The disintegrin and metalloproteinase domain-containing protein 32 (ADAM32; UniProt ID: (Human)Q8TC27; Entrez Gene ID: (Human)203102) gene is a protein-coding gene. ADAM32 belongs to the ADAM protein family and is a transmembrane protein containing a binding domain for metalloproteinases and integrins in its extracellular space; it is a catalytic-like protein. ADAM32 is highly expressed in the testes and may play a role in sperm development and fertilization. Summary of the Invention

[0004] In response to the current clinical need for drugs targeting novel targets for the prevention and treatment of lung adenocarcinoma, the purpose of this invention is to provide the use of ADAM32 in screening drugs for the treatment of lung adenocarcinoma, which can effectively solve the problem of difficulty in screening suitable drugs for the prevention and treatment of lung adenocarcinoma in the prior art.

[0005] This invention provides, in one aspect, the application of a biomarker detection reagent in the preparation of products for diagnosing lung adenocarcinoma, wherein the biomarker is the ADAM32 gene. This invention establishes for the first time the relationship between the ADAM32 gene and lung adenocarcinoma, demonstrating that inhibiting ADAM32 expression can delay the progression of lung adenocarcinoma and prolong patient lifespan. This invention provides an effective new approach for the treatment of lung adenocarcinoma.

[0006] In some embodiments, the biomarker detection reagent is a reagent used to determine the expression level of the ADAM32 gene.

[0007] In some embodiments, the reagents for determining the expression level of the ADAM32 gene include reagents for detecting the messenger RNA (mRNA) or fragments thereof of the ADAM32 gene, reagents for detecting the complementary DNA (cDNA) or fragments thereof of the ADAM32 gene, and reagents for detecting the protein or variants thereof encoded by the ADAM32 gene.

[0008] In some implementations, the product is selected from chips, reagent kits, or test strips.

[0009] In another aspect, this invention provides the application of a reagent for inhibiting the expression of a biomarker, namely the ADAM32 gene, in the preparation of a drug for treating lung adenocarcinoma. This invention establishes for the first time the relationship between the ADAM32 gene and lung adenocarcinoma, demonstrating that inhibiting ADAM32 expression can slow the progression of lung adenocarcinoma and prolong patient lifespan. This invention provides an effective new approach for the treatment of lung adenocarcinoma.

[0010] In some embodiments, the reagents for inhibiting marker expression include siRNA specific to the ADAM32 gene, antisense oligonucleotides specific to the ADAM32 gene, ribozymes specific to the ADAM32 gene, and inhibitory antibodies against the ADAM32 gene.

[0011] In some embodiments, the inhibitory antibody against the ADAM32 gene is a monoclonal antibody, a polyclonal antibody, or a multispecific antibody. In some embodiments, the inhibitory antibody against the ADAM32 gene is a polyclonal antibody, and the polyclonal antibody antigen is CHO-derived recombinant human ADAM32 Ser17-Thr476. In some embodiments, the antibody is Thermo Fisher Scientific's ADAM32 antibody (catalog number: PA5-47731).

[0012] In another aspect, the present invention provides a drug screening system, the drug screening system comprising: (1) a detection module for detecting the inhibitory effect of a drug on the expression of the ADAM32 gene; and (2) an analysis module for outputting the therapeutic effect of the drug on lung adenocarcinoma based on the data output by the detection module.

[0013] In another aspect, the present invention provides a lung adenocarcinoma diagnostic system, the diagnostic system comprising: (1) a detection device, wherein the detection device is used to detect the expression level of the ADAM32 gene; and (2) an analysis device, wherein the analysis device is used to output a diagnostic result of lung adenocarcinoma based on the ADAM32 gene expression level data output by the detection device.

[0014] This invention offers the following advantages and effects compared to existing technologies: It establishes for the first time the relationship between ADAM32 and lung adenocarcinoma, demonstrating that inhibiting ADAM32 can prevent, alleviate, or treat lung adenocarcinoma. Based on the close association between ADAM32 and lung adenocarcinoma, it can serve as a novel target for drugs treating lung adenocarcinoma, thereby enabling the screening of suitable new treatments or drugs for lung adenocarcinoma and effectively addressing the difficulty in drug screening in existing technologies. Inhibitors of ADAM32 can be used to prepare drugs for treating lung adenocarcinoma. Attached Figure Description

[0015] Figure 1 This is the survival analysis result of the ADAM32 gene in the TCGA-HNSC dataset of this invention;

[0016] Figure 2 This invention verifies the interference effect of the interference fragment of the ADAM32 gene in NCI-H1299 cells (quantitative real-time PCR).

[0017] Figure 3 This is the experimental result of the effect of ADAM32 gene interference on the proliferation of NCI-H1299 cells in this invention;

[0018] Figure 4 This is the experimental result of the effect of ADAM32 gene interference on the proliferation of NCI-H1395 cells in this invention;

[0019] Figure 5 This is the experimental result of the effect of ADAM32 gene interference on NCI-H1299 cell scratching in this invention;

[0020] Figure 6 This is the experimental result of the effect of ADAM32 gene interference on NCI-H1395 cell scratching in this invention;

[0021] Figure 7 This is the experimental result of the effect of ADAM32 gene interference on apoptosis in NCI-H1395 cells according to the present invention;

[0022] Figure 8 This is the experimental result of the impact of ADAM32 gene interference on the NCI-H1395 cell cycle in this invention;

[0023] Figure 9 This is the experimental result of the effect of ADAM32 gene interference on NCI-H1395 cell invasion according to the present invention;

[0024] Figure 10 The results of the invention are the results of the IC50 assay of the ADAM32 gene in NCI-H1395 cells inhibiting proliferation by incubation with different concentrations of antibody.

[0025] Figure 11 This is the experimental result of how the ADAM32 gene affects cell migration in NCI-H1395 cells after incubation with 10 μg / mL antibody according to the present invention;

[0026] Figure 12 This is the experimental result of how the ADAM32 gene of this invention affects cell invasion in NCI-H1395 cells after incubation with 10 μg / mL antibody;

[0027] Figure 13 Preliminary mechanism analysis - results of flow cytometry detection of ROS;

[0028] Figure 14 This is a preliminary mechanism analysis – the results of flow cytometry detection of mitochondrial membrane potential. Detailed Implementation

[0029] To better illustrate the objectives, technical solutions, and advantages of the present invention, the invention will be further described below with reference to the accompanying drawings and specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0030] Ohnolog gene

[0031] With the development of evolutionary biomedicine, accumulated evolutionary knowledge has been successfully applied to elucidating the pathogenesis of various diseases and identifying pathogenic genes. Since gene evolution is closely related to the occurrence and progression of many diseases (especially cancer), multiple studies have shown that successful drug targets often share common evolutionary characteristics. Whole genome duplication is generally considered a significant evolutionary event in vertebrates. The human ancestral genome underwent two whole genome duplications during the early vertebrate period. To commemorate Ohno's outstanding contributions to related research, genes associated with these two whole genome duplication events are called "Ohnologs." Ohnolog genes play an important role in the development and transcriptional regulation of organisms. Previous studies have shown that due to the high dose sensitivity of Ohnolog genes (phenotype changes occur with changes in gene copy number), they are closely related to the occurrence and development of human phenotypes or diseases (including cancer). Because a qualified drug target gene must be closely related to the disease phenotype and sensitive to drug stimulation, the dose-sensitive nature of Ohnolog genes makes them promising drug targets. Furthermore, it is well known that cancer cells can escape cell division and programmed cell death control through "rapid" evolution, leading to rapid cancer spread. Numerous studies have also discovered that cancer-related genes have unique evolutionary origins. Therefore, tracing the origins of cancer genes and inhibiting or slowing their evolutionary process is an effective approach to reduce cancer cell adaptability and thus combat cancer progression. Studies have shown that cancer driver genes are significantly enriched in genes originating from four stages: cellular organisms, eukaryotes, opisthokonta, and eumetazoans. Our research team discovered that ADAM32 not only belongs to the Ohnolog gene family but also originates from the eumetazoan origin stage associated with cancer. However, there are currently no reports on the association of ADAM32 with lung adenocarcinoma or its use in screening drugs for the prevention and treatment of lung adenocarcinoma.

[0032] Example 1. Survival analysis results of the ADAM32 gene in the TCGA-HNSC dataset

[0033] Specifically, the process includes the following:

[0034] (1) Analysis of the evolutionary characteristics of the ADAM32 gene: The results show that ADAM32 belongs to the Ohnolog gene; ADAM32 originated from the evolutionary stage of the eumeteozoic origin. ADAM32 conforms to the evolutionary characteristics of cancer genes.

[0035] (2) By querying the DriverDBv3 database of cancer driver genes, ADAM32 is a cancer driver gene.

[0036] (3) By querying the TissGDB database of cancer tissue-specific expression genes, ADAM32 is a cancer tissue-specific expression gene and is specifically highly expressed in cancer tissues.

[0037] (4) By querying the UniProt database, the subcellular location of the ADAM32 encoded protein is the cell membrane.

[0038] (5) Download multi-omics data of lung adenocarcinoma patients (TCGA-HNSC) from the TCGA database (The Cancer Genome Atlas, https: / / www.cancer.gov / tcga). The data includes patient gene expression data and clinical data. The clinical data includes survival status and survival time. The gene expression data is the gene expression level FPKM obtained by RNA-seq sequencing.

[0039] (6) The TCGA-HNSC data from step (5) were preprocessed to obtain the gene expression matrix of human genes in lung adenocarcinoma patients, and a Cox proportional hazards regression model was established to correlate the FPKM expression level of the ADAM32 gene with the prognostic survival time of patients. The results showed that the FPKM expression level of the ADAM32 gene was significantly correlated with the survival time of lung adenocarcinoma patients (P-value = 4.77E-03), and the hazard ratio = 6.6679. Figure 1 This result indicates that high expression of ADAM32 is a risk factor threatening the survival time of patients with lung adenocarcinoma, meaning that inhibiting ADAM32 can alleviate lung adenocarcinoma recurrence and prolong patient life.

[0040] Example 2.

[0041] Three siRNAs and one NC sequence targeting the ADAM32 gene were designed and synthesized, as shown in Table 1. Human cell lines were transfected, and 24 hours later, blank cells were added to form a total of 5 groups with no replicates. The optimal sequence ADAM32-Homo-738 was selected using PCR. Figure 2NCI-H1299 and NCI-H1395 cells were selected and revived using NCI-H1299 and NCI-H1395 cell culture media, respectively. When the cell density reached 80%, the cells were passaged. Next, cells in the logarithmic growth phase and in good growth condition were used for cell transfection experiments, and parallel controls were set up. NCI-H1299 cell group: (1) NCI-H1299 cell normal control, (2) NCI-H1299 cell + NC siRNA (NC group), (3) NCI-H1299 cell + ADAM32-Homo-738 (ADAM32 siRNA); NCI-H1395 cell group: (1) NCI-H1395 cell normal control, (2) NCI-H1395 cell + NC siRNA (NC group), (3) NCI-H1395 cell + ADAM32-Homo-738 (ADAM32 siRNA). Cell CCK8 assay and cell scratch assay were performed 24 h after transfection. NCI-H1299 (human non-small cell lung cancer cells) were purchased from Procell Biotech. NCI-H1299 cells were derived from a lymph node metastasis in a patient who had received initial radiotherapy. NCI-H1299 cells uniformly exhibit partial deletion of p53 protein and lack p53 protein expression. NCI-H1299 cells can synthesize NMB protein at 0.1 pmol / mg protein, but do not synthesize gastrin-releasing peptide (GRP). NCI-H1395 cells (human lung adenocarcinoma cells) were purchased from Procell Biotech. NCI-H1395 cells were established in April 1986; the tissue provider was a smoker (15 packs per year). NCI-BL1395 [BL1395] cells are a lymphoblastoid cell line derived from the same patient.

[0042] Table 1

[0043]

[0044] CCK8 assay: After cell culture, add 10 μL of CCK8 to each well and incubate at 37℃ for 1-2 h; measure the absorbance (OD) of each well using a microplate reader. 450 The experimental results are as follows: Figure 4 , Figure 5 .

[0045] Cell scratch assay: Two parallel control groups were conducted using the two cell lines described above. Using a marker pen, and guided by a ruler, evenly drew horizontal lines across the back of the 6-well plate, approximately every 0.5–1 cm, passing through the wells with 3 lines per well. Cells were then digested with 0.25% trypsin at a concentration of 2 × 10⁶ cells per well. 6Cells were seeded into 6-well plates and cultured at 37°C and 5% CO2 saturated humidity for 24 hours. When the cell density reached approximately 90%, covering about the bottom of the 6-well plate, a pipette tip was used to make incisions perpendicular to the horizontal line on the back of the plate, using the same pipette tip for each well. The cells were washed three times with PBS to remove the incised cells, and serum-free medium was added. A 0-hour photograph was taken at the same time. The plate was then placed in a 37°C, 5% CO2 incubator and cultured for 24 hours. Photos were provided: 100x magnification, 3 photos per group. The results of the scratch inhibition experiment were compiled as follows: Figure 5 and 6 As shown.

[0046] Experimental results showed that NCI-H1299 and NCI-H1395 cells that underwent interference / inhibition of the ADAM32 gene had reduced cell proliferation capacity (e.g., Figure 3 and 4 (as shown) and migration capabilities (such as) Figure 5 and 6 As shown in the figure, the levels were all reduced compared to control cells where the ADAM32 gene was not interfered with.

[0047] NCI-H1395 cells were removed from liquid nitrogen and quickly placed in a 37°C water bath. The cryovials were gently shaken to thaw the cryopreservation solution. After thawing, the cells were transferred to centrifuge tubes containing 5 ml of culture medium, centrifuged to collect the cells, and centrifuged at 1000 rpm for 5 min at room temperature. The supernatant was discarded. The cells were resuspended in complete culture medium containing 10% fetal bovine serum, seeded into culture dishes, and gently mixed by pipetting. The cells were cultured at 37°C and 5% CO2 saturated humidity. For cell passage and expansion culture, when the cell density reached 80%, the cells were passaged, the culture medium was discarded, and the cells were washed once with PBS. 1-2 ml of 0.25% trypsin was added to digest the cells. Under a microscope, digestion was observed for 1-2 min until the cells separated and became rounded, indicating that digestion was complete. The trypsin was quickly discarded, complete culture medium was added, and the cells were pipetted to prepare a single-cell suspension. The cells were passaged at a ratio of 1:3 and expanded cultured at 37°C and 5% CO2 saturated humidity. Cell treatment: NCI-H1395 cells in the logarithmic growth phase and in good growth condition were prepared into a single-cell suspension using 1640 medium and cultured at a density of 2 × 10⁶ cells per well. 5 The cells were evenly seeded into 6-well plates and incubated overnight at 37°C and 5% CO2 saturated humidity. Two hours before transfection, the medium was replaced with serum-free 1640 medium. Transfection was performed according to the following experimental groups:

[0048] 1) NCI-H1395 cell control group;

[0049] 2) ADAM32 siRNA interference control group;

[0050] 3) ADAM32 siRNA interference group;

[0051] Transfection Procedure: For each transfected sample, prepare as follows: Dilute 10 μL of the small fragment (concentration 20 μM) with 100 μL of serum-free opti-MEM, gently mix with a pipette tip, and incubate at room temperature for 5 minutes; before use, gently mix Lipofectamine™ 2000, then take 5 μL of Lipofectamine™ 2000 and dilute it in 100 μL of opti-MEM, and incubate at room temperature for 5 minutes; after incubating at room temperature for 5 minutes, mix Lipofectamine™ 2000 and the dilution buffer of the small fragment (total volume 200 μL), gently mix, and incubate at room temperature for 20 minutes; add 200 μL of the mixture to each well, and gently shake the cell culture plate back and forth to mix the mixture with the culture medium in the plate; incubate the cells in a 37℃ 5% CO2 incubator, and after 6 h, aspirate the transfection solution and replace it with normal culture medium; continue incubation in a 37℃ 5% CO2 incubator for 24 h before subsequent detection.

[0052] Apoptosis detection: Cells were digested with 0.25% trypsin without EDTA. After digestion, the processed cells were collected, centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in PBS. Cells were washed twice with PBS and centrifuged at 1500 rpm for 5 min. Apoptosis was detected using the Annexin V-APC / 7-AAD apoptosis detection kit: 5 μl of 7-AAD staining solution was added to 50 μl of Binding Buffer and mixed. The collected cells were then mixed with the above 7-AAD staining solution and incubated at room temperature in the dark for 5–15 min. After the reaction, 450 μl of Binding Buffer was added and mixed. 5 μl of Annexin V-APC was added and mixed. The reaction was incubated at room temperature in the dark for 5–15 min. Flow cytometry analysis was performed, and the experimental results are shown below. Figure 7 As shown.

[0053] Cell cycle detection: Pre-treated cells were digested with 0.25% trypsin (without EDTA). After digestion, cells were collected and incubated at 1000 rpm for 5 min. The supernatant was discarded, and the cells were resuspended in PBS and washed twice. Cells were then incubated at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 100 μl of PBS. 700 μl of pre-chilled 80% ethanol was slowly added to bring the final ethanol concentration to 70%. Cells were fixed at 4℃ for at least 4 h. Cells were then incubated at 1000 rpm for 5 min and washed twice with pre-chilled PBS. 100 μl of RNase (50 μg / ml) was added, and the cells were incubated at 37℃ for 30 min. 400 μl of PI (50 μg / ml) was added, and the cells were stained at 4℃ in the dark for 30 min. Flow cytometry analysis was performed. The experimental results are shown in Table 2 and below. Figure 8 As shown.

[0054] Table 2

[0055]

[0056] NCI-H1395 cells were selected and resuscitated. When the cell density reached 80%, the cells were passaged. Next, NCI-H1395 cells in the logarithmic growth phase and in good growth condition were collected and prepared into single-cell suspensions using 1640 medium. Cells were then distributed at a density of 2 × 10⁶ cells per well. 5 The cells were evenly seeded into 6-well plates and incubated overnight at 37°C and 5% CO2 saturated humidity. Two hours before transfection, the medium was replaced with serum-free 1640 medium. Transfection was performed according to the following experimental groups:

[0057] 1) Normal cell control group;

[0058] 2) ADAM32 siRNA interference control group;

[0059] 3) ADAM32 siRNA interference group;

[0060] 4) ADAM32 antibody concentration group: 10ug / mL (treatment for 24h);

[0061] Transfection Procedure: For each transfected sample, prepare as follows: Dilute 10 μL of the small fragment (concentration 20 μM) with 100 μL of serum-free opti-MEM, gently mix with a pipette tip, and incubate at room temperature for 5 minutes; before use, gently mix Lipofectamine™ 2000, then take 5 μL of Lipofectamine™ 2000 and dilute it in 100 μL of opti-MEM, and incubate at room temperature for 5 minutes; after incubating at room temperature for 5 minutes, mix Lipofectamine™ 2000 and the dilution buffer of the small fragment (total volume 200 μL), gently mix, and incubate at room temperature for 20 minutes; add 200 μL of the mixture to each well, and gently shake the cell culture plate back and forth to mix the mixture with the culture medium in the plate; incubate the cells in a 37℃ 5% CO2 incubator, and after 6 h, aspirate the transfection solution and replace it with normal culture medium; continue incubation in a 37℃ 5% CO2 incubator for 24 h before subsequent detection.

[0062] Take the processed NCI-H1395 cells, wash them with 3 ml of PBS, digest them with 0.25% trypsin, centrifuge at 1000 rpm for 5 min, discard the supernatant, wash twice with PBS to remove residual serum. Resuspend the cells in serum-free 1640 medium, count them using a cell counting chamber, and dilute the cell concentration to 3 × 10⁻⁶ cells in serum-free 1640 medium. 5 / ml, for later use. Thaw Matrigel at 4°C one day in advance; pre-chill the transwell chamber, 24-well plate, and pipette tip overnight at -20°C; dilute Matrigel to a final concentration of 1 mg / ml with serum-free medium, and operate on ice; add 800 μl of pre-chilled 10% FBS at 4°C to the 24-well plate. 1640 medium (containing antibiotics) was used to prepare transwell chambers. 100 μl of Matrigel (final concentration 1 mg / ml) was vertically added to the center of the bottom of the upper chamber. The chambers were incubated at 37°C for 4–5 h until they dried into a gel. After the Matrigel dried, 200 μl of each group's cell suspension was inoculated into the upper chamber of the transwell. The cells were then incubated at 37°C in a 5% CO2 incubator for 24 h. The transwells were removed, and the chambers were carefully washed once with PBS. Cells were fixed with 70% ice-cold ethanol solution for 1 h. The cells were stained with 0.5% crystal violet solution, incubated at room temperature for 20 min, washed with PBS, and the unmigrated cells on one side of the upper chamber were wiped clean with a clean cotton ball. Microscopic observation and photography were then performed. The experimental results regarding the effect of interference on invasion are as follows: Figure 9 As shown.

[0063] NCI-H1395 cells were selected and resuscitated. When the cell density reached 80%, the cells were passaged. Next, cells in the logarithmic growth phase and in good growth condition were seeded into plates at a concentration of 5 × 10⁻⁶ cells / cells. 3 Cells were inoculated into 96-well plates, with a control group included. Cells were incubated overnight at 37°C (100 μl of sterile PBS was added to the wells surrounding the cells). Cells were treated according to the following groups: NCI-H1395 normal group; NCI-H1395 cells + ADAM32 antibody 1.5625 μg / mL; NCI-H1395 cells + ADAM32 antibody 3.125 μg / mL; NCI-H1395 cells + ADAM32 antibody 6.25 μg / mL; NCI-H1395 cells + ADAM32 antibody 12.5 μg / mL; NCI-H1395 cells + ADAM32 antibody 25 μg / mL; NCI-H1395 cells + ADAM32 antibody 50 μg / mL. Incubation time: 24 h. For cell CCK8 assay, after the required cell culture time, add 10 μl of CCK8 to each well and incubate at 37℃ for 1-4 h; measure the absorbance (OD 450) of each well using a microplate reader.

[0064] Cell scratch assay: Using a marker pen, draw evenly spaced horizontal lines on the back of a 6-well plate, approximately every 0.5–1 cm, passing through the wells, with 3 lines per well; digest cells with 0.25% trypsin at a concentration of 2 × 10⁶ cells per well. 6Cells were seeded in six-well plates and cultured at 37°C and 5% CO2 saturated humidity for 24 hours. When the cell density reached approximately 90%, covering about the bottom of the six-well plate, a pipette tip was used to make incisions perpendicular to the horizontal line on the back of the plate, using the same pipette tip for incisions between different wells. Cells were washed three times with PBS to remove the incised cells, and serum-free medium was added. A 0-hour photograph was taken simultaneously. The cells were then treated as follows: 1) NCI-H1395 cells (normal control); 2) NCI-H1395 cells + ADAM32 antibody (10 μg / mL). The cells were incubated at 37°C and 5% CO2 for 24 hours, and photographed. Photos are provided: 100x magnification, 3 photos per set. The ADAM32 Antibody (PA5-47731) was purchased from Thermo Fisher Scientific and is a polyclonal antibody for antigen immunization. The antigen is CHO-derived recombinant human ADAM32 Ser17-Thr476.

[0065] The cell invasion assay procedure is the same as above. The effect of antibody incubation on invasion is as follows: Figure 12 As shown.

[0066] Experimental results showed that the proliferation rate of lung cancer cells incubated with ADAM32 antibody decreased, and the IC50 results were as follows: Figure 10 As shown; incubation with an antibody concentration of 10 μg / mL resulted in a decrease in the proliferation rate of NCI-H1395 cells of approximately 20% after incubation with ADAM32 antibody. Figure 10 The migration rate of NCI-H1395 cells after incubation with ADAM32 antibody decreased by approximately 20%. Figure 11 The invasion ability of NCI-H1395 cells after incubation with ADAM32 antibody decreased by approximately 20%. Figure 12 The specific ADAM32 antibody can inhibit the proliferation, migration, and invasion of lung adenocarcinoma, thus achieving the effect of treating lung adenocarcinoma.

[0067] Next, we conducted a preliminary analysis of the target mechanism. Through RNA-seq transcriptome sequencing before and after ADAM32 gene interference, and KEGG pathway enrichment analysis, we found that ADAM32 gene interference led to abnormal intracellular oxidative phosphorylation pathways, upregulation of genes involved in mitochondrial oxidative phosphorylase, respiratory electron transport, and chemiosmotic coupling to ATP synthesis, and preliminarily analyzed whether this was cancer cell death mediated by mitochondrial apoptosis. Mitochondrial apoptosis refers to the opening of mitochondrial membrane permeability transition pores mediated by reactive oxygen species, resulting in structural changes, rupture, and fragmentation of the mitochondrial membrane system, with severe consequences leading to cell death. To preliminarily prove our hypothesis, we used flow cytometry to detect ROS and mitochondrial membrane potential to preliminarily explain the apoptosis mechanism, and conducted cellular ROS detection experiments and JC-1 cell experiments.

[0068] Remove NCI-H1395 cells from liquid nitrogen and quickly place them in a 37°C water bath. Gently shake the cryovial to thaw the cryopreservation solution. After thawing, transfer the cells to a centrifuge tube containing 5 ml of culture medium, centrifuge to collect the cells, centrifuge at 1000 rpm for 5 min at room temperature, and discard the supernatant. Resuspend the cells in complete culture medium containing 10% fetal bovine serum, seed them into culture dishes, gently pipette to mix, and culture at 37°C under saturated humidity of 5% CO2.

[0069] Cell passage and scale-up culture:

[0070] When the cell density reaches 80%, passage the cells: discard the culture medium and wash once with PBS; add 1-2 ml of 0.25% trypsin to digest the cells, observe under a microscope, digest for 1-2 minutes, and you can see the cells separating and becoming round, indicating that digestion is complete; quickly discard the trypsin, add complete culture medium, pipette the cells to make a single-cell suspension, passage at a ratio of 1:3, and expand the culture at 37℃ and 5% CO2 saturated humidity.

[0071] Cell treatment:

[0072] NCI-H1395 cells in the logarithmic growth phase and in good growth condition were collected and prepared into single-cell suspensions using 1640 medium. The suspensions were then divided into groups of 2 × 10⁶ cells per well. 5 The cells were evenly seeded into 6-well plates and incubated overnight at 37°C and 5% CO2 saturated humidity. Two hours before transfection, the medium was replaced with serum-free 1640 medium. Transfection was performed according to the following experimental groups:

[0073] 1) Normal cell control group;

[0074] 2) ADAM32 siRNA interference control group;

[0075] 3) ADAM32 siRNA interference group;

[0076] 4) ADAM32 antibody concentration group: 10ug / mL (treatment for 24h);

[0077] Transfection steps:

[0078] For each transfected sample, prepare it as follows: dilute 10 μL of the small fragment (concentration of 20 μM) with 100 μL of serum-free opti-MEM, mix gently with a pipette tip, and let stand at room temperature for 5 minutes.

[0079] Before use, gently mix Lipofectamine™ 2000. Then, take 5 μL of Lipofectamine™ 2000 and dilute it in 100 μL of opti-MEM. Let it stand at room temperature for 5 minutes. After standing at room temperature for 5 minutes, mix Lipofectamine™ 2000 with the dilution buffer for the small fragment (total volume 200 μL), gently mix, and let it stand at room temperature for 20 minutes. Add 200 μL of the mixture to each well and gently shake the cell culture plate back and forth to mix the mixture with the culture medium in the plate. Incubate the cells in a 37°C 5% CO2 incubator. After 6 hours, aspirate the transfection solution and replace it with normal culture medium. Continue incubating in a 37°C 5% CO2 incubator for another 24 hours before subsequent detection.

[0080] Cellular ROS detection:

[0081] Cells were digested with 0.25% trypsin without EDTA, collected after digestion, centrifuged at 1500 rpm for 5 min, supernatant was discarded, and cells were resuspended in PBS. Cells were washed twice with PBS at 1500 rpm for 5 min. The DCFH-DA cell ROS assay kit was used for detection: 1 mL of diluted DCFH was added to the PBS. Cells were incubated at 37°C for 20 min, mixing every 3 min. Cells were washed three times with serum-free medium. Cells were resuspended in 500 μL of PBS. Cells were then analyzed by flow cytometry.

[0082] JC-1 cell detection:

[0083] Digest cells with 0.25% trypsin without EDTA. After digestion, collect the cells, centrifuge at 1200 rpm for 5 min, discard the supernatant, and resuspend in 0.5 ml of cell culture medium, which may contain serum and phenol red. Add 0.5 ml of JC-1 staining working solution, invert several times to mix, and incubate at 37°C for 20 min in a cell culture incubator. During incubation, prepare an appropriate amount of JC-1 staining buffer (1X) by adding 4 ml of distilled water to 1 ml of JC-1 staining buffer (5X) and place it on ice. After incubation at 37°C, centrifuge at 1200 rpm for 3 min at 4°C and discard the supernatant. Wash twice with JC-1 staining buffer (1X). Resuspend the cells in 1 ml of JC-1 staining buffer (1X), centrifuge at 1200 rpm for 3 min at 4°C, and discard the supernatant. Add 1 ml of JC-1 staining buffer (1X) to resuspend the cells, centrifuge at 1200 rpm at 4℃ for 3 minutes, and discard the supernatant; resuspend the cells in 500 μl of JC-1 staining buffer (1X) and run them on a flow cytometer.

[0084] Experimental results showed that the drug / gene interference ADAM32 affected mitochondrial function in lung adenocarcinoma cells, leading to an increase in intramitochondrial ROS. Figure 13 ), abnormal membrane potential ( Figure 14 ).

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention. SEQUENCE LISTING <110> Huazhong Agricultural University <120> Application of ADAM32 gene in the diagnosis and treatment of lung adenocarcinoma <130> 2021-12-14 <160> 8 <170> PatentIn version 3.5 <210> 1 <211> twenty one <212> DNA <213> ADAM32-Homo-383 Chain of Justice <400> 1 gcucuggacu aagaggaaut t 21 <210> 2 <211> twenty one <212> DNA <213> ADAM32-Homo-383 antisense chain <400> 2 auuccucuua guccagagct t 21 <210> 3 <211> twenty one <212> DNA <213> ADAM32-Homo-738 Justice Chain <400> 3 gcugucauca uuggaguuat t 21 <210> 4 <211> twenty one <212> DNA <213> ADAM32-Homo-738 antisense chain <400> 4 uaacuccaau gaugacagct t 21 <210> 5 <211> twenty one <212> DNA <213> ADAM32-Homo-1395 Chain of Justice <400> 5 gccgaaagca cauccugaat t 21 <210> 6 <211> twenty one <212> DNA <213> ADAM32-Homo-1395 antisense chain <400> 6 uucaggaugu gcuuucggct t 21 <210> 7 <211> twenty one <212> DNA <213> NC Sequence Justice Chain <400> 7 uucuccgaac gugucacgut t 21 <210> 8 <211> twenty one <212> DNA <213> NC sequence antisense chain <400> 8 acgugacacg uucggagaat t 21

Claims

1. Use of a marker detection reagent in the manufacture of a prognosis product for lung adenocarcinoma, characterized in that, The marker is ADAM32 a gene.

2. Use according to claim 1, characterized in that, The marker detection reagent is a reagent for measuring ADAM32 the amount of gene expression.

3. Use according to claim 2, characterized in that, The assay ADAM32 Reagents for the amount of gene expression include detection ADAM32 Reagents for the messenger RNA of the gene, detection ADAM32 Reagents for the complementary DNA of the gene, detection ADAM32 Reagents for the protein encoded by the gene.

4. Use according to claim 1, characterized in that, The product is selected from a chip or a kit.

5. Use of an agent that inhibits expression of a marker in the manufacture of a medicament for the treatment of lung adenocarcinoma, characterized in that, The marker is ADAM32 gene; The agent that inhibits the expression of the marker includes an siRNA specific to ADAM32 a gene of the marker. The siRNA is ADAM32-Homo-738. The sequence of the sense strand of ADAM32-Homo-738 is shown as SEQ ID NO: 3, and the sequence of the antisense strand is shown as SEQ ID NO:

4.

6. A drug screening system characterized by, The drug screening system comprises: (1) a detection module for detecting the inhibitory effect of a drug on ADAM32 gene expression; The analysis module is used for outputting the effect of the drug for treating lung adenocarcinoma according to the detection module output data.

7. A lung adenocarcinoma prognosis system, characterized by, The prognosis system comprises: (1) a detection device, wherein the detection device is used for detecting ADAM32 gene expression amount; (2) an analysis device, wherein the analysis device is configured to output a prognosis result of lung adenocarcinoma based on the data of the gene expression amount output from the detection device ADAM32 the analysis device is configured to output a prognosis result of lung adenocarcinoma based on the data of the gene expression amount output from the detection device

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