ALOX15 as lung adenocarcinoma biomarker and application

Abstract

The invention discloses ALOX15 as a lung adenocarcinoma biomarker and application, the lung adenocarcinoma biomarker in the application is an ALOX15 gene or protein, and the expression of the ALOX15 gene or protein in a lung adenocarcinoma patient is reduced. Meanwhile, the reagent for detecting the lung adenocarcinoma biomarker is applied to preparation of a product for lung adenocarcinoma diagnosis and / or prognosis evaluation, and the reagent is used for detecting the expression level of the ALOX15 gene or protein. According to the application, ALOX15 is used as a marker, and related kits and drugs provide a new tool for diagnosis, prognosis evaluation and treatment of LUAD, and have the potential of improving the diagnosis accuracy, the treatment effect and the life quality of a patient.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to ALOX15 as a biomarker for lung adenocarcinoma and its application. Background Art

[0002] In some populations, chronic irritation and chronic inflammation of the lungs can develop into chronic obstructive pulmonary disease or even lung cancer. Lung cancer is indeed a very common malignant tumor in clinical practice, and its morbidity and mortality rates have remained high. Due to the rich blood vessels in the lungs, lung cancer will locally infiltrate blood vessels and lymphatic vessels in the early stages, so distant metastasis through blood vessels often occurs in the early stages of lung cancer. When local infiltration is quite severe or metastasis occurs, it usually leads to an increased cancer recurrence rate and shortens the patient's survival. Lung adenocarcinoma (LUAD) is a common type of lung cancer, which is more common in women and the incidence rate is still rising. Despite the continuous advancement of medical technology in recent years, most patients with lung adenocarcinoma have already developed to the middle and late stages when they are diagnosed, and the 5-year survival rate of most LUAD patients is low. Therefore, identifying new biomarkers for early diagnosis and targeted therapy is very important for the prevention and treatment of LUAD.

[0003] Studies have found that arachidonic acid (ALOX15) affects the occurrence, metastasis and apoptosis of cancer. In colon cancer, esophageal cancer, breast cancer, pancreatic cancer and other cancers, the expression level of ALOX15 is reduced, thus affecting the development of cancer. ALOX15 is associated with many aspects of cancer development, such as angiogenesis, inflammation and metastasis. The metabolite of ALOX15, 13(S)-HODE, can increase the activity of epidermal growth factor-dependent mitogen-activated protein kinase in lung cancer, thereby promoting the activity of peroxisome proliferator-activated receptor γ and preventing the formation of lung tumors. However, the mechanism by which ALOX15 inhibits the progression of LUAD still needs further verification. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide an ALOX15 as a biomarker for lung adenocarcinoma and its application, aiming to explore the expression pattern of ALOX15 in lung adenocarcinoma (LUAD) and its effect on LUAD cell proliferation, migration and apoptosis, so as to disclose the possibility of ALOX15 as a potential biomarker and therapeutic target, thereby providing new ideas and methods for the early diagnosis and treatment of LUAD.

[0005] The technical problem to be solved by the present invention is achieved through the following technical solutions:

[0006] The first purpose of the present application is to provide a lung adenocarcinoma biomarker, wherein the biomarker is the ALOX15 gene or protein, and the expression of the ALOX15 gene or protein is decreased in patients with lung adenocarcinoma.

[0007] The second objective of the present application is to provide a reagent for detecting a biomarker for lung adenocarcinoma, wherein the reagent is used to detect the expression level of the ALOX15 gene or protein.

[0008] The third object of the present application is to provide a reagent for detecting a lung adenocarcinoma biomarker for use in the preparation of a product for diagnosis and / or prognosis evaluation of lung adenocarcinoma, wherein the biomarker is the ALOX15 gene or protein, and the reagent is used to detect the expression level of the ALOX15 gene or protein.

[0009] Preferably, in the above technical solution, the product is a kit, a medicine or a device. The test sample of the product is blood.

[0010] The fourth object of the present application is to provide a kit for detecting lung adenocarcinoma, wherein the kit comprises reagents for detecting the expression level of ALOX15 gene or protein.

[0011] The fifth object of the present application is to provide a drug for preventing or treating lung adenocarcinoma, wherein the drug contains a substance that can increase the expression level of ALOX15 gene or protein.

[0012] Preferably, in the above technical solution, the substance contains anti-inflammatory factors, which can promote the overexpression of ALOX15, thereby upregulating the mRNA level of PPAR-γ, and then promoting tumor cell apoptosis through the activation of PPAR-γ to inhibit cancer progression.

[0013] Preferably, in the above technical solution, the substance contains anti-inflammatory factors such as IL-4 and / or IL-13.

[0014] The above technical solution of the present invention has the following beneficial effects:

[0015] In this application, ALOX15 as a marker, as well as related kits and drugs, provide new tools for the diagnosis, prognosis assessment and treatment of LUAD, and have the potential to improve diagnostic accuracy, treatment efficacy and patient quality of life. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

[0017] Figure 1 This is a flow chart of the study on the expression of ALOX15 in LUAD of Example 1, wherein: A is the TCGA database analysis; B is the clinical sample analysis.

[0018] Figure 2 This is a flow chart of the study on the effect of ALOX15 on LUAD cells in Example 2.

[0019] Figure 3 This is a flow chart of the study on the potential mechanism of ALOX15 affecting LUAD in Example 3, wherein: A is transcriptome sequencing; B is a blocking experiment.

[0020] Figure 4 Figure 2 is the expression of ALOX15 in LUAD, where: Figure A shows that the expression level of ALOX15 in LUAD tumor tissue is lower than that in normal tissue; Figure B shows the expression level of ALOX15 in clinical stage I-IV LUAD; Figure C shows the expression of ALOX15 in LUAD T stage; Figure D shows the survival analysis of the relationship between ALOX15 expression and the survival rate of LUAD patients; Figure E shows the diagnostic ROC curve of ALOX15 in LUAD model; Figure F shows the expression of ALOX15 in LUAD N stage and M stage; Figure G shows the expression of ALOX15 in LUAD M stage expression; stage Ⅰ-Ⅳ: the higher the number, the deeper the cancer progresses and the more it spreads to the surrounding tissues, T: the range and size of the primary tumor, T1-T4 indicates the size of the primary tumor and its increasing impact on the surrounding tissues, N0 represents no lymph node involvement, N1 and N2 represent the degree and range of lymph node involvement, M0 represents no remote metastasis, and M1 represents remote metastasis, n=3, ns-no significant difference, *(P<0.05), **(P<0.01), ***(P<0.001), ****(P<0.0001).

[0021] Figure 5 Figure 1 is the result of immunohistochemical staining, where: Figure A is HE staining of tumor and adjacent tumor tissue; Figure B is the result of immunohistochemical staining, showing that the expression of ALOX15 was reduced in lung cancer tissue; Figure C is the positive area ratio of ALOX15, calculated as positive area / tissue area; Figure D is the expression difference of ALOX15 in T1 and T2 stage LUAD tissue; Figure E is the result of immunohistochemical staining, showing the expression difference of ALOX15 in T1 and T2 stage LUAD tissue; Adjacent Tumor: adjacent tumor tissue; Tumor: cancer tissue; Positive Area: positive area ratio, n=3, ns-no significant difference, *(P<0.05), **(P<0.01), ***(P<0.001), ****(P<0.0001).

[0022] Figure 6 Figure 1 is an immunofluorescence staining diagram, in which: Figure A shows the expression of ALOX15 in the cell nucleus; Figures B and C are WB experimental results, indicating that in clinical samples with lymph node metastasis, the expression level of ALOX15 is significantly lower than that in samples without lymph node metastasis (P < 0.05).

[0023] Figure 7The expression levels of ALOX15 in different lung adenocarcinoma cell lines, where: Figure A is an electrophoresis diagram, and Figure B is a bar graph.

[0024] Figure 8 Figure 2 is the effect of anti-inflammatory factors on ALOX15 expression, where: Figure A and Figure B respectively show that IL-4 and IL-13 significantly induced ALOX15 upregulation in A549 cells; Figure C and Figure D respectively show that IL-4 and IL-13 significantly induced ALOX15 upregulation in NCI-H1944 cells; Figure E and Figure F respectively show that ALOX15 expression in PC9 cells induced by IL-4 and IL-13 did not increase or decrease significantly; n=3, ns-no significant difference, *(P<0.05), **(P<0.01), ***(P<0.001), ****(P<0.0001).

[0025] Fig. 9 Figure 2 is the effect of ALOX15 overexpression on LUAD cell proliferation, where: Figure A shows that ALOX15 overexpression inhibits the proliferation of A549 adenocarcinoma cells; Figure B shows that ALOX15 overexpression inhibits the proliferation of NCI-H1944 adenocarcinoma cells; Figure C shows that overexpression of ALOX15 inhibits the proliferation of PC9 adenocarcinoma cells, oe-ALOX15: overexpression of ALOX15, n=3, ns-no significant difference, *(P<0.05), **(P<0.01), ***(P<0.001), ****(P<0.0001).

[0026] Fig.10 Figure 2 shows the effect of ALOX15 overexpression on LUAD cell migration, where: Figure A and Figure B detect the migration ability of A549 cells overexpressing ALOX15 through wound healing experiment; Figure C and Figure D show that overexpression of ALOX15 inhibits the migration ability of NCI-H1944 cells; Figure E and Figure F show that overexpression of ALOX15 inhibits the migration ability of PC9 cells, oe-ALOX15: overexpression of ALOX15, n=3, ns-no significant difference, *(P<0.05), **(P<0.01), ***(P<0.001), ****(P<0.0001).

[0027] Fig.11 Figure 5 shows the changes in the number of apoptotic cells in A549 cells overexpressing ALOX15; Figures C, D, and E show the changes in the expression levels of Caspase-9 and Bcl-2 proteins after overexpression of ALOX15.

[0028] Fig.12Figure 5 shows the changes in the number of apoptotic cells in NCI-H1944 cells overexpressing ALOX15; Figures C, D, and E show the changes in the expression levels of Caspase-9 and Bcl-2 proteins after overexpression of ALOX15.

[0029] Fig.13 Figure 5 shows the changes in the number of apoptotic cells in PC9 cells overexpressing ALOX15; Figures C, D, and E show the changes in the expression levels of Caspase-9 and Bcl-2 proteins after overexpression of ALOX15.

[0030] Fig.14 This is the gene expression profile detection of lung adenocarcinoma cells after overexpression of ALOX15, where: Figure A shows the experimental design and workflow; Figure B shows the correlation detection of gene expression levels between samples; Figures C, D, and E show the differences in gene expression profiles between the experimental group and the control group.

[0031] Fig.15 The following are the analysis diagrams of signaling pathways and protein interactions affected by ALOX15 overexpression, where Figures A and B use scatter plots to represent pathway entries with differential gene enrichment based on KEGG annotation results and official classifications; Figure C is a protein-protein interaction (PPI) network established using the STRING database and visualized in the Cytoscape software.

[0032] Fig.16 Figure 2 shows the effect of ALOX15 overexpression on PPAR-γ, where: Figure A and Figure B respectively show that ALOX15 overexpression upregulates the mRNA level (P < 0.001) and protein level (P < 0.01) of PPAR-γ; Figure C shows that ALOX15 overexpression has no significant effect on the total protein level of ERK1 / 2; ALOX15 overexpression has no significant effect on the total protein level of ERK1 / 2, but upregulates the expression of phosphorylated ERK1 / 2 (P < 0.01).

[0033] Fig.17 To inhibit the effect of PPAR-γ on the proliferation and apoptosis of LUAD cells induced by ALOX15, Figures A and B show that after inhibiting PPAR-γ (GW9662), the total protein level of ERK1 / 2 was relatively reduced (P < 0.0001); Figures C and D show that after inhibiting PPAR-γ (GW9662), the phosphorylation level of ERK1 / 2 was relatively reduced (P < 0.01); Figures E and F show that after inhibiting ERK1 / 2 phosphorylation (PD98059), the expression of PPAR-γ was relatively reduced (P < 0.001).

[0034] Fig.18To detect whether the effect of ALOX15 on LUAD proliferation and apoptosis is through the PPAR-γ-ERK1 / 2 axis, among which: Figures A, B and C show that after inhibiting PPAR-γ and ERK1 / 2 phosphorylation in A549 cells overexpressing ALOX15, the inhibitory ability of ALOX15 on cell proliferation was significantly reduced (P < 0.05); Figures C, D and E show that after inhibiting PPAR-γ and ERK1 / 2 phosphorylation in A549 cells expressing ALOX15, the promoting effect of ALOX15 on cell apoptosis was significantly reduced (P < 0.001).

[0035] Fig.19 Schematic diagram of the potential mechanism of action of ALOX15 in the progression of LUAD. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that the relative arrangement of components and steps, numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present invention unless otherwise specifically stated.

[0037] Unless otherwise specified, the reagents used in this application are all commercially available or obtained through commercial channels, or can be prepared by referring to existing chemical methods.

[0038] Example 1 Expression of ALOX15 in LUAD

[0039] 1. TCGA database analysis:

[0040] A total of 59 clinical normal lung tissues and 490 LUAD tissue samples were collected from TCGA (https: / / portal.gdc.cancer.gov) for statistical analysis. Figure 1 Figure A.

[0041] 2. Clinical sample analysis:

[0042] Normal tissue and tumor tissue samples adjacent to tumors were collected from 33 patients with LUAD in the Second Affiliated Hospital of Shandong First Medical University, China. HE staining, immunofluorescence (IF), and immunohistochemistry (IHC) were used to detect the expression of ALOX15 protein in LUAD tissues and adjacent tumor tissues in 29 patients after diagnosis. Western Blot (WB) was performed in 4 cases to detect the differential expression of ALOX15 in LUAD tissues and normal tissues adjacent to tumor tissues. The specific steps are shown in Figure 1 Figure B.

[0043] 2.1 HE staining

[0044] (1) The steps of paraffin section dewaxing are as follows: first, the tissue sections are placed in xylene I (15 min) → xylene II (15 min) to remove the paraffin, then placed in anhydrous ethanol (5 min) → anhydrous ethanol II (5 min) to wash away the residual xylene, then placed in 95% alcohol (10 min) → 90% alcohol (10 min) → 85% alcohol (10 min) → 80% alcohol (10 min) → 75% alcohol (10 min), and finally washed with distilled water 3 times, 5 min each time;

[0045] (2) Hematoxylin staining of cell nuclei: After 3-5 minutes of hematoxylin staining, wash the sections with distilled water to remove residual dye. Immerse the tissue sections in hematoxylin differentiation solution for 10-60 seconds (observe the degree of staining under a microscope), wash with distilled water to stop differentiation;

[0046] (3) Bluing: Use 0.2% ammonia water to soak and bluing, the time is determined by the effect of observation under the microscope;

[0047] (3) Eosin staining of cytoplasm: stain with eosin solution for 1 to 2 minutes, and then wash off the remaining stain with water;

[0048] (4) Dehydration: The tissue sections were first placed in 75% ethanol (3 s) → 85% ethanol (3 s) → 95% ethanol (3 s) → anhydrous ethanol I (3 s) → anhydrous ethanol II (2 min), and then placed in xylene I (10 min) → xylene II (10 min) for dehydration and transparency;

[0049] (5) After removing the tissue sections from xylene, seal the sections with neutral gum, taking care to avoid bubbles during the sealing process;

[0050] (6) Microscope observation, image acquisition and analysis.

[0051] 2.2 Immunohistochemistry

[0052] (1) The steps of paraffin section dewaxing are as follows: first, the tissue sections are placed in xylene I (15 min) → xylene II (15 min) to remove the paraffin, then placed in anhydrous ethanol (5 min) → anhydrous ethanol II (5 min) to wash away the residual xylene, then placed in 95% alcohol (10 min) → 90% alcohol (10 min) → 85% alcohol (10 min) → 80% alcohol (10 min) → 75% alcohol (10 min), and finally washed with distilled water 3 times, 5 min each time;

[0053] (2) Antigen repair: Dilute the Tris-EDTA antigen repair solution, heat it in a microwave oven, heat it to boiling point, take it out and cool it at room temperature for 2 minutes, put it in the slice, and heat it in the microwave oven again for repair. Repeat 3 times, 5 minutes each time, and cool it for 2 minutes after each repair before repairing. After the repair is completed, cool it to room temperature and wash it three times with PBS, 5 minutes each time. Draw circles with an immunohistochemistry pen, add 3% H2O2 to the tissue in a wet box, incubate it for 20 minutes, and then wash it three times with PBS, 5 minutes each time;

[0054] (3) Permeabilization: The tissue sections were permeabilized in 0.2% Tween-100 solution for 30 min and then washed three times with PBS, 5 min each time;

[0055] (4) Blocking: 5% bovine serum albumin blocking for 2 h;

[0056] (5) Incubation with primary antibody: After blocking, dilute the primary antibody with 5% BSA solution until it covers the tissue on the slice. Then place the tissue slice in a humidified box and place it in a 4°C refrigerator overnight.

[0057] (6) Incubate with secondary antibody: The next day, remove the wet box from the 4°C refrigerator and rewarm for 30 minutes. After rewarming, wash with PBS three times, 5 minutes each time, and then incubate with secondary antibody in a dark room for 1 hour. Then develop with DAB for 5 to 10 minutes, and then rinse with PBS three times, 5 minutes each time;

[0058] (7) Re-staining, dehydrating and transparent mounting: Use hematoxylin staining solution for 5 minutes, then immediately rinse with water. First, place the slices in 75% ethanol (5 minutes) → 80% ethanol (5 minutes), then 90% ethanol (5 minutes) → 95% ethanol (5 minutes) → 100% ethanol (5 minutes), followed by xylene I (5 minutes) → xylene II (5 minutes), dehydrate and make transparent, and finally mount the slices with neutral gum;

[0059] (8) Image acquisition: The slides were scanned using the Pannoramic DESK system. The positively stained cells in three fields of view per slide were analyzed using ImageScope 9.1 software (Aperio Technologies, USA). The stained slides were examined by a pathologist with more than 15 years of experience, and the percentage of positive area was calculated.

[0060] 2.3Western Blot (WB)

[0061] 2.3.1 Protein extraction from clinical samples

[0062] (1) Tissue weighing and homogenization: 100 mg of tissue was placed in each EP tube, and 1 mL of protein lysis mixture (RIPA:PMSF was prepared in a ratio of 100:1) was added to homogenize the tissue;

[0063] (2) Lysis: 4°C for 30 min;

[0064] (3) Centrifugation: Place the EP tube in a 4°C centrifuge and centrifuge at 12,000 g for 10 min. Use a pipette to carefully aspirate the upper layer of liquid, and try not to aspirate the lower layer of sediment.

[0065] 2.3.2 Total protein extraction from adherent cells

[0066] (1) Rinse the adherent cells twice with pre-cooled PBS and then remove any residual liquid in the cell culture plate;

[0067] (2) Add 100 μL of protein lysis buffer (RIPA:PMSF in a ratio of 100:1) to the cell culture plate and shake to ensure full contact between the reagent and the cells;

[0068] (3) The cell lysis solution has a short action time. Use a cell scraper to scrape off the liquid on the plate and transfer it to a 1.5 mL centrifuge tube. Place the cells in an ice box and lyse for 30 minutes. Shake the cells on a shaker every 10 minutes.

[0069] (4) Place the EP tube in a 4°C centrifuge and centrifuge at 12,000 g for 10 min. Use a pipette to carefully aspirate the upper layer of liquid, and try not to aspirate the lower layer of sediment.

[0070] 2.3.3 Protein concentration determination

[0071] Follow the steps in the instruction manual.

[0072] 2.3.4SDS-PAGE electrophoresis

[0073] (1) Sample processing: adjust the loading volume of each well according to the concentration of the sample to make the loading volume of each well fixed and consistent. Add 1 / 3 volume of 4× protein sample buffer to the test tube containing the protein sample and mix well, then place it in a 100℃ metal bath for 10 minutes, take it out and cool it to room temperature for further experiments or store it at -20℃;

[0074] (2) Gel preparation: According to the instructions, a certain volume of separation gel preparation reagents are mixed according to the required separation gel concentration, and the gel is quickly poured. ddH20 is added to the upper layer to flatten the gel surface. After 30 minutes, ddH20 is removed. A certain volume of concentrated gel preparation reagents are mixed and the concentrated gel is quickly filled to fill the remaining space, and a comb is inserted.

[0075] (3) Electrophoresis: Place the electrophoresis table in the electrophoresis tank, add an appropriate amount of electrophoresis buffer to the tank, gently pull out the comb for making gel, use a syringe to remove the gel thread in the loading well, add the samples to the loading wells according to the experimental sequence, and connect the power supply. The DC voltage for concentrated gel electrophoresis is 80V, the time is 30min, and the DC voltage for separation gel electrophoresis is 120V, the time is 60min~90min. Adjust the separation gel electrophoresis time according to the experimental requirements, and perform the electrophoresis on ice throughout the process.

[0076] 2.3.5 Transfer

[0077] (1) Activate the PVDF membrane with methanol for 2 minutes and then quickly place it in a container containing electrotransfer solution;

[0078] (2) Soak the transfer sponge and filter paper completely in the electrotransfer solution and place them in the correct order. Be sure to remove all bubbles during the process.

[0079] (3) Transfer membrane at 400 mA constant current for 30 min.

[0080] 2.3.6 Closure

[0081] Dissolve skim milk powder in TBST solution in advance and mix thoroughly to complete the configuration and preparation. After transfer, block with the previously prepared blocking solution (5% skim milk powder) at room temperature for 2 hours. Immerse the PVDF membrane completely in the blocking solution, ensuring that the blocking solution completely covers the surface of the membrane. Blocking for 2 hours at room temperature is usually sufficient for the blocking solution to fully absorb.

[0082] 2.3.7 Antibody incubation

[0083] (1) After blocking, aspirate the blocking solution and wash with TBST three times, 10 min each time;

[0084] (2) Add primary antibody and incubate at 4°C overnight. The volume of antibody should completely cover the PVDF membrane.

[0085] (3) On the second day, remove the antibody incubation box from 4°C and allow it to warm up at room temperature for 30 min. Then add an appropriate volume of TBST to the antibody incubation box and wash three times, 10 min each time.

[0086] (4) Add diluted secondary antibody and incubate for 60 min at room temperature;

[0087] (5) Wash three times with TBST, 10 min each time.

[0088] 2.3.8 Chemiluminescence detection

[0089] (1) Use a high-sensitivity ECL chemiluminescence kit to prepare an ECL mixed solution;

[0090] (2) Add ECL mixture dropwise to the membrane protein side;

[0091] (3) Expose the sample in a light-proof state, then develop and fix it, save the results and use Image J software for data analysis.

[0092] Result analysis:

[0093] (1) ALOX15 expression was significantly downregulated in LUAD clinical samples, and the expression level was correlated with clinical stage and tumor diameter. ALOX15 also has a certain diagnostic significance for LUAD models, and low levels of ALOX15 may be an unfavorable factor affecting the prognosis of LUAD patients.

[0094] like Figure 4 As shown in Figure A, the expression level of ALOX15 in LUAD tumor tissues was significantly lower than that in normal tissues, indicating that the expression of ALOX15 in LUAD was downregulated. Figures B and C, the expression level of ALOX15 was correlated with the clinical stage and tumor diameter (T stage) of LUAD, indicating that ALOX15 may play a role in the progression of LUAD. Figure D, survival analysis of the relationship between ALOX15 expression and survival rate of LUAD patients showed that the expression level of ALOX15 was correlated with the prognosis of patients. Conclusion: ALOX15 is lowly expressed in LUAD clinical samples, and its expression level is correlated with clinical indices such as tumor diameter and clinical stage in clinical samples. Low levels of ALOX15 may be an unfavorable factor affecting the prognosis of LUAD patients.

[0095] like Figure 5 As shown in the results, ALOX15 expression was reduced in lung cancer tissues, and the expression level of ALOX15 in cancer tissues was lower than that in paracancerous tissues (P < 0.01). In addition, the expression level of ALOX15 in T2 LUAD cancer tissues was significantly lower than that in T1 (P < 0.05), indicating that the expression level of ALOX15 was related to the tumor stage. Conclusion: The results of immunohistochemical staining showed that the expression level of ALOX15 in cancer tissues was lower than that in paracancerous tissues, and the expression level of ALOX15 in T2 LUAD cancer tissues was significantly lower than that in T1.

[0096] like Figure 6 As shown, ALOX15 is expressed in the nucleus (e.g. Figure 6 The results of WB experiments showed that the expression level of ALOX15 in clinical samples with lymph node metastasis was significantly lower than that in samples without lymph node metastasis (P < 0.05) (Figure 2A). Figure 6 Combining the results of the database with those of clinical samples, it was shown that the expression of ALOX15 was downregulated in LUAD, and its expression level was correlated with the tumor diameter (T) in clinical samples.

[0097] (2) The expression level of ALOX15 is closely related to the differentiation degree of lung adenocarcinoma cells.

[0098] like Figure 7 As shown in the figure, the expression level of PC9 in well-differentiated adenocarcinoma was higher than that in A549 and NCI-H1944 (P<0.05) (Figure A and Figure B). The expression level of ALOX15 was PC9 (well-differentiated) > A549 (well-differentiated) > NCI-H1944 (poorly differentiated). Conclusion: The expression level of ALOX15 is closely related to the degree of differentiation of lung adenocarcinoma cells. The higher the degree of differentiation, the higher the expression level of ALOX15.

[0099] (3) Anti-inflammatory factors can prevent the downregulation of ALOX15 and may have a certain preventive effect on LUAD.

[0100] like Figure 8 As shown in the figure, in order to illustrate the correlation between ALOX15 expression and inflammation using anti-inflammatory factors, different LUAD cell lines were treated with different concentrations of anti-inflammatory factors IL-4 and IL-13. WB results showed that in the well-differentiated adenocarcinoma cell A549 (P < 0.05) ( Figure 8 Figures A and B) and poorly differentiated adenocarcinoma cells NCI-H1944 (P < 0.001) ( Figure 8 In Figures C and D of Figure 2, the expression level of ALOX15 in cells induced by IL-4 and IL-13 was significantly increased, and the induction effect of IL-13 on ALOX15 was concentration-dependent, with statistically significant differences. However, in the highly differentiated adenocarcinoma cells PC9, the expression of ALOX15 did not change significantly ( Figure 8 This suggests that inhibiting inflammation can upregulate the activity of ALOX15 and may have the opportunity to prevent inflammation from developing into cancer.

[0101] Example 2 Effect of ALOX15 on LUAD cells

[0102] 1. Selection of cell lines and detection of ALOX15 expression: Based on literature search and database analysis of lung adenocarcinoma cell lines related to ALOX15, three human lung adenocarcinoma cell lines with different degrees of differentiation were selected: A549, PC9, and NCI-H1944. Western Blot (WB) technology was used to detect the difference in ALOX15 expression in the three cell lines.

[0103] 2. Construct a stable LUAD cell line overexpressing ALOX15 and detect the effect of ALOX15 on the growth and apoptosis of LUAD cells.

[0104] 2.1 Stable lentivirus transfection

[0105] 2.1.1 Screening of optimal concentration of puromycin

[0106] (1) Cell plating: Exponentially growing A549, NCI-H1944, and PC9 cells with good morphology were inoculated into different 24-well cell culture plates according to the experimental groups. Use a pipette to add 500 μL of cell suspension (1×105 cells / mL) to each well of the well plate, and mix by cross-cross method to ensure that the cells are evenly distributed in the cell culture plate. Then, we put the well plate into a constant temperature incubator to ensure that the cells are cultured in an environment of 5% CO2 and 37°C;

[0107] (2) Prepare screening medium: prepare complete medium containing different concentrations of puromycin: 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL;

[0108] (3) After incubating the cells overnight, different concentrations of screening medium were added and the cells were incubated;

[0109] (4) Replace the screening medium with fresh one every 2 to 3 days;

[0110] (5) Observe cell growth daily;

[0111] (6) Determine the optimal screening medium concentration for all cells: the lowest puromycin concentration that kills all cells starting from the addition of puromycin is the optimal screening medium concentration.

[0112] 2.1.2 Lentivirus infection MOI pre-experiment

[0113] (1) Day 1: Cell inoculation. Exponentially growing, morphologically well-formed A549, NCI-H1944, and PC9 cells were inoculated into different 24-well cell culture plates according to the experimental groups. Use a pipette to add 500 μL of cell suspension (1×105 cells / mL) to each well of the well plate, and cross-mix to ensure that the cells are evenly distributed in the cell culture plate. Subsequently, we placed the well plate in a constant temperature incubator to ensure that the cells were cultured in an environment of 5% CO2 and 37°C. MOI gradients were set for each cell type: 0, 20, 40, 60, 80, 100;

[0114] (2) The next day: add virus. The amount of virus to be added to each well needs to be calculated based on the pre-set MOI value (multiplicity of infection). We observe the cells under a microscope. If the cells in the well plate are in good condition, evenly distributed, and the cell density reaches 30% to 40%, a certain volume of virus stock solution is added to the culture medium for infection. The cells are then placed in a cell culture incubator for culture;

[0115] (3) Day 3: Change medium. 12 to 16 hours after adding the virus, remove the medium in each experimental group and replace it with fresh complete cell medium, and continue to culture in the incubator;

[0116] (4) Day 5: Observe and take photos under a fluorescence microscope to determine the optimal MOI value for lentivirus infection of cells. Observe under a fluorescence microscope 3 days after virus infection to determine the optimal MOI (infection efficiency reaches about 80%).

[0117] 2.1.3 Screening of stable cell lines by lentiviral infection

[0118] (1) Day 1: Cell inoculation. Exponentially growing, morphologically well-formed A549, NCI-H1944, and PC9 cells were inoculated into different 24-well cell culture plates according to the experimental groups. Use a pipette to add 500 μL of cell suspension (1×105 cells / mL) to each well of the well plate, and mix by cross-cross method to ensure that the cells are evenly distributed in the cell culture plate. Subsequently, we placed the well plate in a constant temperature incubator to ensure that the cells were cultured in an environment of 5% CO2 and 37°C;

[0119] (2) The next day: viral infection. According to the conclusion of the preliminary experiment, select the appropriate MOI and infection conditions to calculate the virus volume required for each well and perform viral infection. Observe the cells under a microscope. If the cells are growing well and evenly distributed, and the cell density reaches 30% to 40%, add lentivirus for infection;

[0120] (3) Day 3: Change the medium. 12 to 16 hours after virus infection, replace the medium with fresh one according to the cell status. Discard the old medium in the well plate and add 500 μL of newly prepared complete medium to each well. If the cell status is not good under the microscope, the medium should be replaced immediately. If the cell status is good under the microscope, the medium can be replaced within 24 hours according to the cell status.

[0121] (4) Day 5: Observe the expression. Within 72 hours after infection, the cells can be cultured and expanded by replacing the original complete medium with the puromycin selection medium.

[0122] (5) Replace the freshly prepared screening medium every 48 hours;

[0123] (6) Monitor cells daily and observe the growth ratio of live cells, as well as the expression level and proportion of GFP and other proteins. A GFP expression level higher than 80% indicates that a stable strain has been formed.

[0124] 2.2 Cell proliferation ability detection method (CCK8 method)

[0125] 2.2.1 Cell plating

[0126] A549, NCI-H1944, PC9 cells with exponential growth and good morphology and A549, NCI-H1944, PC9 cells overexpressing ALOX15 were collected. 100 μL of cell suspension (6×104 cells / mL) was added to each well of the 96-well plate, and the cells were grouped and marked according to the experimental requirements. The cross-mixing method was used to ensure that the cells were evenly distributed in the cell culture plate. Then, we placed the well plate in a constant temperature incubator to ensure that the cells were cultured at 5% CO2 and 37°C;

[0127] 2.2.2 Add CCK-8

[0128] 24 h after cells were plated, 10 μL of CCK-8 solution was added to each well using a pipette and incubated in an incubator for 1 to 3 h;

[0129] 2.2.3 Absorbance measurement

[0130] The absorbance of cells at a wavelength of 450 nm was measured every 30 min using an enzyme-labeled instrument, and the data were entered into a computer for analysis.

[0131] 2.3 Scratch test

[0132] 2.3.1 Cell plating

[0133] Use a marker pen on the back of the six-well plate to draw even horizontal lines along the ruler through all the wells. Collect exponentially growing, well-morphological A549, NCI-H1944, PC9 cells and A549, NCI-H1944, PC9 cells overexpressing ALOX15. Add 2 mL of cell suspension (1×105 cells / mL) to each well of the 6-well plate, group the cells according to experimental requirements and write labels, and mix them by cross-cross method to ensure that the cells are evenly distributed in the cell culture plate. Then, we put the well plate in a constant temperature incubator to ensure that the cells are cultured at 5% CO2 and 37°C;

[0134] 2.3.2 Scratches

[0135] When the confluence of the cell culture reaches 80% to 90%, use the tip of a 200 μL pipette to hold the ruler, and try to make the tip perpendicular to the horizontal line on the back of the well plate. Then rinse the cells 3 times with PBS to wash off the scraped cells;

[0136] 2.3.3 Microscope observation

[0137] Cell scratch photos were taken under a microscope at 0h, 12h, 24h, 36h, 48h, 60h, and 72h in the same field of view, and the percentage of scratch healing area was calculated using ImageJ 1.52a software to represent the cell migration ability.

[0138] 2.4 AnnexinV-PE / 7AAD flow cytometry double staining assay

[0139] 2.4.1 Cell plating

[0140] A549, NCI-H1944, PC9 cells with exponential growth and good morphology and A549, NCI-H1944, PC9 cells overexpressing ALOX15 were collected. 2 mL of cell suspension (1×105 cells / mL) was added to each well of a 6-well plate, and the cells were grouped and labeled according to experimental requirements. The cross-mixing method was used to ensure that the cells were evenly distributed in the cell culture plate. Then, we placed the well plate in a constant temperature incubator to ensure that the cells were cultured at 5% CO2 and 37°C;

[0141] 2.4.2 Cell sample collection

[0142] (1) First, rinse twice with PBS and then add an appropriate amount of EDTA-free trypsin to the well plate. After digestion for 5 minutes, add culture medium to terminate digestion and gently blow down all cells with a pipette to mix evenly.

[0143] (2) Transfer the cell suspension to a 1.5 mL sterile centrifuge tube. Centrifuge at 1000 rpm for 5 min and then carefully remove the upper layer of liquid using a pipette. Try not to completely remove the upper layer of culture medium.

[0144] (3) Add 1 mL of 4°C PBS to each centrifuge tube, remix the cells with a pipette, centrifuge again at 1000 rpm for 5 min, and carefully remove the upper layer of liquid with a pipette.

[0145] (4) Dilute the binding buffer in the kit with sterile ddH2O at a ratio of 1:3. Add an appropriate volume of the diluted 1× binding buffer to the centrifuge tube and re-pipette to mix the cells so that the cell concentration is between 1×106 cells / mL and 5×106 cells / mL.

[0146] (5) Transfer 100 μL of the cell suspension to a labeled 1.5 mL light-proof centrifuge tube. Add 5 μL of Annexin V / PE to each tube and mix thoroughly by pipetting. Incubate at room temperature in the dark for 5 min.

[0147] (6) Add 10 μL of 7AAD (20 ug / mL) to each centrifuge tube, and immediately add 400 μL of PBS. Mix well by pipetting and perform flow cytometry. Use Kaluza to analyze the results.

[0148] Result analysis:

[0149] (1) ALOX15 overexpression inhibits LUAD cell proliferation and migration:

[0150] like Fig. 9 As shown in the results of CCK-8 experiments, the overexpression of ALOX15 significantly inhibited the proliferation of A549 cells (P < 0.01) ( Fig. 9 Figure A), NCI-H1944 cells (P<0.01) ( Fig. 9 B) and PC9 cells (P<0.001) ( Fig. 9 (C) The proliferation capacity of

[0151] like Fig.10 As shown in the figure, the results of the scratch experiment showed that overexpression of ALOX15 also inhibited the proliferation of A549 cells (P<0.01) ( Fig.10 Figures A and B), NCI-H1944 cells (P<0.01) ( Fig.10 C and D) and PC9 cells (P < 0.01) ( Fig.10 These data indicate that ALOX15 can inhibit the activity of LUAD cells, thereby inhibiting the occurrence and development of LUAD.

[0152] (2) ALXO15 induces cell apoptosis by upregulating Caspase-9 and downregulating Bcl-2 protein:

[0153] To investigate the mechanism by which ALOX15 inhibits LUAD cell proliferation and migration, flow cytometry was used to detect the number of apoptotic cells.

[0154] In A549 cells overexpressing ALOX15 (P<0.001) ( Fig.11 Figures A and B), NCI-H1944 cells (P < 0.001) ( Fig.12 A and B) and PC9 cells (P < 0.0001) ( Fig.13 In Figures A and B of Figure 2, the number of early apoptotic cells increased significantly. These data indicate that ALOX15 overexpression can promote early apoptosis of LUAD cells. The experiment further determined which apoptotic proteins were involved in ALOX15-induced cell apoptosis.

[0155] WB results showed that after overexpression of ALOX15, LUAD cells A549 (P<0.05) ( Fig.11C and D), NCI-H1944 (P < 0.001) ( Fig.12 C and D) and PC9 (P < 0.001) ( Fig.13 The expression level of Caspase-9 protein in LUAD cells A549 (P<0.01) was significantly increased after overexpression of ALOX15. Fig.11 CE images), NCI-H1944 (P<0.01) ( Fig.12 CE graph) and PC9 (P < 0.0001) ( Fig.13 The expression levels of Bcl-2 proteins in the CE images were significantly decreased. These results indicate that the apoptosis induced by ALOX15 overexpression is related to the changes in the activities of Caspase-9 and Bcl-2 proteins.

[0156] ALOX15 overexpression promotes apoptosis in LUAD cells: Fig.11 , Fig.12 and Fig.13 As shown, the number of apoptotic cells in LUAD cells overexpressing ALOX15 was observed to increase by AnnexinV-PE / 7AAD flow cytometry double staining detection, indicating that ALOX15 may promote the apoptosis of LUAD cells.

[0157] In summary, ALOX15 may play the role of a tumor suppressor gene in LUAD, and its overexpression can inhibit the proliferation and migration of LUAD cells and promote cell apoptosis. These findings provide a scientific basis for ALOX15 as a potential target for the treatment of LUAD.

[0158] Example 3 Potential mechanism of ALOX15 affecting LUAD

[0159] The main purpose was to explore the mechanism of ALOX15's potential impact on LUAD, including transcriptome sequencing and blocking experiments.

[0160] 1. Screening of differentially expressed genes of ALOX15 by transcriptome sequencing.

[0161] 1.1 Information Analysis

[0162] RNA samples of A549 cells and A549 cells overexpressing ALOX15 were selected for transcriptome resequencing. RNA samples were quality controlled in terms of total amount, purity, and integrity. After passing the test, subsequent library construction was carried out. After completing library construction, sequencing was performed using the Illumina platform. First, the RNA sequencing results were screened using software to remove low-quality reads with a quality lower than the set threshold, and corresponding data processing was also performed for possible cleavage phenomena to ensure the integrity and accuracy of the data, and finally clean reads were obtained. Clean reads were aligned with reference genome or transcriptome sequences using alignment tools. The alignment results can identify new transcripts that may exist in the sample, and single nucleotide variations (SNP detection) in the sample can be identified. In addition to single nucleotide variations, longer insertion or deletion variations (Indel detection) can also be detected. For the selected differentially expressed genes (DEGs), quantitative analysis was first performed, and then differential expression analysis was performed on genes with significant changes in expression levels in different sample sets, and functional GO analysis, pathway function analysis, cluster analysis, and protein interaction network analysis of DEGs were analyzed.

[0163] 1.2 Screening of differentially expressed genes

[0164] DEseq2 algorithm was used for DEG detection. DEseq2 method is based on the negative binomial distribution principle and uses Log2 fold change (FC) to evaluate the degree of gene expression difference. The fold change of differentially expressed genes meets the requirement of fold change ≥ 2.00 and P ≤ 0.05.

[0165] According to the detection results of different genetic differences, we performed hierarchical clustering analysis using the pheatmap function in R software, and visualized the identified DEGs by generating volcano plots and heat maps using R packages.

[0166] 1.3GO functional analysis and pathway functional analysis

[0167] According to the results of GO annotation and official classification and KEGG annotation and official classification, we used the phyper function of R software to perform functional classification and signal pathway enrichment analysis on DEGs. Then, the P value was corrected for FDR, and the results with FDR ≤ 0.01 were usually considered significant.

[0168] 1.4PPI Construction

[0169] The string DB package of R software was used to perform protein-gene interaction differential network analysis on the interactions in the STRING protein interaction database (http: / / string-db.org / ). Then, the differential gene-protein interaction network data file was imported into Cytoscape software for visual editing.

[0170] 2. ALOX15 overexpression leads to PPAR-γ upregulation

[0171] 2.1 Effects of PPAR-γ antagonist GW9662 on proliferation and apoptosis of lung adenocarcinoma cells

[0172] 2.1.1 Cell grafting

[0173] A549, NCI-H1944, PC9 lung adenocarcinoma cells with exponential growth and good morphology and A549, NCI-H1944, PC9 cells overexpressing ALOX15 were collected, and 100 μL of cell suspension (6×104 cells / mL) was added to each well of the 96-well plate, and 100 μL of cell suspension (2×105 cells / mL) was added to each well of the 12-well plate. At the same time, the cells were grouped and marked accordingly, and the cross-cross method was used to mix to ensure that the cells were evenly distributed in the cell culture plate. Subsequently, we placed the well plate in a constant temperature incubator to ensure that the cells were cultured in an environment of 5% CO2 and 37°C.

[0174] 2.1.2 GW9662 induction

[0175] After 24 h of cell culture, GW9662 at a concentration of 20 μM was added to the blocking group, and the same volume of DMSO was added to the blank group. Subsequently, we placed the well plate in a constant temperature incubator to ensure that the cells were cultured in an environment of 5% CO2 and 37°C.

[0176] 2.1.3 Cell proliferation ability detection

[0177] The cell proliferation ability detection method is the same as the CCK-8 experiment above.

[0178] 2.1.4 Detection of apoptotic cell count

[0179] AnnexinV-PE / 7AAD flow cytometry double staining detection method is as above.

[0180] 2.2 Effects of ERK1 / 2 phosphorylation inhibitor PD98059 on proliferation and apoptosis of lung adenocarcinoma cells

[0181] 2.2.1 Cell plating

[0182] A549, NCI-H1944, PC9 lung adenocarcinoma cells with exponential growth and good morphology and A549, NCI-H1944, PC9 cells overexpressing ALOX15 were collected. 100 μL of cell suspension (6×104 cells / mL) was added to each well of the 96-well plate, and 100 μL of cell suspension (2×105 cells / mL) was added to each well of the 12-well plate. The cells were grouped and marked according to the experimental requirements, and the cross-cross method was used to mix to ensure that the cells were evenly distributed in the cell culture plate. Then, we placed the well plate in a constant temperature incubator to ensure that the cells were cultured at 5% CO2 and 37°C.

[0183] 2.2.2 PD98059 induction

[0184] After 24 hours of cell culture, PD98059 at a concentration of 10 μM was added to the cells in the blocking group, and the same volume of DMSO was added to the blank group. Subsequently, we placed the well plate in a constant temperature incubator to ensure that the cells were cultured in an environment of 5% CO2 and 37°C.

[0185] 2.2.3 Cell proliferation ability detection

[0186] The cell proliferation ability detection method is the same as the CCK-8 experiment above.

[0187] 2.2.4 Detection of apoptotic cell numbers

[0188] AnnexinV-PE / 7AAD flow cytometry double staining detection method is as above.

[0189] Result analysis:

[0190] (1) Detection of gene expression profiles in lung adenocarcinoma cells after overexpression of ALOX15

[0191] To further investigate the mechanism by which ALOX15 affects the progression of lung adenocarcinoma, RNA was extracted from A549 cells and A549 cells overexpressing ALOX15 for transcriptome sequencing comparison.

[0192] Experimental design and workflow (e.g. Fig.14 As shown in Figure A, RNA samples were extracted and tested for RNA. After passing the test, the library was constructed and sequenced using the Illumina platform. In the gene expression level correlation test between samples, it was found that the correlation coefficient between biological replicates of the control group and the experimental group was greater than 0.8, which indicated that the gene expression levels of samples within the group were well correlated (as shown in Figure A). Fig.14 The experimental data showed that there were significant differences in the gene expression profiles between the experimental group and the control group. Compared with the control group, the expression levels of 151 genes increased, while the expression levels of 54 genes decreased (Figure 2A). Fig.14According to the KEGG annotation results and official classification, a scatter plot is used to represent the pathway entries of differentially enriched genes (such as Fig.15 Subsequently, in order to explore DEPs in more depth, a protein-protein interaction (PPI) network was established using the STRING database and visualized in the Cytoscape software (see Figure 2A and Figure 2B). Fig.15 Figure C).

[0193] The above analysis showed that ALOX15 overexpression would affect Bcl-2 gene and PPAR-γ gene, and Bcl-2 and PPAR-γ also play a key role in cancer-related pathways such as cell growth and death (Cell growth and death), Cancer Overview (Cancer:Overview).

[0194] (2) Inhibition of PPAR-γ and ERK1 / 2 phosphorylation partially abolished the anticancer effect of ALOX15 in A549 cells

[0195] Transcriptome sequencing results showed that ALOX15 overexpression affects apoptotic proteins Bcl-2 and PPAR-γ. Because the metabolites of ALOX15 are the activating ligands of PPAR-γ. Troglitazone activates PPAR-γ, which can induce apoptosis in cancer cells and lead to reduced expression of Bcl-2 and increased expression of pro-apoptotic Caspase-9 protein. In addition, the study also found that PPAR-γ agonists can rapidly activate the MEK / ERK pathway in vascular smooth muscle cells. The function of ERK is affected by its phosphorylation subcellular location. ERK1 / 2 phosphorylation forms dimers, which can enter the nucleus through active transport across the nuclear membrane to perform a large number of intracellular functions. Under specific DNA damage conditions, the activation of ERK1 / 2 can enhance the activity of some pro-apoptotic signaling molecules and further induce cell apoptosis. Therefore, the potential role of PPAR-γ and ERK1 / 2 pathways in ALOX15-induced growth arrest of LUAD cells was studied.

[0196] Overexpression of ALOX15 upregulated the mRNA level of PPAR-γ (P < 0.001) (e.g. Fig.16 A) and protein levels (P < 0.01) (e.g. Fig.16 ALOX15 overexpression had no significant effect on the total protein level of ERK1 / 2 (Figure B). Fig.16 C), but up-regulated the expression of phosphorylated ERK1 / 2 (P < 0.01) (Fig. Fig.16 This indicates that ERK1 / 2 phosphorylation activation plays a specific role in ALOX15 inhibition of LUAD.

[0197] We further examined whether the inhibitory effect of ALOX15 on LUAD cells was mediated by the PPAR-γ-ERK1 / 2 signaling axis. A549 cells overexpressing ALOX15 were treated with a PPAR-γ specific antagonist (GW9662) and an ERK1 / 2 phosphorylation inhibitor (PD98059). The experimental results showed that after inhibiting PPAR-γ (GW9662), the total protein level of ERK1 / 2 was also relatively reduced (P < 0.0001) (e.g. Fig.17 Figures A and B) and the ERK1 / 2 phosphorylation level was also relatively decreased (P < 0.01) (e.g. Fig.17 These results indicate that inhibition of PPAR-γ prevents ALOX15 from promoting ERK1 / 2 phosphorylation. After inhibition of ERK1 / 2 phosphorylation (PD98059), the expression of PPAR-γ was also relatively reduced (P < 0.001) (Figure 2). Fig.17 These results suggest that ALOX15 inhibits cancer progression by promoting ERK1 / 2 phosphorylation after activating PPAR-γ, and that inhibition of ERK1 / 2 phosphorylation in turn leads to decreased PPAR-γ expression.

[0198] In order to further detect whether the effect of ALOX15 on LUAD proliferation and apoptosis is through the PPAR-γ-ERK1 / 2 axis. The CCK-8 kit was used to detect the cell proliferation ability at different time points. The experiment showed that after the A549 cells overexpressing ALOX15 inhibited PPAR-γ and ERK1 / 2 phosphorylation, the inhibitory ability of ALOX15 on cell proliferation was significantly reduced (P < 0.05) (such as Fig.18 AnnexinV-PE / 7AAD flow cytometry double staining experiments showed that after inhibiting PPAR-γ and ERK1 / 2 phosphorylation, A549 cells overexpressing ALOX15 also significantly reduced the promotion of ALOX15 on cell apoptosis (P < 0.001) (Figure 2A and Figure 2B). Fig.18 Figures C and D of the study).

[0199] In conclusion, the expression of ALOX15 is significantly downregulated in the early stages of LUAD. The expression of ALOX15 is closely related to the differentiation of lung cancer cells, and cytokines IL-4 and IL-13 can induce significant upregulation of ALOX15. Overexpression of ALOX15 inhibits the proliferation and migration of LUAD and promotes apoptosis of LUAD. In this pathway, ALOX15-induced ERK1 / 2 phosphorylation plays an important role in its inhibition of LUAD, and acts in a PPAR-γ-dependent manner. The experiment found that the potential mechanism by which ALOX15 affects the progression of LUAD (such as Fig.19), the interaction between PPAR-γ and ERK1 / 2 may form a self-regulatory and positive feedback loop, thereby enhancing the tumor suppressor effect of ALOX15. ALOX15 is a potential novel tumor suppressor gene in LUAD and may be a promising therapeutic target.

[0200] Although the present invention has been disclosed as above by the embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various choices and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention is limited by the claims and their equivalents.

Claims

1. A lung adenocarcinoma biomarker, characterized in that: The biomarker is the ALOX15 gene or protein, and the expression of the ALOX15 gene or protein is decreased in patients with lung adenocarcinoma.

2. A reagent for detecting a biomarker for lung adenocarcinoma, characterized in that: The reagent is used to detect the expression level of ALOX15 gene or protein.

3. Use of a reagent for detecting a lung adenocarcinoma biomarker in the preparation of a product for lung adenocarcinoma diagnosis and / or prognosis assessment, characterized in that: The biomarker is the ALOX15 gene or protein, and the reagent is used to detect the expression level of the ALOX15 gene or protein.

4. The use according to claim 3, characterized in that: The product is a kit, a drug or a device.

5. The use according to claim 3, characterized in that: The test sample of the product is blood.

6. A kit for detecting lung adenocarcinoma, characterized in that: The kit comprises the reagent according to claim 3, and the reagent is used to detect the expression level of ALOX15 gene or protein.

7. A drug for preventing or treating lung adenocarcinoma, characterized in that: The medicine contains a substance capable of increasing the expression level of ALOX15 gene or protein.

8. The drug for preventing or treating lung adenocarcinoma according to claim 7, characterized in that: The substance contains anti-inflammatory factors, which can promote the overexpression of ALOX15, thereby upregulating the mRNA level of PPAR-γ, and then promoting tumor cell apoptosis through the activation of PPAR-γ to inhibit cancer progression.

9. The drug for preventing or treating lung adenocarcinoma according to claim 8, characterized in that: The substance contains anti-inflammatory factors such as IL-4 and / or IL-13.

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