15-hydroxyprostaglandin dehydrogenase

Abstract

The invention relates to application of a 15-hydroxyprostaglandin dehydrogenase detection reagent in preparation of a lung adenocarcinoma prognosis evaluation kit. According to the invention, the 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is determined as a lung adenocarcinoma metastasis / prognosis diagnosis / evaluation reagent for the first time, and the expression level of the 15-PGDH is significantly related to the metastasis risk and survival outcome of a patient; the invention deeply discloses a negative regulation mechanism of a tumor hypoxia microenvironment on 15-PGDH expression, and the biological rationality and clinical interpretation capability of metastasis judgment are remarkably improved; besides, the application potential of the 15-PGDH serving as a new target for research and development of the lung adenocarcinoma metastasis resisting medicine is determined, important theoretical support is provided for development of targeted treatment strategies, and the 15-PGDH has important conversion application value in the field of precise medical treatment.

Description

Technical Field

[0001] This invention relates to the medical field, and more particularly to 15-hydroxyprostaglandin dehydrogenase. Background Technology

[0002] Lung cancer is one of the leading causes of cancer-related morbidity and mortality worldwide, with non-small cell lung cancer (NSCLC) accounting for approximately 80% of all lung cancer cases. Despite continuous improvements in treatment methods, the high mortality rate of lung cancer remains primarily due to its highly aggressive nature and tendency to metastasize. Clinical data shows that approximately 15-20% of patients with very early-stage (T1-2 stage) lung cancer already have distant micrometastases that cannot be detected by conventional clinical methods at the time of diagnosis. Lung cancer metastasis is a complex biological process involving multiple steps and factors, including key stages such as in situ invasion, intravascular infiltration, circulating survival, distant colonization, and proliferation. Currently, traditional treatments such as surgical resection, chemotherapy, and radiotherapy have limited effectiveness in controlling lung cancer metastasis, resulting in extremely poor prognoses for patients with metastatic lung cancer.

[0003] The tumor microenvironment plays a crucial regulatory role in lung cancer metastasis, with hypoxia considered a significant initiating factor driving malignant tumor progression. Solid tumors commonly exhibit regions with significantly lower oxygen partial pressures than normal tissues due to abnormal vascular structure and vigorous metabolism. Hypoxia promotes metastasis through multiple mechanisms: hypoxia-inducible factor-1α (HIF-1α) upregulates pro-metastatic molecules such as vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs); simultaneously, the loss of p53 function under hypoxic conditions enhances the anti-apoptotic ability and metastatic potential of tumor cells. Although the association between hypoxia and tumor metastasis is widely recognized, the molecular mechanisms by which the hypoxic microenvironment regulates lung cancer metastasis remain incompletely elucidated, and therapeutic targets targeting this process are still scarce.

[0004] Prostaglandin E2 (PGE2) is a key lipid mediator in the inflammatory and tumor microenvironment, and has been shown to promote the metastasis of various tumors, including lung cancer and colorectal cancer, by activating the EP4 receptor and its downstream signaling pathways such as PI3K / AKT and EGFR. In vivo, PGE2 is produced from arachidonic acid via cyclooxygenase (COX), and the termination of its biological effects mainly depends on the degradation by 15-hydroxyprostaglandin dehydrogenase (15-PGDH, encoded by the HPGD gene), which converts PGE2 into 15-keto-PGE2 with significantly reduced biological activity. However, the changes in 15-PGDH expression and its clinical significance in the process of lung cancer metastasis remain unclear, and its interaction mechanism with the hypoxic microenvironment requires further investigation.

[0005] In current technologies, prognostic assessment of metastatic non-small cell lung cancer (NSCLC) mainly relies on TNM staging, imaging examinations, and traditional tumor markers (such as CEA and CYFRA21-1). However, these methods lack sufficient sensitivity and specificity in early prediction of metastasis risk and assessment of individualized prognosis. Particularly for lung adenocarcinoma, a major subtype of NSCLC, there is still a lack of reagents capable of efficiently predicting metastatic progression, making it difficult to implement effective interventions in the early stages of the disease and missing the optimal treatment window. Therefore, there is an urgent need in this field to develop novel and reliable prognostic reagents (kits) for lung cancer metastasis and to establish a molecular mechanism-based assessment system to improve precision diagnosis and treatment and survival outcomes for lung cancer patients. Summary of the Invention

[0006] This invention aims to overcome the shortcomings of the prior art, and to this end, the following technical solution is adopted: The first aspect of the present invention is to provide the use of a 15-hydroxyprostaglandin dehydrogenase detection reagent in the preparation of a prognostic assessment kit for lung adenocarcinoma.

[0007] A second aspect of the present invention is to provide the use of a 15-hydroxyprostaglandin dehydrogenase detection reagent in the preparation of a diagnostic kit for lung adenocarcinoma metastasis.

[0008] A third aspect of the present invention is to provide the application of a combined detection reagent for 15-hydroxyprostaglandin dehydrogenase and hypoxia microenvironment markers in the preparation of a diagnostic kit for lung adenocarcinoma metastasis.

[0009] Preferably, the hypoxic microenvironment marker includes carbonic anhydrase 9.

[0010] A fourth aspect of the present invention is to provide the use of a 15-hydroxyprostaglandin dehydrogenase activator in the preparation of a medicament for treating metastatic lung adenocarcinoma.

[0011] Compared with the prior art, the beneficial effects of this invention are reflected in: This invention is the first to clearly define 15-hydroxyprostaglandin dehydrogenase (15-PGDH) as a diagnostic / prognostic reagent for lung adenocarcinoma metastasis, and its expression level is significantly correlated with patient metastasis risk and survival outcome. This application further reveals the negative regulatory mechanism of 15-PGDH expression by the tumor hypoxic microenvironment, significantly improving the biological rationale and clinical interpretability of metastasis assessment. In addition, this invention establishes the application potential of 15-PGDH as a new target for the development of anti-lung adenocarcinoma metastasis drugs, providing important theoretical support for the development of targeted therapy strategies, and has significant translational application value in the field of precision medicine. Attached Figure Description

[0012] Figure 1 Box plots showing the expression level and translocation of 15-PGDH in Example 1 of this application are shown; Figure 2 The results of Kaplan-Meier survival analysis in Embodiment 1 of this application are shown; Figure 3 Immunohistochemical staining images and statistical analysis results from Example 2 of this application are shown; Figure 4 The results of Kaplan-Meier survival analysis of the clinical cohort in Example 2 of this application are shown; Figure 5 The bar chart shows the experimental results of ELISA detection of PGE2 content in tumor cells in Example 3 of this application; Figure 6 Images of transwell invasion staining in Embodiment 4 of this application are shown; Figure 7 A line graph showing the change of HPGD mRNA levels over time in Example 5 of this application is shown; Figure 8 The following is a Western blot plot showing the change in 15-PGDH protein level over time in Example 6 of this application; Figure 9 An immunofluorescence staining image of tumor tissue in Example 6 of this application is shown; Figure 10 The following are in vivo imaging images and statistical results from Example 7 of this application. Detailed Implementation

[0013] The specific embodiments of the present invention will be described in detail below.

[0014] Unless otherwise defined, the technical or scientific terms used in the claims and description shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0015] The word "comprising" or similar terms used in the specification and claims of this patent application mean that the objects preceding "comprising" include the objects listed after "comprising" or their equivalents, and do not exclude other objects.

[0016] In this invention, the T1-2 stage lung adenocarcinoma subtype refers to the lung adenocarcinoma subtype with a smaller primary tumor (T1-2); 15-PGDH refers to 15-hydroxyprostaglandin dehydrogenase, a member of the prostaglandin E2 (PGE2) metabolic enzyme family; the hypoxic microenvironment refers to a region within a solid tumor where the oxygen concentration is significantly lower than that of normal tissue. It is not a uniform state, but rather a heterogeneous and dynamic pathological feature caused by abnormal tumor structure and function. Generally, when the oxygen partial pressure is lower than 1%-2% (normal tissue is about 2%-9%), it can be identified as a hypoxic region. This environment is one of the key driving factors for malignant tumor progression and treatment resistance.

[0017] Example 1 Transcriptome data of non-small cell lung cancer patients were obtained using the Kaplan-Meier Plotter online database. Cases were divided into a non-metastasis (Non-metastasis, T...) group based on TNM staging criteria. 1-4 N0M0, n=628) and metastasis (T) n N ≥1 M ≥1 (n=212), compare the differences in 15-PGDH expression levels between the two groups; such as Figure 1 As shown, the expression level of 15-PGDH in the metastasis group was significantly lower than that in the non-metastasis group, with a Logrank test P value of 8.73 × 10⁻⁶. -9 This indicates that low expression of 15-PGDH is statistically significantly associated with tumor metastasis.

[0018] Further, using the median 15-PGDH expression level as a threshold, all patients were divided into a high-expression group (High, n=1082) and a low-expression group (Low, n=1084), Kaplan-Meier survival curves were plotted, and hazard ratios were calculated; Figure 2 As shown, the hazard ratio (HR) for the 15-PGDH high expression group was 0.72, with a 95% confidence interval of 0.64–0.81, and the Logrank test p-value was 3.6 × 10⁻⁶. -8 The survival curve of the high-expression group was above that of the low-expression group, confirming that high expression of 15-PGDH is significantly associated with a good prognosis in patients with non-small cell lung cancer.

[0019] Example 2 Thirty lung adenocarcinoma tissue specimens of stage T1N0M0 (without lymph node metastasis) and stage T1N2M0 (with lymph node metastasis) diagnosed by postoperative pathology were collected from Shanghai Pulmonary Hospital. The staging of all cases was determined according to the 8th edition of the AJCC TNM staging criteria.

[0020] Lung or tumor tissue was fixed with 4% formaldehyde solution, followed by washing three times in phosphate buffer for 5 minutes each time. The tissue was then treated sequentially with 70%, 80%, and 90% ethanol solutions for 30 minutes each, then with 95% and 100% ethanol solutions twice each for 20 minutes each. Next, it was treated with a 1:1 mixture of 100% ethanol and xylene for 15 minutes, and finally treated with xylene alone until the tissue became transparent. Molten paraffin was poured into a prepared container, and the paraffin-impregnated tissue was quickly transferred into this container. After cooling, a paraffin block was obtained. The paraffin block was fixed in a microtome, sectioned to a standard thickness of 3 mm, flattened in warm water, quickly lifted with a glass slide, and dried on a 45°C slide dryer.

[0021] The slides were heated in a 60°C oven for 2 hours, then dewaxed using a series of xylene solutions, with the ethanol concentration gradually reduced to 70%, and finally washed with phosphate buffer. The slides were treated with 3% hydrogen peroxide solution for 20 minutes to block endogenous peroxidase activity, followed by washing with phosphate buffer. The samples were then heated in boiling EDTA buffer (1×) for 20 minutes, allowed to cool naturally to room temperature, and finally washed with phosphate buffer. Blocking was performed using blocking buffer containing 5% bovine serum albumin and 0.5% Triton X-100, and incubated at room temperature for 1 hour. Primary antibody solution was added, and the slides were incubated overnight in a humidified chamber. After returning to room temperature, the slides were washed with phosphate buffer and the corresponding secondary antibody solution was added, incubated at room temperature for 1 hour, and then washed again with phosphate buffer. DAB chromogenic solution was added for staining, and the staining was stopped with double-distilled water after observing a brownish-yellow positive reaction. Hematoxylin staining was performed for approximately 1 minute, followed by rinsing with tap water. After treatment with differentiation solution for 30 seconds, the sections were rinsed with tap water to proceed with differentiation. Dehydration was then carried out using ethanol solutions of varying concentrations, followed by treatment with xylene. After drying, the sections were mounted with neutral resin.

[0022] like Figure 3 As shown, the expression level of 15-PGDH in tumor tissues of the T1N2M0 group with lymph node metastasis was significantly lower than that in the T1N0M0 group without metastasis (weakened brown-yellow positive signal). Kaplan-Meier survival analysis was performed on the aforementioned clinical cohorts, and the results are as follows: Figure 4 As shown, the Logrank test P value for the difference in survival rate between the high-expression group and the low-expression group of 15-PGDH was 0.016, indicating that the survival rate of the high-expression group was significantly better than that of the low-expression group, further verifying the positive correlation between 15-PGDH expression and the prognosis of lung adenocarcinoma patients.

[0023] Example 3 Lentiviral-mediated short hairpin RNA technology was employed. The shRNA specifically targeting the HPGD gene (shHPGD) and the non-specific control shNC lentiviral particles were both purchased from Beijing Qingke Biotechnology Co., Ltd. The nucleotide sequence of the shRNA targeting the HPGD gene is 5'-GGCATCATTATCAATATGT-3' (SEQ ID NO: 1) or / and 5'-GTTGGCTGCTAATCTTATG-3' (SEQ ID No: 2).

[0024] 1. Construct a stable cell line with knocked-down HPGD gene (shHPGD) and a control cell line (shNC). The specific process is as follows: 1) Determine the lentiviral multiplicity of infection (MOI): Select the A549 cell line as the target for lentiviral transfection. When the cells are in good growth condition, prepare a single-cell suspension after digestion and continue to culture. When the cell density reaches 20% to 30%, change the culture medium, add different titers of virus and corresponding infection enhancement solution, and observe the GFP fluorescence intensity under a microscope.

[0025] 2) Determine the screening concentration of puromycin.

[0026] 3) Construction of stable HPGD gene-silenced cell lines via lentiviral infection: Cell lines with the best interference effect were screened by observing GFP fluorescence intensity and detecting HPGD expression levels using qPCR and Western Blot. The screening criteria were: qPCR showed a decrease in HPGD mRNA expression level of at least 70% compared to the shNC group; Western Blot results, analyzed using ImageJ software, showed that 15-PGDH protein expression was downregulated by at least 60% compared to the internal control β-tubulin; and infection efficiency was also considered based on GFP fluorescence observation. Cells with stable and significant interference effects were selected for subsequent experiments.

[0027] The HPGD primer sequences used in qPCR detection are as follows: upstream primer: 5'-TGTTCATCCAGTGCGATGTGG-3' (SEQ ID NO: 3); downstream primer: 5'-GGGCATGAGTCCTGCTAAAGA-3' (SEQ ID NO: 4); the primer sequences for the internal reference gene Actin are as follows: upstream primer: 5'-ACCTTCTACAATGAGCTGCGTGT-3' (SEQ ID NO: 5); downstream primer: 5'-ACAGGGATAGCACAGCCTGG-3' (SEQ ID NO: 6).

[0028] 2. ELISA detection of intracellular PGE2 levels The PGE2 detection ELISA kit was purchased from Shanghai Sangon Biotech Co., Ltd. The specific detection process is as follows: 1) Sample Preparation: Tumor cells were collected into centrifuge tubes using trypsin digestion. The cells were washed once with pre-chilled phosphate-buffered saline (1×PBS), centrifuged at 4°C and 1000×g for 5 minutes, and the supernatant was discarded to remove serum proteins and other impurities from the culture medium. A cell suspension was then prepared using 100 μL of PBS. The centrifuge tubes containing the cell suspension were sealed and immersed in a -80°C freezer for 10 minutes until the sample was completely frozen. After freezing, the sample was removed from the cryogenic environment and immediately placed in a warm environment and gently mixed to accelerate thawing. This cycle was repeated three times. After all freeze-thaw cycles were completed, the lysate was centrifuged at 4°C and 12,000×g for 15 minutes, and 50 μL of the supernatant was collected for analysis.

[0029] 2) Incubation: Add 50 μL of the sample to be tested to the corresponding well, and set up duplicate wells and blank wells (add only PBS). Add 50 μL of biotin-labeled PGE2 antigen to each well, seal and incubate at 37°C for 45 minutes.

[0030] 3) Second incubation: Discard the liquid in the wells, wash 5 times with PBST, dilute horseradish peroxidase-labeled streptavidin to the working concentration with diluent, add 100 μL of diluted HRP-streptavidin to each well, seal and incubate at 37°C in the dark for 30 minutes.

[0031] 4) Color Development: Discard the liquid in the wells, wash 5 times with PBST, add 100 μL of freshly prepared TMB substrate solution to each well, and develop the color at room temperature in the dark for 15-20 minutes, closely observing the color change. When the high-concentration wells of the standard show a clear blue color and a good gradient, add 50 μL of 2M H2SO4 stop solution to each well; the solution color will then change from blue to yellow.

[0032] 5) Detection: The absorbance of each well was measured at a wavelength of 450 nm using an ELISA reader, and calibrated using 620 nm as a reference wavelength.

[0033] like Figure 5 As shown, after stable knockdown of the HPGD gene, the PGE2 content in tumor cells was significantly higher than that in the control group (shNC), indicating that 15-PGDH has the function of degrading PGE2 under physiological conditions.

[0034] Example 4 After melting Matrigel overnight at 4°C, it was diluted 1:10 with pre-cooled serum-free medium. 200 μL of this diluted solution was used to evenly coat the polycarbonate membrane at the bottom of each Transwell chamber. The membrane was then incubated at 37°C with 5% CO2 for 2 hours to allow polymerization. Logarithmic growth phase A549 cells were trypsinized to prepare single-cell suspensions, and then cultured at 8 × 10⁸ cells per chamber. 4Cells were seeded in the upper chamber, and 700 μL of culture medium containing 10% fetal bovine serum was added to the lower chamber as a chemokine. The cells were incubated at 37°C and 5% CO2 for 48 hours. The chambers were then removed, washed with phosphate-buffered saline, fixed with 4% paraformaldehyde for 20 minutes, stained with 0.1% crystal violet for 15 minutes, and the uninvaded cells on the membrane surface were wiped away with a moistened cotton swab. Five cells were randomly selected at 200x magnification under a microscope to count the number of cells that had penetrated the matrix gel and membrane. Figure 6 As shown, treatment with the 15-PGDH small molecule inhibitor SW033291 or stable knockdown of the HPGD gene significantly increased the number of invasive cells compared with the control group, indicating that inhibiting the expression or function of 15-PGDH can significantly enhance the metastatic ability of lung adenocarcinoma cells.

[0035] Example 5 Lung adenocarcinoma cells were cultured under hypoxic and normoxic conditions, respectively. Total RNA was extracted using Trizol reagent, ensuring an RNA purity 260 / 280 ratio between 1.8 and 2.0. 800 ng of total RNA was used to synthesize cDNA using a reverse transcription kit at 37°C for 15 minutes and then inactivated at 85°C for 5 seconds. Real-time quantitative PCR was performed using SYBR Green Mix. The primer sequences for the HPGD gene are as follows: upstream primer SEQ ID NO: 3, downstream primer SEQ ID NO: 4. The primer sequences for the internal control gene Actin are as follows: upstream primer SEQ ID NO: 5, downstream primer SEQ ID NO: 6. 2... -ΔΔCt The relative expression level of HPGD mRNA was calculated using this method. Figure 7 As shown, the expression level of HPGD mRNA under hypoxic conditions was significantly downregulated compared to normal oxygen conditions. P <0.05 indicates a negative regulatory effect of the hypoxic microenvironment on HPGD transcription.

[0036] Example 6 Lung adenocarcinoma cells were treated under hypoxic and normoxic environments, respectively. Lysis was then performed on ice for 30 minutes using RIPA lysis buffer containing protease and phosphatase inhibitors. The supernatant was collected by centrifugation at 14,000×g for 15 minutes at 4°C. Protein concentration was determined using the BCA method and adjusted to 40 μg / μL. Denaturation was then performed at 95°C for 15 minutes. SDS-PAGE electrophoresis was performed using a 10% separating gel and a 5% stacking gel at 80V for 30 minutes, followed by a change to 120V for continued electrophoresis. Proteins were transferred to a PVDF membrane using wet transfer and blotting at a constant current of 300mA for 90 minutes. After blocking with 5% skim milk powder at room temperature for 1 hour, the membrane was incubated overnight at 4°C with 15-PGDH-specific primary antibody. After washing with TBST buffer, the membrane was incubated at room temperature with horseradish peroxidase-labeled secondary antibody for 1 hour. Chemiluminescent substrate imaging was then performed. Figure 8As shown, the 15-PGDH protein level was significantly reduced in the hypoxia treatment group. Simultaneously, 4 μm thick frozen sections of lung adenocarcinoma tissue were prepared, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and subjected to microwave thermal retrieval of the antigen using sodium citrate buffer (pH 6.0). The tissue was blocked with 5% normal serum for 30 minutes, incubated overnight at 4°C with 15-PGDH primary antibody, incubated for 1 hour at room temperature with fluorescently labeled secondary antibody, stained with DAPI, and observed under a confocal microscope. Figure 9 As shown, 15-PGDH is mainly expressed in the normoxic region of tumors, and its expression is weakened in the region positive for the hypoxia marker CA9. The spatial distribution suggests that the hypoxic microenvironment has a negative regulatory effect on the expression of 15-PGDH protein.

[0037] Example 7 An adeno-associated virus vector carrying the 15-PGDH gene expression cassette was constructed. The coding sequence of the 15-PGDH gene is as follows: 5'--3' (SEQ ID NO: 7). The construction of stable transfected cell lines was carried out in accordance with the steps described in Example 1. In this example, the A549 cell line that stably overexpressed the luciferase reporter gene was constructed in Example 1.

[0038] Establishment of a lung metastasis model in immunodeficient mice 1) Laboratory Animals: Sixteen healthy male Balb / c nude mice, aged 4-6 weeks, were purchased from the Shanghai Laboratory Animal Center. All laboratory animals were housed in the specific pathogen-free facility of the Laboratory Animal Center of Shanghai Pulmonary Hospital affiliated with Tongji University. Before the experiment, the nude mice were randomly divided into two groups of eight each: a control group (AAV-Vector) and an experimental group (AAV-15-PGDH), and underwent a one-week environmental acclimatization period.

[0039] 2) Cell suspension preparation: Cells were digested and collected on the day of tumor cell inoculation, and a suspension was prepared with a concentration of 2×10⁻⁶. 7 Cell suspension per mL.

[0040] 3) Tail vein injection: 100 μL of the above cell suspension was drawn using an insulin syringe and injected into the tail vein of nude mice. After the injection, the needle was slowly withdrawn and the injection site was pressed for a few seconds to prevent leakage of the liquid.

[0041] 4) AAV administration: On the day following the first injection of cell suspension, 100 μL of AAV-Vector or AAV-15-PGDH was drawn using an insulin syringe and administered to immunodeficient mice via intraperitoneal injection.

[0042] 5) Tumor metastasis monitoring: Starting from the first month after inoculation, tumor growth was observed using a small animal in vivo imaging system, and tumor metastasis dynamics were recorded weekly. A549 cell lines overexpressing the luciferase reporter gene were injected into nude mice via tail vein injection and treated with AAV. Tumor formation was detected in the first month, and tumor metastasis was monitored weekly. On day 60, the nude mice were sacrificed and the tumor tissue was removed.

[0043] like Figure 10 As shown, the tumor metastasis level in the control group (AAV-Vector) was significantly stronger than that in the 15-PGDH overexpression group (AAV-15-PGDH).

[0044] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the description and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

Application of 1,15-hydroxyprostaglandin dehydrogenase detection reagent in the preparation of a prognostic assessment kit for lung adenocarcinoma. Application of 2,15-hydroxyprostaglandin dehydrogenase detection reagent in the preparation of a diagnostic kit for lung adenocarcinoma metastasis. Application of a combined detection reagent for 3,15-hydroxyprostaglandin dehydrogenase and hypoxia microenvironment markers in the preparation of a diagnostic kit for lung adenocarcinoma metastasis.

4. The application according to claim 3, characterized in that, The markers of the hypoxic microenvironment include: carbonic anhydrase 9. Application of 5,15-hydroxyprostaglandin dehydrogenase activator in the preparation of drugs for treating lung adenocarcinoma metastasis.

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