Library construction method based on high-throughput sequencing endometrial cancer molecular typing

Abstract

The invention relates to a library construction method based on high-throughput sequencing endometrial cancer molecular typing, and relates to the technical field of medical data process.The library construction method includes the steps that a clinical index-molecular feature linked dynamic typing system is established, and sequencing data of peripheral blood LDL concentration and other clinical metabolic indexes and tumor tissue pathway genes are integrated; a multi-dimensional data analysis model is established, double typing of metabolic phenotypes and molecular pathways of endometrial cancer is realized, a basis is provided for matching differentiated treatment schemes for patients with different subtypes, the typing specificity and accuracy are improved, a high-throughput sequencing library of targeted LDL metabolism and JAK / STAT pathway genes is constructed, and a high-throughput sequencing library of targeted LDL metabolism and JAK / STAT pathway genes is established. Nucleic acid fragments of key genes such as JAK2, STAT3 and LDL receptors are enriched through a specific probe, irrelevant nucleic acid interference is eliminated, the detection sensitivity and typing accuracy of LDL-JAK / STAT pathway abnormal endometrial cancer are improved, and the technical blank of special subtype molecular typing is filled.

Description

Library Construction Methods for Molecular Subtyping of Endometrial Cancer Based on High-Throughput Sequencing Technical Field

[0001] This invention relates to the field of medical data processing technology, and in particular to a method for constructing libraries based on molecular typing of endometrial cancer using high-throughput sequencing. Background Technology

[0002] Current Status of Endometrial Cancer Diagnosis and Treatment: Endometrial cancer is a common malignant tumor of the female reproductive system, with an increasing incidence rate and a trend towards younger patients. The recurrence and mortality rates for advanced-stage patients remain high. Traditional diagnostic and treatment methods primarily rely on pathological biopsy and imaging assessments. While these methods can clarify the pathological stage, they cannot achieve precise subtyping based on molecular mechanisms, leading to poor treatment outcomes for some patients due to insufficient targeted treatment plans. Furthermore, research has confirmed that endometrial cancer patients often have lipid metabolism disorders. Low-density lipoprotein can promote cancer cell proliferation, migration, and invasion by activating the JAK / STAT signaling pathway. Patients with these unique pathogenesis mechanisms require differentiated treatment strategies, but current technologies lack corresponding molecular subtyping systems to support this approach.

[0003] High-throughput sequencing technology has been widely used in tumor molecular subtyping research, enabling high-throughput detection of gene expression and mutations in tumor tissues, providing a core tool for disease molecular subtyping and targeted therapy target screening. However, for endometrial cancer, existing sequencing library construction methods are mostly generalized, failing to incorporate its unique molecular characteristics such as lipid metabolism abnormalities and JAK / STAT pathway activation. This results in insufficient differentiation of specific subtypes in sequencing data, making it difficult to meet the needs of precision diagnosis and treatment.

[0004] Tumor molecular subtyping can break through the limitations of traditional pathological staging and achieve "different treatments for the same disease". Currently, mature molecular subtyping systems have been established for tumors such as breast cancer and lung cancer and guide clinical treatment. However, molecular subtyping research on endometrial cancer is still in the exploratory stage, especially lacking a subtyping model that integrates lipid metabolism indicators and pathway gene expression. Targeted library construction technology is urgently needed to promote the improvement of the subtyping system.

[0005] The closest existing technical solutions can be mainly divided into the following two categories:

[0006] I. General-Purpose High-Throughput Sequencing Library Construction Technology for Tumors: This technology is a fundamental tool for tumor molecular subtyping. The overall process involves sample nucleic acid extraction, end repair, adapter ligation, PCR amplification, and sequencing. For example, for solid tumor tissue, genomic DNA or RNA is first extracted using conventional methods, a library is constructed using a kit, and then whole-genome or transcriptome sequencing is performed using platforms such as Illumina. Finally, molecular subtyping is performed based on gene expression profiles or mutation characteristics. This technology has a mature library construction process and standardized operating system, enabling high-throughput detection of large-scale samples; however, it has significant limitations. It does not target and enrich specific molecular markers for endometrial cancer (such as JAK2, STAT3, and LDL receptor-related genes), resulting in a high proportion of irrelevant information in the sequencing data. It has weak identification ability for LDL-JAK / STAT pathway abnormalities in endometrial cancer and does not integrate clinical indicators such as peripheral blood LDL concentration, making it impossible to establish a correlation between "clinical phenotype and molecular characteristics."

[0007] II. Research Techniques Related to Molecular Subtyping of Endometrial Cancer: Some studies have attempted molecular subtyping of endometrial cancer. The techniques primarily involve collecting tumor tissue samples and using first-generation sequencing or microarray technology to detect mutations or expression levels of specific genes (such as PTEN and TP53), then classifying subtypes based on pathological characteristics. Other studies utilize conventional transcriptome sequencing libraries to analyze gene expression differences and perform subtyping, exploring differences in prognosis and treatment sensitivity among different subtypes. A preliminary research framework for molecular subtyping of endometrial cancer has been established, confirming its value in prognostic assessment. However, it has not incorporated the specific clinical characteristic of abnormal lipid metabolism, has not included LDL concentration and JAK / STAT pathway genes in the subtyping indicators, and the library construction methods used lack targeting, failing to efficiently capture expression information of key pathway genes. Therefore, the subtyping results are insufficient to guide precise treatment for patients with lipid metabolism disorders.

[0008] To address the aforementioned technical deficiencies, a solution is proposed. Summary of the Invention

[0009] The purpose of this invention is to improve the specificity and accuracy of subtyping, construct a high-throughput sequencing library targeting LDL metabolism and JAK / STAT pathway genes, enrich nucleic acid fragments of key genes such as JAK2, STAT3, and LDL receptor through specific probes, eliminate irrelevant nucleic acid interference, improve the detection sensitivity and subtyping accuracy of LDL-JAK / STAT pathway abnormal endometrial cancer, and fill the technical gap in molecular subtyping of this special subtype.

[0010] To achieve the above objectives, the present invention employs the following technical solution: a library construction method for endometrial cancer based on high-throughput sequencing molecular subtyping, comprising the following steps:

[0011] S1. Establish a correlation mechanism at the clinical sample level: Endometrial cancer patients showed obvious lipid metabolism abnormalities and pathway activation characteristics. Their peripheral blood LDL concentration was significantly higher than that of patients with benign endometrial hyperplasia. At the same time, the expression levels of phosphorylated JAK2 and phosphorylated STAT3 proteins in tumor tissue were abnormally elevated, suggesting that there is a direct clinical correlation between abnormal accumulation of LDL and activation of the JAK / STAT pathway.

[0012] S2. Verify the mechanism of action at the cellular level: including the dose-dependent oncogenic effect of LDL and the reversal effect of pathway inhibitors;

[0013] S3. Validation mechanism at the animal level: A nude mouse subcutaneous endometrial cancer xenograft model was constructed. In the model, it was verified that after LDL injection via the tail vein, the volume and weight of the xenograft increased compared with the control group. The expression levels of p-JAK2, p-STAT3 and the proliferation marker Ki-67 in the tumor tissue were increased, while the expression of the apoptosis-related protein caspase-3 was decreased. This verified that LDL can activate the JAK / STAT pathway in vivo, promote the proliferation of tumor tissue and inhibit its apoptosis, accelerate tumor growth, and clarify the role of the LDL-JAK / STAT pathway axis in the progression of endometrial cancer.

[0014] Furthermore, the dose-dependent oncogenic effect of LDL is specifically demonstrated by the fact that when endometrial cancer cells are stimulated by different concentrations of LDL, the uptake of LDL by the cells increases with the increase of LDL concentration. At the same time, the expression levels of p-JAK2 and p-STAT3 are upregulated in a dose-dependent manner, the JAK / STAT pathway is activated, and the proliferation, migration and invasion of cancer cells are enhanced along with the activation of the pathway. This indicates that LDL can promote the malignant phenotype of cancer cells by activating the JAK / STAT pathway.

[0015] Furthermore, the reversal effect of the pathway inhibitor is specifically demonstrated by pretreatment of cancer cells with the JAK2-specific inhibitor SD-1029, which blocks LDL-induced JAK / STAT pathway activation. Simultaneously, LDL-mediated cancer cell proliferation, migration, and invasion are significantly inhibited, confirming that the JAK / STAT pathway is the core signaling pathway for LDL to exert its oncogenic effect.

[0016] Furthermore, the specific process for establishing the association mechanism at the clinical sample level is as follows:

[0017] S101, Clinical Trials:

[0018] Fifty patients with endometrial cancer and 50 patients with benign endometrial hyperplasia due to other benign diseases underwent surgery. Surgical tissue samples and peripheral blood were collected, and clinical information was collected. The LDL level in peripheral blood and the expression and activation of JAK and STAT3 in tumor tissue were detected. Informed consent forms were signed by all patients.

[0019] Specific inclusion criteria: 1. No history of metabolic diseases and no use of any hormone drugs in the past 3 months; 2. Patients with good follow-up conditions and good medical compliance; 3. Exclusion criteria: 1. Patients with a history of hormone replacement therapy; 2. Patients who have been taking hormone drugs for a long time due to other systemic diseases; 3. Patients with liver and kidney dysfunction; 4. Patients who have been taking statins to lower blood lipids; 5. Patients who have been taking oral medications for metabolic diseases such as diabetes.

[0020] Blood samples, LDL samples, and endometrial samples were collected from the patient. The endometrial samples were retrieved during the operation, fixed, and sent to the central laboratory for processing. The specific experimental steps were the same as the in vitro experimental procedures below. The degree of activation of the JAK2 / STAT3 pathway in patients with endometrial cancer at different stages was measured and compared, and all results were recorded in detail.

[0021] S102, Laboratory Experiment;

[0022] S1021, In vitro experiments:

[0023] Ishikawa cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum, 1×10⁵ U / L penicillin, and 1×10⁵ / L streptomycin, and incubated at 37°C in a 5% CO₂ incubator. When the cells reached 80%–90% confluence, they were passaged. When the cells had essentially covered 80%–90% of the bottom of the culture flask, they were passaged again.

[0024] S1022, Detecting cell proliferation:

[0025] Collect cells in the logarithmic growth phase and prepare a single-cell suspension;

[0026] After the passaged cells adhered and grew, they were starved in serum-free DMEM for 24 hours to synchronize the cells.

[0027] LDL at concentrations of 0, 10, 50, 100, and 150 ng / ml were added, and the cells were cultured for 6 h, 12 h, and 24 h, respectively. The maximum effective dose of LDL was preset. The cells cultured with the maximum effective dose of LDL were used in the following processes.

[0028] The JAK2 pathway-specific inhibitor AG490 of LDL was added at the maximum effective dose and cultured for 6h, 12h and 24h respectively.

[0029] The cells were cultured for 6 h, 12 h, and 24 h after adding the maximum effective dose of the STAT3 pathway-specific inhibitor JSI-124 for LDL, respectively.

[0030] The optical density of each well was measured at a wavelength of 570 nm using an ELISA reader.

[0031] Cell proliferation rate (%) = (OD value of experimental group - OD value of 0 ng / ml LDL group in the same time period) / OD value of 0 ng / ml LDL group in the same time period × 100%

[0032] S1023, Cell Scratch Test:

[0033] Seed Ishikawa cells into a 6-well plate with three evenly drawn horizontal lines on the back. After the cells covered the plate, use a pipette tip to draw lines perpendicular to the drawn horizontal lines.

[0034] Wash twice with 1 mL PBS, then add 2 mL serum-free 1640.

[0035] Observed under an inverted microscope at 0, 12, and 24 hours.

[0036] S1024, Transwell cell invasion assay:

[0037] Melt Matrigel overnight at 4°C;

[0038] Dilute Matrigel with serum-free culture medium pre-cooled to 4°C. The ratio of Matrigel gel to serum-free 1640 is 1:7.

[0039] Add 70 μL of diluted Matrigel to the upper chamber and incubate at 37°C for 4-5 hours.

[0040] Digest the cells, centrifuge at 800 r / min for 5 min, resuspend the cells, count them, and adjust the cell count to 2 × 10⁵ / mL;

[0041] Add 800 μL of culture medium containing 10% serum to the lower chamber, and add 100 μL of cell suspension, Ishikawa cells treated with LDL only, Ishikawa cells treated with LDL+AG490, and Ishikawa cells treated with LDL+JSI-124 to the upper chamber. Continue to culture in a 37°C incubator for 24 h.

[0042] Remove the chamber, gently rinse and soak it several times with clean water, remove the chamber, absorb the liquid, and wipe away the cells on the membrane surface at the bottom of the chamber with a damp cotton swab;

[0043] Fixation was performed using 4% paraformaldehyde, followed by staining with crystal violet. After cleaning and wiping, images were taken under a microscope and statistically analyzed.

[0044] S1025. Detection of JAK2 / STAT3 pathway expression and activation:

[0045] The expression and activation levels of the JAK2 / STAT3 pathway in Ishikawa cells, LDL+AG490 cells, and LDL+JSI-124 cells were detected by immunoblotting and qPCR.

[0046] 1) Prepare the relevant reagents;

[0047] 2) Extract total protein from cells;

[0048] 3) Determine protein concentration;

[0049] 4) Electrophoresis, membrane transfer, and immunochromatographic analysis;

[0050] S103, In vivo experiments:

[0051] S1031. Establishment of animal models:

[0052] After centrifuging to collect Ishikawa cells, they were resuspended in PBS. The cell suspension was then mixed with Matrigel at a 1:1 ratio to adjust the cell density to 7.5 × 10¹⁰ / L.

[0053] Nude mice were placed in a clean bench, and after the skin was disinfected, 200 μL of cell suspension (approximately 1.5 × 10⁷ cells) was injected subcutaneously into the right back scapula of the nude mice. The mice were housed in an SPF environment, and the condition of the nude mice, the size and hardness of the tumors at the injection site were observed daily. After one week, all nude mice were observed to have subcutaneous xenografts (diameter > 5 mm) on the right back scapula.

[0054] S1032, Grouping of animal models:

[0055] Tumor-bearing nude mice were randomly divided into four groups: experimental group 1 (LDL + AG490), experimental group 2 (LDL + JSI-124), control group (LDL), and blank group (physiological saline), with eight mice in each group. Each group received the corresponding drug via tail vein injection once every 3 days for a total of 3 weeks.

[0056] S1033, Observation Indicators:

[0057] Before each administration of medication, the major diameter (a) and minor diameter (b) of the tumor in nude mice were measured with vernier calipers. The tumor volume was calculated according to the formula (V=ab2 / 2), and a tumor growth curve was plotted.

[0058] After treatment, all nude mice were euthanized by cervical dislocation. The tumors were dissected in a clean bench, and after removing fat and blood, their weight was measured. The tumor inhibition rate was calculated as follows: Tumor inhibition rate (%) = (Tumor weight of control group - Tumor weight of treatment group) / Tumor weight of control group × 100%.

[0059] Furthermore, the clinical sample-level correlation mechanism is specifically verified by comparing serum and tissue samples from the case group and the control group, simultaneously verifying the synchronous abnormalities of LDL concentration and JAK / STAT pathway activation level, establishing the clinical correlation between the two and EC, and laying a clinical foundation for subsequent verification of the molecular mechanism by which LDL activates the JAK / STAT pathway to promote EC proliferation, migration, and invasion at the cellular and animal levels.

[0060] Furthermore, the specific process for constructing a nude mouse subcutaneous endometrial cancer xenograft model is as follows:

[0061] S301. Selection and Feeding of Laboratory Animals:

[0062] Animal breeds and specifications: Twenty male C57BL / 6 nude mice aged 4-6 weeks were selected and purchased from Beijing Vital River Laboratories Co., Ltd.

[0063] Housing environment: Nude mice were housed in a specific pathogen-free (SPF) environment to ensure that the experimental environment met the requirements of animal experimentation ethics;

[0064] S302, Cell Preparation:

[0065] Cell lines: Two endometrial cancer cell lines were selected—Ishikawa and RL95-2 (both purchased from the American Type Culture Collection Center (ATCC)).

[0066] Cell treatment: Two types of cells were prepared into cell suspensions with a concentration of 2 × 10⁻⁶. 7 100 μL of cells were suspended in dimethyl sulfoxide for later use.

[0067] S303, Model Construction Steps:

[0068] Cell inoculation: The prepared Ishikawa cell suspension and RL95-2 cell suspension were injected subcutaneously into the subcutaneous tissue of nude mice to complete the tumor cell inoculation.

[0069] S304. Grouping: After inoculation, nude mice were divided into 4 groups of 5 mice each, as follows:

[0070] Ishikawa control group: Ishikawa cells were injected with an equal amount of DMSO, without any additional treatment;

[0071] IshikawaLDL group: LDL intervention was given after injection of Ishikawa cells;

[0072] RL95-2 control group: only RL95-2 cells were injected with an equal amount of DMSO, with no additional treatment;

[0073] RL95-2LDL group: LDL intervention was given after injection of RL95-2 cells;

[0074] Intervention measures: After tumor cell inoculation, LDL was intravenously injected into nude mice in the LDL group every 3 days; nude mice in the control group were injected with an equal amount of DMSO as a control.

[0075] S305, Model Monitoring and Sample Collection:

[0076] Tumor volume monitoring: Once the tumor is visible to the naked eye, the tumor volume is measured every 7 days. The calculation formula is: Tumor volume (mm³) = Long diameter (L) × Short diameter (W)² / 2, where L is the longest diameter of the tumor and W is the shortest diameter of the tumor.

[0077] Experimental endpoint determination and sample processing: When the tumor diameter reached 2 cm, the nude mice were euthanized, the tumor tissue was completely dissected, the tumor weight was measured, and the tumor tissue was further analyzed.

[0078] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0079] 1. This library construction method based on high-throughput sequencing for molecular subtyping of endometrial cancer improves the specificity and accuracy of subtyping. It constructs a high-throughput sequencing library targeting LDL metabolism and JAK / STAT pathway genes, enriches nucleic acid fragments of key genes such as JAK2, STAT3, and LDL receptor with specific probes, eliminates irrelevant nucleic acid interference, improves the detection sensitivity and subtyping accuracy of LDL-JAK / STAT pathway abnormal endometrial cancer, and fills the technical gap in molecular subtyping of this special subtype.

[0080] 2. This library construction method based on high-throughput sequencing for molecular subtyping of endometrial cancer establishes a "clinical indicator-molecular characteristic" linkage subtyping system. It integrates clinical metabolic indicators such as peripheral blood LDL concentration with sequencing data of tumor tissue pathway genes, establishes a multi-dimensional data analysis model, and realizes dual subtyping of endometrial cancer based on "metabolic phenotype + molecular pathway", providing a basis for matching differentiated treatment plans for patients with different subtypes.

[0081] 3. This library construction method based on high-throughput sequencing for molecular subtyping of endometrial cancer enhances the clinical translational value of the library, optimizes the library construction process, introduces sample-specific barcodes to achieve multi-sample mixed sequencing, improves detection efficiency and reduces costs, and forms a standardized technical system of "sample preprocessing-targeted enrichment-library construction-subtyping modeling", which promotes the transformation of molecular subtyping results from laboratory research into clinically applicable diagnostic and treatment tools, and helps the precision diagnosis and treatment of endometrial cancer. Attached Figure Description

[0082] Figure 1 shows a schematic diagram of the overall method flow of the present invention. Detailed Implementation

[0083] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0084] Example:

[0085] As shown in Figure 1, the library construction method based on high-throughput sequencing molecular subtyping of endometrial cancer includes the following steps:

[0086] S1. Establish a correlation mechanism at the clinical sample level: Endometrial cancer patients showed obvious lipid metabolism abnormalities and pathway activation characteristics. Their peripheral blood LDL concentration was significantly higher than that of patients with benign endometrial hyperplasia. At the same time, the expression levels of phosphorylated JAK2 and phosphorylated STAT3 proteins in tumor tissue were abnormally elevated, suggesting that there is a direct clinical correlation between abnormal accumulation of LDL and activation of the JAK / STAT pathway.

[0087] The specific process for establishing a correlation mechanism at the clinical sample level is as follows:

[0088] S101, Clinical Trials:

[0089] Fifty patients with endometrial cancer and 50 patients with benign endometrial hyperplasia due to other benign diseases underwent surgery. Surgical tissue samples and peripheral blood were collected, and clinical information was collected. The LDL level in peripheral blood and the expression and activation of JAK and STAT3 in tumor tissue were detected. Informed consent forms were signed by all patients.

[0090] Specific inclusion criteria: 1. No history of metabolic diseases and no use of any hormone drugs in the past 3 months; 2. Patients with good follow-up conditions and good medical compliance; 3. Exclusion criteria: 1. Patients with a history of hormone replacement therapy; 2. Patients who have been taking hormone drugs for a long time due to other systemic diseases; 3. Patients with liver and kidney dysfunction; 4. Patients who have been taking statins to lower blood lipids; 5. Patients who have been taking oral medications for metabolic diseases such as diabetes.

[0091] Blood samples, LDL samples, and endometrial samples were collected from the patient. The endometrial samples were retrieved during the operation, fixed, and sent to the central laboratory for processing. The specific experimental steps were the same as the in vitro experimental procedures below. The degree of activation of the JAK2 / STAT3 pathway in patients with endometrial cancer at different stages was measured and compared, and all results were recorded in detail.

[0092] S102, Laboratory Experiment;

[0093] S1021, In vitro experiments:

[0094] Ishikawa cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum, 1×10⁵ U / L penicillin, and 1×10⁵ / L streptomycin, and incubated at 37°C in a 5% CO₂ incubator. When the cells reached 80%–90% confluence, they were passaged. When the cells had essentially covered 80%–90% of the bottom of the culture flask, they were passaged again.

[0095] S1022, Detecting cell proliferation:

[0096] Collect cells in the logarithmic growth phase and prepare a single-cell suspension;

[0097] After the passaged cells adhered and grew, they were starved in serum-free DMEM for 24 hours to synchronize the cells.

[0098] LDL at concentrations of 0, 10, 50, 100, and 150 ng / ml were added, and the cells were cultured for 6 h, 12 h, and 24 h, respectively. The maximum effective dose of LDL was preset. The cells cultured with the maximum effective dose of LDL were used in the following processes.

[0099] The JAK2 pathway-specific inhibitor AG490 of LDL was added at the maximum effective dose and cultured for 6h, 12h and 24h respectively.

[0100] The cells were cultured for 6 h, 12 h, and 24 h after adding the maximum effective dose of the STAT3 pathway-specific inhibitor JSI-124 for LDL, respectively.

[0101] The optical density of each well was measured at a wavelength of 570 nm using an ELISA reader.

[0102] Cell proliferation rate (%) = (OD value of experimental group - OD value of 0 ng / ml LDL group in the same time period) / OD value of 0 ng / ml LDL group in the same time period × 100%

[0103] S1023, Cell Scratch Test:

[0104] Seed Ishikawa cells into a 6-well plate with three evenly drawn horizontal lines on the back. After the cells covered the plate, use a pipette tip to draw lines perpendicular to the drawn horizontal lines.

[0105] Wash twice with 1 mL PBS, then add 2 mL serum-free 1640.

[0106] Observed under an inverted microscope at 0, 12, and 24 hours.

[0107] S1024, Transwell cell invasion assay:

[0108] Melt Matrigel overnight at 4°C;

[0109] Dilute Matrigel with serum-free culture medium pre-cooled to 4°C. The ratio of Matrigel gel to serum-free 1640 is 1:7.

[0110] Add 70 μL of diluted Matrigel to the upper chamber and incubate at 37°C for 4-5 hours.

[0111] Digest the cells, centrifuge at 800 r / min for 5 min, resuspend the cells, count them, and adjust the cell count to 2 × 10⁵ / mL;

[0112] Add 800 μL of culture medium containing 10% serum to the lower chamber, and add 100 μL of cell suspension, Ishikawa cells treated with LDL only, Ishikawa cells treated with LDL+AG490, and Ishikawa cells treated with LDL+JSI-124 to the upper chamber. Continue to culture in a 37°C incubator for 24 h.

[0113] Remove the chamber, gently rinse and soak it several times with clean water, remove the chamber, absorb the liquid, and wipe away the cells on the membrane surface at the bottom of the chamber with a damp cotton swab;

[0114] Fixation was performed using 4% paraformaldehyde, followed by staining with crystal violet. After cleaning and wiping, images were taken under a microscope and statistically analyzed.

[0115] S1025. Detection of JAK2 / STAT3 pathway expression and activation:

[0116] The expression and activation levels of the JAK2 / STAT3 pathway in Ishikawa cells, LDL+AG490 cells, and LDL+JSI-124 cells were detected by immunoblotting and qPCR.

[0117] 1) Prepare the relevant reagents;

[0118] 2) Extract total protein from cells;

[0119] 3) Determine protein concentration;

[0120] 4) Electrophoresis, membrane transfer, and immunochromatographic analysis;

[0121] S103, In vivo experiments:

[0122] S1031. Establishment of animal models:

[0123] After centrifuging to collect Ishikawa cells, they were resuspended in PBS. The cell suspension was then mixed with Matrigel at a 1:1 ratio to adjust the cell density to 7.5 × 10¹⁰ / L.

[0124] Nude mice were placed in a clean bench, and after the skin was disinfected, 200 μL of cell suspension (approximately 1.5 × 10⁷ cells) was injected subcutaneously into the right back scapula of the nude mice. The mice were housed in an SPF environment, and the condition of the nude mice, the size and hardness of the tumors at the injection site were observed daily. After one week, all nude mice were observed to have subcutaneous xenografts (diameter > 5 mm) on the right back scapula.

[0125] S1032, Grouping of animal models:

[0126] Tumor-bearing nude mice were randomly divided into four groups: experimental group 1 (LDL + AG490), experimental group 2 (LDL + JSI-124), control group (LDL), and blank group (physiological saline), with eight mice in each group. Each group received the corresponding drug via tail vein injection once every 3 days for a total of 3 weeks.

[0127] S1033, Observation Indicators:

[0128] Before each administration of medication, the major diameter (a) and minor diameter (b) of the tumor in nude mice were measured with vernier calipers. The tumor volume was calculated according to the formula (V=ab2 / 2), and a tumor growth curve was plotted.

[0129] After treatment, all nude mice were euthanized by cervical dislocation. The tumors were dissected in a clean bench, and after removing fat and blood, their weight was measured. The tumor inhibition rate was calculated as follows: Tumor inhibition rate (%) = (Tumor weight of control group - Tumor weight of treatment group) / Tumor weight of control group × 100%.

[0130] The clinical sample-level correlation mechanism was specifically established by comparing serum and tissue samples from the case group and the control group, and simultaneously verifying the synchronous abnormalities in LDL concentration and JAK / STAT pathway activation level. This established the clinical correlation between the two and EC, laying a clinical foundation for subsequent verification at the cellular and animal levels of the molecular mechanism by which LDL activates the JAK / STAT pathway to promote EC proliferation, migration, and invasion.

[0131] S2. Verify the mechanism of action at the cellular level: including the dose-dependent oncogenic effect of LDL and the reversal effect of pathway inhibitors;

[0132] The dose-dependent oncogenic effect of LDL is specifically demonstrated by the fact that when endometrial cancer cells are stimulated by different concentrations of LDL, the uptake of LDL by the cells increases with the increase of LDL concentration. At the same time, the expression levels of p-JAK2 and p-STAT3 are upregulated in a dose-dependent manner, the JAK / STAT pathway is activated, and the proliferation, migration and invasion of cancer cells are enhanced along with the activation of the pathway. This indicates that LDL can promote the malignant phenotype of cancer cells by activating the JAK / STAT pathway.

[0133] The reversal effect of the pathway inhibitor was specifically demonstrated by pretreatment of cancer cells with the JAK2-specific inhibitor SD-1029, which blocked LDL-induced JAK / STAT pathway activation. At the same time, LDL-mediated cancer cell proliferation, migration, and invasion were significantly inhibited, confirming that the JAK / STAT pathway is the core signaling pathway for LDL to exert its oncogenic effect.

[0134] S3. Validation mechanism at the animal level: A nude mouse subcutaneous endometrial cancer xenograft model was constructed. In the model, it was verified that after LDL injection via the tail vein, the volume and weight of the xenograft increased compared with the control group. The expression levels of p-JAK2, p-STAT3 and the proliferation marker Ki-67 in the tumor tissue were increased, while the expression of the apoptosis-related protein caspase-3 was decreased. This verified that LDL can activate the JAK / STAT pathway in vivo, promote the proliferation of tumor tissue and inhibit its apoptosis, accelerate tumor growth, and clarify the role of the LDL-JAK / STAT pathway axis in the progression of endometrial cancer.

[0135] The specific process for constructing a subcutaneous endometrial cancer xenograft model in nude mice is as follows:

[0136] S301. Selection and Feeding of Laboratory Animals:

[0137] Animal breeds and specifications: Twenty male C57BL / 6 nude mice aged 4-6 weeks were selected and purchased from Beijing Vital River Laboratories Co., Ltd.

[0138] Housing environment: Nude mice were housed in a specific pathogen-free (SPF) environment to ensure that the experimental environment met the requirements of animal experimentation ethics;

[0139] S302, Cell Preparation:

[0140] Cell lines: Two endometrial cancer cell lines were selected—Ishikawa and RL95-2 (both purchased from the American Type Culture Collection Center (ATCC)).

[0141] Cell treatment: Two types of cells were prepared into cell suspensions with a concentration of 2 × 10⁻⁶. 7 100 μL of cells were suspended in dimethyl sulfoxide for later use.

[0142] S303, Model Construction Steps:

[0143] Cell inoculation: The prepared Ishikawa cell suspension and RL95-2 cell suspension were injected subcutaneously into the subcutaneous tissue of nude mice to complete the tumor cell inoculation.

[0144] S304. Grouping: After inoculation, nude mice were divided into 4 groups of 5 mice each, as follows:

[0145] Ishikawa control group: Ishikawa cells were injected with an equal amount of DMSO, without any additional treatment;

[0146] IshikawaLDL group: LDL intervention was given after injection of Ishikawa cells;

[0147] RL95-2 control group: only RL95-2 cells were injected with an equal amount of DMSO, with no additional treatment;

[0148] RL95-2LDL group: LDL intervention was given after injection of RL95-2 cells;

[0149] Intervention measures: After tumor cell inoculation, LDL was intravenously injected into nude mice in the LDL group every 3 days; nude mice in the control group were injected with an equal amount of DMSO as a control.

[0150] S305, Model Monitoring and Sample Collection:

[0151] Tumor volume monitoring: Once the tumor is visible to the naked eye, the tumor volume is measured every 7 days. The calculation formula is: Tumor volume (mm³) = Long diameter (L) × Short diameter (W)² / 2, where L is the longest diameter of the tumor and W is the shortest diameter of the tumor.

[0152] Experimental endpoint determination and sample processing: When the tumor diameter reached 2 cm, the nude mice were euthanized, the tumor tissue was completely dissected, the tumor weight was measured, and the tumor tissue was further analyzed.

[0153] The above formulas are all dimensionless calculations. The formulas are derived from software simulations based on a large amount of collected data to obtain the most recent real-world results. The preset parameters in the formulas are set by those skilled in the art according to the actual situation.

[0154] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A library construction method for endometrial cancer based on high-throughput sequencing molecular subtyping, characterized in that, Includes the following steps: S1. Establish a correlation mechanism at the clinical sample level: Endometrial cancer patients showed obvious lipid metabolism abnormalities and pathway activation characteristics. Their peripheral blood LDL concentration was significantly higher than that of patients with benign endometrial hyperplasia. At the same time, the expression levels of phosphorylated JAK2 and phosphorylated STAT3 proteins in tumor tissue were abnormally elevated, suggesting that there is a direct clinical correlation between abnormal accumulation of LDL and activation of the JAK / STAT pathway. S2. Verification of the mechanism of action at the cellular level: including the dose-dependent oncogenic effect of LDL and the reversal effect of pathway inhibitors; S3. Verification of the mechanism at the animal level: a nude mouse subcutaneous endometrial cancer xenograft model was constructed. In the model, it was verified that after LDL was injected via the tail vein, the volume and weight of the xenograft increased compared with the control group. The expression levels of p-JAK2, p-STAT3 and the proliferation marker Ki-67 in the tumor tissue were increased, while the expression of the apoptosis-related protein caspase-3 was decreased. This verifies that LDL can activate the JAK / STAT pathway in vivo, promote the proliferation of tumor tissue and inhibit its apoptosis, accelerate tumor growth, and clarify the role of the LDL-JAK / STAT pathway axis in the progression of endometrial cancer.

2. The library construction method for endometrial cancer based on high-throughput sequencing molecular subtyping according to claim 1, characterized in that, The dose-dependent oncogenic effect of LDL is specifically demonstrated by the fact that when endometrial cancer cells are stimulated by different concentrations of LDL, the uptake of LDL by the cells increases with the increase of LDL concentration. At the same time, the expression levels of p-JAK2 and p-STAT3 are upregulated in a dose-dependent manner, the JAK / STAT pathway is activated, and the proliferation, migration and invasion of cancer cells are enhanced along with the activation of the pathway. This indicates that LDL can promote the malignant phenotype of cancer cells by activating the JAK / STAT pathway.

3. The library construction method for endometrial cancer based on high-throughput sequencing molecular subtyping according to claim 1, characterized in that, The reversal effect of the pathway inhibitor is specifically demonstrated by pretreatment of cancer cells with the JAK2-specific inhibitor SD-1029, which blocks LDL-induced JAK / STAT pathway activation. Simultaneously, LDL-mediated cancer cell proliferation, migration, and invasion are significantly inhibited, confirming that the JAK / STAT pathway is the core signaling pathway for LDL to exert its oncogenic effect.

4. The library construction method for endometrial cancer based on high-throughput sequencing molecular subtyping according to claim 1, characterized in that, The specific process for establishing a clinical sample-level association mechanism is as follows: S101, Clinical Experiment: 50 patients with endometrial cancer and 50 patients who underwent surgery for benign endometrial hyperplasia due to other benign diseases were selected. Surgical tissue samples and peripheral blood were collected, and clinical information was gathered. The LDL level in peripheral blood and the expression and activation of JAK and STAT3 in tumor tissue were detected. Informed consent was obtained from all patients. Blood LDL samples and endometrial samples were collected from the patients. The endometrial samples were retrieved during surgery, fixed, and sent to the central laboratory for processing. The specific experimental steps are the same as the in vitro experimental procedure below. The degree of activation of the JAK2 / STAT3 pathway in patients with endometrial cancer at different stages was measured and compared, and all results were recorded in detail; S102, Laboratory Experiment; S1021, In vitro experiment: Ishikawa cells were cultured in high glucose DMEM medium containing 10% fetal bovine serum and 1×105 U / L penicillin and 1×105 / L streptomycin, and placed in a constant temperature incubator at 37℃ and 5% CO2. When the cells reached 80%~90% confluence, they were passaged. When the cells basically covered the bottom of the culture flask at 80%~90%, they were passaged again.S1022. Cell proliferation detection: Cells in logarithmic growth phase were prepared into single-cell suspensions. After passage and adherence, cells were starved in serum-free DMEM for 24 hours to synchronize them. LDL at concentrations of 0, 10, 50, 100, and 150 ng / ml were added, and cells were cultured for 6 hours, 12 hours, and 24 hours respectively, using the preset maximum effective dose. Cells cultured with the maximum effective dose of LDL were used in the following procedures. The JAK2 pathway-specific inhibitor AG490 of LDL at the maximum effective dose was added, and cells were cultured for 6 hours, 12 hours, and 24 hours respectively. 4h; Add the maximum effective dose of the STAT3 pathway-specific inhibitor JSI-124 of LDL and continue culturing for 6h, 12h, and 24h respectively; Measure the optical density of each well at 570nm wavelength using a microplate reader; Cell proliferation rate = (OD value of experimental group - OD value of 0ng / ml LDL group in the same time period) / OD value of 0ng / ml LDL group in the same time period × 100% S1023, Cell scratch assay: Seed Ishikawa cells into 6-well plates with 3 evenly drawn horizontal lines on the back. After the cells cover the entire plate, use a pipette tip perpendicular to the drawn horizontal lines... Draw lines; wash twice with 1 mL PBS, add 2 mL serum-free 1640; observe under an inverted microscope at 0, 12, and 24 h; S1024 and Transwell cell invasion assays: melt Matrigel overnight at 4°C; dilute Matrigel with pre-cooled serum-free culture medium at 4°C (Matrigel:serum-free 1640 = 1:7); add 70 μL of diluted Matrigel to the upper chamber and incubate at 37°C for 4-5 h; digest cells, centrifuge at 800 r / min for 5 min, and resuspend cells. Count the cells and adjust the cell count to 2×10⁵ / mL. Add 800 μL of culture medium containing 10% serum to the lower chamber, and add 100 μL of cell suspension, Ishikawa cells treated with LDL only, Ishikawa cells treated with LDL+AG490, and Ishikawa cells treated with LDL+JSI-124 to the upper chamber. Continue to incubate at 37°C for 24 h. Remove the chamber, gently rinse and soak it several times with water, remove the chamber, aspirate the liquid, and wipe the cells off the membrane surface at the bottom of the chamber with a damp cotton swab. Fix with 4% paraformaldehyde and stain with crystal violet.After cleaning and wiping, photographs were taken under a microscope and statistics were compiled; S1025, Detection of JAK2 / STAT3 pathway expression and activation: Immunoblotting and qPCR were used to detect the expression and activation of the JAK2 / STAT3 pathway in Ishikawa cells treated with LDL, LDL+AG490, and LDL+JSI-124; 1) Prepare relevant reagents; 2) Extract total protein from cells; 3) Measure protein concentration; 4) Electrophoresis, transfer, and immunochromatographic reaction; S103, In vivo experiments: S1031, Establishment of animal model: Ishikawa cells were collected by centrifugation and resuspended in PBS. The cell density was adjusted to 7.5 × 10¹⁰ / L by mixing the solution with Matrigel at a 1:1 ratio. Nude mice were placed in a clean bench, and after skin disinfection, 200 μL of the cell suspension was injected subcutaneously into the right back scapula of the nude mice. The mice were housed in an SPF environment, and the condition of the nude mice, the size and hardness of the tumor at the injection site were observed daily. After one week, the subcutaneous transplanted tumors on the right back scapula of all nude mice were observed. S1032. Grouping of animal models: Tumor-bearing nude mice were randomly divided into experimental group 1 (LDL + AG490), experimental group 2 (LDL + JSI-124), control group (LDL), and blank group (physiological saline), with 8 mice in each group. Each group was injected with the corresponding drug via the tail vein, once every 3 days for a total of 3 weeks; S1033, Observation indicators: Tumor growth in nude mice. Before each administration, the long diameter (a) and short diameter (b) of the tumor were measured with calipers, and the tumor volume was calculated according to the formula (V=ab2 / 2) to plot the tumor growth curve; After treatment, all nude mice were euthanized by cervical dislocation, and the tumor was dissected in a clean bench. After removing fat and blood, the tumor was weighed and the tumor inhibition rate was calculated: Tumor inhibition rate (%) = (Tumor weight of control group - Tumor weight of treatment group) / Tumor weight of control group × 100%.

5. The library construction method for endometrial cancer based on high-throughput sequencing molecular subtyping according to claim 1, characterized in that, The clinical sample-level correlation mechanism is specifically established by comparing serum and tissue samples from the case group and the control group, and verifying the synchronous abnormalities of LDL concentration and JAK / STAT pathway activation level. This establishes the clinical correlation between the two and EC, laying a clinical foundation for subsequent verification of the molecular mechanism by which LDL activates the JAK / STAT pathway to promote EC proliferation, migration, and invasion at the cellular and animal levels.

6. The library construction method for endometrial cancer based on high-throughput sequencing molecular subtyping according to claim 1, characterized in that, The specific process for constructing a subcutaneous endometrial cancer xenograft model in nude mice is as follows: S301. Selection and rearing of experimental animals: Animal breed and specifications: Twenty male C57BL / 6 nude mice aged 4-6 weeks were selected and purchased from Vital River Laboratories, Beijing; Rearing environment: The nude mice were kept in a specific pathogen-free (SPF) environment to ensure that the experimental environment met the ethical requirements for animal experiments; S302. Cell preparation: Cell lines: Two endometrial cancer cell lines were selected—Ishikawa and RL95-2 (both purchased from the American Type Culture Collection Center (ATCC)); Cell treatment: The two cell lines were prepared into cell suspensions with a concentration of 2×10⁻⁶. 7 100 μL of cells / cell, suspended in dimethyl sulfoxide for later use; S303, Model construction steps: Cell seeding: Ishikawa cell suspension and RL95-2 cell suspension were injected subcutaneously into the subcutaneous tissue of nude mice to complete tumor cell seeding; S304, Grouping and treatment: After seeding, nude mice were divided into 4 groups of 5 mice each, as follows: Ishikawa control group: Ishikawa cells + equal volume of DMSO were injected only, without additional treatment; Ishikawa LDL group: LDL intervention was given after Ishikawa cell injection; RL95-2 control group: RL95-2 cells + equal volume of DMSO were injected only, without additional treatment. Therapeutic methods; RL95-2LDL group: After injection of RL95-2 cells, LDL intervention was given; Intervention measures: After tumor cell inoculation, LDL was injected intravenously into nude mice in the LDL group every 3 days; nude mice in the control group were injected with an equal amount of DMSO as a control; S305, Model monitoring and sample collection: Tumor volume monitoring: When tumor formation can be observed with the naked eye, the tumor volume is measured every 7 days. The calculation formula is: Tumor volume (mm³) = long diameter (L) × short diameter (W)² / 2, where L is the longest diameter of the tumor and W is the shortest diameter of the tumor; Experimental endpoint determination and sample processing: When the tumor diameter reaches 2cm, the nude mice are sacrificed, the tumor tissue is completely dissected, the tumor weight is measured, and the tumor tissue is analyzed subsequently.

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