Application of KYAT1 protein and its modification sites as biomarkers in the preparation of products for breast cancer auxiliary diagnosis and prognostic assessment

Abstract

This invention relates to the field of biomedical detection and molecular diagnostics, specifically to the application of KYAT1 protein and its modification sites as biomarkers in the preparation of products for the auxiliary diagnosis and prognostic assessment of breast cancer. The invention aims to reveal the positive correlation between high expression of the KYAT1 protein and the hydroxylation status of its proline at position 352 with the malignant biological behavior of breast cancer, and to use this as a molecular biomarker for the preparation of products to aid in the diagnosis of breast cancer or assess its perioperative prognostic risk.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection and molecular diagnostics, specifically to the application of KYAT1 protein and its modification sites as biomarkers in the preparation of products for auxiliary diagnosis and prognostic assessment of breast cancer. Background Technology

[0002] Breast cancer is the most common malignant tumor among women worldwide, and early diagnosis, accurate staging, and prognostic assessment after treatment are crucial for improving patient survival. Surgical resection is the primary treatment for breast cancer. However, numerous studies have shown that certain perioperative interventions, particularly widely used inhaled anesthetics (such as sevoflurane), may promote the proliferation, migration, and distant metastasis of residual tumor cells by inducing stress responses or altering the tumor microenvironment, thus producing a "pro-cancer effect" and impacting long-term patient prognosis.

[0003] Currently, clinical staging and prognostic assessment of breast cancer mainly rely on pathological morphological indicators (such as tumor size and lymph node metastasis) and a few molecular markers (such as estrogen receptor ER, progesterone receptor PR, human epidermal growth factor receptor 2 HER2, and the proliferation index Ki-67). However, these conventional markers are static indicators of inherent tumor characteristics and cannot dynamically and sensitively reflect the immediate impact of perioperative acute stress or environmental changes (such as anesthetic exposure and metastasis accumulation) on the invasive ability of tumor cells. They also make it difficult to accurately identify patients with high postoperative recurrence / metastasis risk, resulting in the inability to conduct targeted close follow-up or intensive adjuvant therapy. In addition, existing biomarker research is mostly limited to the mRNA transcription level, but there is often a huge difference between gene expression and the actual protein level that performs the function, and the decisive influence of post-translational modifications on protein stability and its biological function is ignored, resulting in limited accuracy of mRNA-based predictive models. Summary of the Invention

[0004] The technical problem this invention aims to solve is to provide the KYAT1 (also known as CCBL1) protein and its modification sites as biomarkers for the preparation of products for the auxiliary diagnosis and prognostic assessment of breast cancer. Specifically, this invention aims to reveal the positive correlation between high expression of the KYAT1 protein and the hydroxylation modification status of its proline 352 (Pro352) residue and the malignant biological behavior (migration, proliferation) of breast cancer, and to use this as a molecular biomarker for the preparation of products for the auxiliary diagnosis of breast cancer or the assessment of its perioperative prognostic risk.

[0005] The technical solution of this invention is as follows:

[0006] Application of reagents for detecting the hydroxylation level of KYAT1 protein and / or its 352nd proline in the preparation of products for breast cancer adjuvant diagnosis and prognostic assessment.

[0007] The prognostic assessment product is for evaluating the perioperative prognostic risk of breast cancer. This product is applicable to the assessment of prognostic risk in breast cancer patients who have received sevoflurane anesthesia, based on the upregulation of KYAT1 protein expression levels or its 352-position proline hydroxylation modification level induced by sevoflurane anesthesia exposure. The prognostic risk refers to the risk of breast cancer recurrence or metastasis.

[0008] The application includes comparing the expression level of KYAT1 protein and / or the hydroxylation level of proline at position 352 in a subject's sample with a reference value. When the expression level or modification level is higher than the reference value, it indicates that the subject has breast cancer or is at high risk of breast cancer recurrence or metastasis. The sample is selected from blood, serum, plasma, or tumor tissue samples.

[0009] A kit for the auxiliary diagnosis or prognostic prediction of breast cancer includes reagents that specifically detect the level of hydroxylation modification of KYAT1 protein and / or its 352-position proline. The kit also includes reagents for detecting the content of kynurenine (a key molecule in the upstream metabolic pathway of KYAT1).

[0010] The kit is used in the preparation of products that assist in the diagnosis of breast cancer or in assessing its perioperative prognostic risks.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] 1. Novel biomarkers and clear mechanisms: For the first time, KYAT1 protein and its specific Pro352 hydroxylation modification have been established as biomarkers for the malignant progression of breast cancer, and a novel mechanism of action has been elucidated: the upregulation of KYAT1 protein is not due to an increase in transcriptional levels, but rather to a slowdown in protein degradation and an increase in stability mediated by hydroxylation modification at the Pro352 site. Compared with biomarkers based solely on mRNA levels, directly detecting the protein and its post-translational modification status is more helpful in reflecting tumor biological characteristics directly and timely at the functional level.

[0013] 2. Facilitates perioperative prognostic risk assessment for breast cancer patients: This invention dynamically correlates molecular markers with specific perioperative stressors (sevoflurane anesthesia), providing a new detection approach for assessing the risk of anesthesia-related breast cancer recurrence or metastasis. It can be used to help identify high-risk patients after surgery and provide a reference for the development of individualized follow-up and adjuvant therapy plans.

[0014] 3. The detection method has a certain basis for clinical translation: The detection target of this invention is KYAT1 protein and its modification level, which has better functional relevance compared with simple gene-level detection. The detection can be carried out in combination with immunohistochemistry, Western blotting, immunoassay or mass spectrometry, etc. The required samples include peripheral blood and tissue samples, which are relatively convenient to obtain, and therefore have the potential for further clinical translation and product development.

[0015] 4. Provide a combined assessment solution: By jointly detecting KYAT1 and its upstream metabolite KYN, a multi-risk factor assessment model can be constructed, thereby further improving the robustness and accuracy of risk assessment. Attached Figure Description

[0016] Figure 1 , seven Figure showing the experimental results of how exposure to halothane promotes tumor growth and distant metastasis in an orthotopic xenograft model of breast cancer;

[0017] Figure 2 Figure 1. Proteomics analysis results of upregulated KYAT1 protein expression in paired blood samples of breast cancer patients before and after anesthesia.

[0018] Figure 3 , seven Figure showing the effect of halothane on KYAT1 protein expression in breast cancer tumor tissue;

[0019] Figure 4 Immunohistochemical results of KYAT1 protein expression distribution in breast cancer tissue;

[0020] Figure 5 Kaplan-Meier survival curve;

[0021] Figure 6 Figure 1 shows the experimental results of the effect of KYAT1 overexpression on the biological function of breast cancer cells.

[0022] Figure 7 , seven Mass spectrometry identification and structural analysis of hydroxylation modification at Pro352 site of KYAT1 protein induced by halothane;

[0023] Figure 8 Figure 1 shows the CHX tracking experiment results showing the effect of Pro352 hydroxylation on the stability of KYAT1 protein.

[0024] Figure 9 The Western blot results of the effect of kynurenine on the upregulation of KYAT1 induced by sevoflurane. Detailed Implementation

[0025] To make the objectives and technical solutions of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings. Unless otherwise specified, the experimental methods described in the following tests are conventional methods; for tests where specific techniques or conditions are not specified, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions; unless otherwise specified, the reagents and materials mentioned are all commercially available.

[0026] Example 1

[0027] To assess the effect of sevoflurane on in vivo breast cancer progression, we constructed an orthotopic xenograft model of breast cancer carrying luciferase-labeled tumors and treated the tumors with 3% sevoflurane three times a week for three weeks, starting one week after inoculation. IVIS in vivo imaging was then performed. Figure 1 The results showed that, compared with the air-treated group, the bioluminescent signal of the in situ tumors in the sevoflurane-treated group increased week by week, indicating an increased tumor burden. Further imaging of ex vivo organs at the endpoint showed that the sevoflurane group exhibited stronger fluorescence signals in distant organs such as the lungs, suggesting that it promoted distant metastasis of breast cancer.

[0028] Example 2

[0029] To screen for potential molecular biomarkers associated with perioperative anesthetic exposure, we performed proteomic analysis on paired blood samples from breast cancer patients before and after anesthesia (sevoflurane general anesthesia). Figure 2 ).like Figure 2 As shown, proteomics analysis revealed that KYAT1 was significantly upregulated in paired blood samples from breast cancer patients before and after anesthesia. Figure 2 Figure A shows a box plot of KYAT1 quantification in blood samples from clinical patients at the Ninth People's Hospital affiliated with Shanghai Jiao Tong University School of Medicine before and after anesthesia. Figure 2 Box plot of KYAT1 quantification in blood samples from clinical patients at Tongji Hospital, affiliated with Tongji Medical College of Huazhong University of Science and Technology, before and after anesthesia. Figure 2 The figure in C represents a quantitative comparison of KYAT1 levels between the two hospitals. These results suggest that KYAT1 may be associated with changes in the tumor microenvironment related to anesthesia exposure and could serve as a candidate molecular biomarker for further research.

[0030] Example 3

[0031] To verify whether in vivo sevoflurane exposure affects KYAT1 expression in tumor tissue, we performed Western blot analysis on in situ tumor tissue. Figure 3 A represents the Western blot results of KYAT1 protein in tumor tissue and the quantitative analysis results of its band gray values ​​after normalization with internal control. Compared with the air control group, the intensity of the KYAT1 protein band in the sevoflurane-treated group was significantly enhanced. Figure 3B shows the DNA electrophoresis results and qPCR results of the two groups of KYAT1 genes. The results show that there is no difference in the mRNA expression level of KYAT1 in the two groups of samples.

[0032] Example 4

[0033] This embodiment analyzes the distribution of KYAT1 using immunohistochemical (IHC) detection of pathological sections from breast cancer patients. The results are as follows: Figure 4 Compared with normal breast tissue, the staining intensity and positive expression area of ​​KYAT1 protein were significantly enhanced in breast cancer tissue, indicating that the expression level of KYAT1 is elevated in breast cancer tissue. Furthermore, KYAT1 maintained a high expression pattern in tumor tissue with lymph node metastasis, suggesting that it may be associated with the malignant progression of breast cancer.

[0034] Example 5

[0035] This study retrospectively analyzed tumor tissue samples from 200 breast cancer patients who underwent radical surgery. Immunohistochemistry (IHC) was used to detect the expression level of KYAT1 protein, and patients were divided into a high-expression group (n=85) and a low-expression group (n=115) based on their expression levels. All patients were followed up long-term, with first recurrence or metastasis as the endpoint. Kaplan-Meier survival curves were plotted. Results are as follows... Figure 5 As shown, during the 10-year follow-up period, the survival probability of patients in the KYAT1 high-expression group was consistently lower than that in the low-expression group, and the difference was statistically significant (p<0.05). These results indicate that high expression of KYAT1 protein in tumor tissue is associated with poorer postoperative prognosis in breast cancer patients, suggesting that KYAT1 protein can serve as a candidate biomarker for assessing the prognostic risk of breast cancer patients, providing experimental evidence for the development and application of the prognostic assessment product described in this invention.

[0036] Example 6

[0037] To clarify the biological function of KYAT1 in breast cancer, this embodiment constructed a KYAT1 overexpressing cell model (KYAT1-OE) and analyzed its phenotypic changes through relevant functional experiments.

[0038] Transwell migration experiment ( Figure 6 A) The results showed that the number of transmembrane cells (purple-stained cells) in the KYAT1 overexpression group (KYAT1-OE) was significantly higher than that in the control group, and the cell count showed that the migration ability of the OE group was significantly enhanced, suggesting that KYAT1 can promote the migration of breast cancer cells. (Scratch healing assay) Figure 6B) The results showed that the wound closure rate (e.g., wound width changes at 12h, 24h, and 48h) in the KYAT1 overexpression group (KYAT1-OE) was significantly faster than that in the control group, indicating that KYAT1 can accelerate the motility (wound healing ability) of breast cancer cells. This was confirmed by the CCK8 assay. Figure 6 C) The results also showed that KYAT1 overexpression significantly improved cell viability (proliferation or survival ability), suggesting that KYAT1 has a promoting effect on the proliferation / survival of breast cancer cells.

[0039] Functional experiments demonstrated the role of KYAT1 in the biological behavior of breast cancer cells, namely, overexpression of KYAT1 significantly enhances cell viability, migration, and wound healing ability.

[0040] Example 7

[0041] Since qPCR did not observe an increase in KYAT1 transcriptional levels in mouse tumor tissues, we hypothesized that sevoflurane might regulate KYAT1 protein stability through post-translational modifications. Therefore, we overexpressed Flag-KYAT1 in 293T cells and treated them with sevoflurane. We then enriched KYAT1 protein using Flag-IP and analyzed the modifications using PTM mass spectrometry.

[0042] Flag-IP authentication ( Figure 7 A) The results showed that the Flag-KYAT1 band was brighter after IP in the sevoflurane-treated group, indicating that sevoflurane can promote the enrichment of Flag-KYAT1 (or improve its protein stability, making it easier for IP to capture). PTM mass spectrometry identification ( Figure 7 B) The results showed that KYAT1 was hydroxylated at position 352 (Pro352), and the abundance of this modification in the sevoflurane-treated group was significantly higher than that in the air control group, directly proving that sevoflurane can induce Pro352 hydroxylation. Figure 7 The upper image in Figure C shows the 3D structural model of KYAT1 wild-type (KYAT1-WT). The lower image marks the hydroxylation modification sites of Pro352 in the wild-type structure, visually demonstrating the location of the modification in the protein structure. (KYAT1 protein domain map) Figure 7 (D) shows that Pro352 is located within a Type I PLP-dependent aspartate aminotransferase-like domain, which belongs to the PLP-dependent transaminase family, suggesting that the modification site is in a functionally critical region. This is compared with the KYAT1 protein sequences of humans, mice, cattle, and zebrafish. Figure 7The sites E and Pro352 (Ile at position 351 and Pro at position 352 in humans; Pro at the corresponding sites in mice, cattle, and zebrafish) are highly conserved in evolution, suggesting that modifications at these sites may have important biological functions.

[0043] This embodiment demonstrates from multiple aspects, including protein enrichment, modification site identification, structural localization, functional domains, and species conservation, that sevoflurane can induce hydroxylation at the Pro352 site of KYAT1, and that this site is located in a key functional region of the protein and is evolutionarily conserved, providing molecular-level experimental evidence for "sebflurane regulating KYAT1 stability through post-translational modification".

[0044] Example 8

[0045] To verify the effect of Pro352 hydroxylation on the stability of the KYAT1 protein, we constructed a Pro352→alanine (P352A) mutant (alanine cannot be hydroxylated, used to simulate the "dehydroxylated" state) and analyzed the protein degradation rate using CHX (cyclohexylimide, a protein synthesis inhibitor). The results are as follows: Figure 8 Wild-type (WT) protein levels showed a slow decline with prolonged CHX treatment (with more protein retained within 0-10 minutes); P352A mutant protein degradation was significantly faster than wild-type (lower protein levels at the same time points, especially at 8-10 minutes). Quantitative analysis further showed that the relative protein levels of WT decreased gradually over time, while the protein levels of P352A decreased rapidly and continuously.

[0046] Hydroxylation at the Pro352 site can delay the degradation of KYAT1 protein and maintain its stability. When this site is mutated to an alanine that cannot be hydroxylated, the protein half-life is significantly shortened, suggesting that Pro352 hydroxylation is one of the key post-translational modifications that regulate the stability of KYAT1 protein.

[0047] Example 9

[0048] To clarify the regulatory role of kynurenine (KYN) on KYAT1, cells were pretreated with specific inhibitors of tryptophan metabolism upstream enzymes: an IDO1 inhibitor and a TDO1 inhibitor, to block the production of the substrate KYN. Cells were then treated with sevoflurane, and samples were collected. Western blot analysis was used to assess the effect of inhibiting substrate production on sevoflurane-induced KYAT1 upregulation. Figure 9 The results showed that KYAT1 levels were significantly reduced after treatment with the upstream enzyme inhibitor, suggesting that the upregulation of KYAT1 levels after sevoflurane treatment may be related to the IDO1-KYN metabolic pathway.

[0049] The experimental materials and methods used in the examples are as follows:

[0050] (1) Laboratory animals

[0051] All mice were housed in specific pathogen-free (SPF) grade animal facilities, in individually ventilated plastic cages (IVCs) with a 12-hour light-dark cycle (lighting on at 8:00 AM), under constant temperature (22±2℃) and humidity (50±10%), and provided with standard feed and water (free access). All animal experiments were conducted strictly in accordance with the guidelines established by the Animal Experimentation Committee of Qingdao University, and the experimental protocols were approved by the Animal Experimentation Ethics Committee of Qingdao University. Female BALB / c mice aged 6-8 weeks were used in this experiment.

[0052] (2) Establishment of an orthotopic xenograft model of breast cancer and sevoflurane treatment

[0053] To establish an orthotopic breast cancer tumor model, stably luciferase-expressing breast cancer cells (4T1-Luc, 1 x 10^6 cells / 50 μL PBS) were injected orally into the mammary fat pads of female mice. One week after inoculation, the mice were randomly divided into groups. Mice in the sevoflurane treatment group were placed in a specially designed anesthesia chamber and inhaled a mixture of gas containing 3% sevoflurane (oxygen-carrying gas) delivered by an anesthesia vaporizer for 2 hours each time, 3 times a week, for 3 consecutive weeks; mice in the control group inhaled an air / oxygen mixture under the same conditions.

[0054] (3) IVIS in vivo imaging and assessment of ex vivo organ transfer

[0055] To observe in situ tumor growth and metastasis, mice underwent in vivo fluorescence imaging (IVIS Spectrum, PerkinElmer) once every week starting with sevoflurane treatment. Ten minutes before imaging, D-luciferin potassium (150 mg / kg) was injected intraperitoneally, and bioluminescent signals were collected under light isoflurane anesthesia. At the end of the fourth week (after the last in vivo imaging), mice were immediately euthanized by cervical dislocation. The heart, liver, spleen, lungs, kidneys, and other major organs of the mice were rapidly isolated and harvested for immediate IVIS stereoscopic imaging of the ex vivo organs to quantitatively assess tumor metastasis in each organ. Subsequently, in situ tumor tissue was collected; one portion was stored at -80°C for molecular biological analysis, and the other portion was fixed in 4% paraformaldehyde for pathological observation.

[0056] (4) Clinical sample collection and proteomics analysis

[0057] Paired blood samples were collected from breast cancer patients treated at our hospital before and after anesthesia (this study was approved by our hospital's ethics committee, and informed consent was obtained from the patients). Serum samples were processed to remove high-abundance proteins and then subjected to Olink proteomics analysis to screen for differentially expressed proteins (specifically KYAT1). In addition, surgical pathology sections from breast cancer patients were collected for subsequent immunohistochemical (IHC) staining to assess the expression abundance of KYAT1 in clinical samples.

[0058] (5) Cell culture

[0059] Human embryonic kidney cell line (HEK-293T) and human breast cancer cell line (MDA-MB-231). All cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin in a humid incubator at 37°C with 5% CO2.

[0060] (6) Plasmid construction and cell transfection

[0061] Wild-type KYAT1 cells were obtained and cloned into a Flag-tagged eukaryotic expression vector (Flag-KYAT1-WT). Using site-directed mutagenesis, proline at position 352 of KYAT1 was mutated to alanine, constructing a mutant plasmid (Flag-KYAT1-P352A). Hydroxylase family plasmids (HA-P4HA1, HA-P4HA2, HA-P4HA3) with HA tags were also purchased. When cell confluence reached 70%-80%, plasmid transfection was performed according to the manufacturer's instructions for Lipofectamine 3000. The medium was replaced with fresh complete culture medium 6 hours after transfection.

[0062] (7) In vitro sevoflurane exposure model and protein crosslinking / mass spectrometry analysis

[0063] 293T cells (cultured in 6cm dishes) transfected with Flag-KYAT1-WT or Flag-KYAT1-P352A plasmids for 24 hours had part of the culture medium aspirated (leaving only a thin layer of liquid to reduce the medium's hindrance to sevoflurane dissolution and diffusion). The culture dishes were placed in a sealed, gas-circulating exposure chamber, and 3% sevoflurane was introduced continuously for 3 hours. Air was introduced into the control group. After treatment, the culture medium was replenished and cultured again. Cell lysates were collected and incubated with Anti-Flag magnetic beads (Flag-beads) at 4°C to enrich the Flag-KYAT1 protein complex. The eluted protein samples were sent for PTM (post-translational modification) analysis and tandem mass spectrometry identification of interacting proteins (hydroxylases).

[0064] (8) Protein half-life (CHX tracking) detection

[0065] To assess the protein stability of KYAT1, Flag-KYAT1-WT and Flag-KYAT1-P352A plasmids were transfected into 293T cells. Twenty-four hours after transfection and medium change, the protein synthesis inhibitor cycloheximide (CHX, final concentration 50 μg / mL) was added to the culture medium. Cell protein samples were collected at 0, 2, 4, 6, 8, and 10 hours after CHX addition, and the degradation rate of KYAT1 was detected by Western blotting.

[0066] (9) Co-immunoprecipitation (Co-IP) detection

[0067] To verify the binding of KYAT1 to specific hydroxylases, plasmid co-transfection experiments were performed in 293T cells. The groups were set as follows: (1) single transfection with Flag-KYAT1 (2 μg); (2) co-transfection with Flag-KYAT1 (2 μg) + HA-P4HA1 / 2 / 3 (2 μg); (3) co-transfection with Flag-KYAT1 (2 μg) + HA-P4HA1 / 2 / 3 (4 μg). After transfection, cells were treated with sevoflurane and collected. Total protein (input) was extracted using NP-40 cell lysis buffer. Cocktail protease inhibitor and PMSF were pre-added to the lysis buffer before use. A portion of the lysis buffer was incubated with Anti-Flag magnetic beads at 4°C overnight for immunoprecipitation (IP). After washing and elution, Western blotting was performed using anti-HA antibody to assess its dose-dependent binding effect.

[0068] (10) Western Blot

[0069] Total protein was extracted from tissues and cells using RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors. Total protein concentration was determined using a BCA protein assay kit. Equal volumes of protein samples were separated by SDS-PAGE (12.5% ​​gel) and transferred to PVDF membranes. After blocking with 5% skim milk at room temperature for 1 hour, primary antibodies were added: anti-KYAT1 (1:2000), anti-Flag (1:2000), anti-HA (1:2000), and internal control anti-GAPDH or β-actin (1:5000), and incubated overnight at 4°C. After washing with TBST, the samples were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 hour. Bands were developed using an enhanced chemiluminescence (ECL) kit, and images were acquired using a chemiluminescence imaging system. Density analysis software was used to normalize the protein to the internal control.

[0070] (11) RNA extraction, quantitative real-time PCR (qPCR) and DNA electrophoresis

[0071] Total RNA was extracted from mouse tumor tissues and cells using TRIzol reagent (Invitrogen) and reverse transcribed into cDNA using a reverse transcription kit. qRT-PCR was performed using SYBR Green on a PCR instrument. GAPDH was used as an internal control gene, and relative expression levels were calculated using the 2^(-ΔΔCt) method. The amplified products were subjected to agarose gel electrophoresis with nucleic acid dyes to verify the amplification specificity at the transcriptional level.

[0072] (12) Immunohistochemistry (IHC) and immunofluorescence (IF)

[0073] For pathological tissues, paraffin sections were dewaxed and hydrated. After antigen retrieval and endogenous peroxidase blocking, they were incubated with anti-KYAT1 antibody. Subsequently, they were incubated with secondary antibody, DAB staining was performed, and counterstaining was done with hematoxylin. The expression distribution of KYAT1 was observed under an optical microscope.

[0074] For immunofluorescence colocalization experiments, co-transfected cells were fixed, permeabilized, and blocked. Mouse anti-Flag and rabbit anti-HA primary antibodies were added and incubated overnight. The following day, cells were incubated at room temperature in the dark with secondary antibodies containing different fluorescent labels (Alexa Fluor 488 and 594). After DAPI staining of the nuclei, colocalization was observed using a laser confocal microscope.

[0075] (13) Cell Biology Functional Experiments

[0076] MDA-MB-231 cell lines with KYAT1 overexpression or knockdown were constructed by transfecting plasmids or siRNA.

[0077] CCK-8 assay: Cells were seeded in 96-well plates, and CCK-8 reagent was added in a time gradient. After incubation, the absorbance at 450 nm was measured using a microplate reader to evaluate cell viability and proliferation.

[0078] Scratch healing assay: After cells grew to a monolayer and fused, a cell-free area was drawn using a pipette tip, and suspended cells were washed away. The scratch width was photographed and recorded at 0, 24, and 48 hours, and the healing rate was calculated to assess cell migration ability.

[0079] Transwell assay: Starved cells were suspended in serum-free medium and seeded into the upper chamber of a Transwell chamber. Medium containing 10% FBS was added to the lower chamber as an inducer. After culturing for 24-48 hours, cells that had not penetrated the membrane were wiped off from the upper chamber. Cells on the lower surface of the membrane were stained with crystal violet, photographed, and counted.

[0080] (14) Upstream metabolic pathway inhibition experiment

[0081] To clarify the regulatory role of kynurenine (KYN) on KYAT1, cells were pretreated with specific inhibitors of tryptophan metabolism upstream enzymes: IDO1 inhibitors and TDO1 inhibitors, to block the production of the substrate KYN. Cells were then treated with sevoflurane, and samples were collected. Western blotting was used to assess the effect of inhibiting substrate production on sevoflurane-induced KYAT1 upregulation.

[0082] (15) Data Analysis

[0083] All statistical analyses were performed using GraphPad Prism 10 software. Independent samples t-tests (Student's t-test) were used for comparisons between two groups, and one-way or two-way ANOVA combined with Tukey or Sidak multiple comparison tests were used for comparisons among multiple groups. All data are expressed as mean ± standard deviation (Mean ± SD) or standard error (SEM). Significance criteria were defined as: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.

Claims

1. Application of reagents for detecting the hydroxylation level of KYAT1 protein and / or its 352nd proline in the preparation of products for auxiliary diagnosis and prognostic assessment of breast cancer.

2. Use according to claim 1, wherein The prognostic assessment product is used to assess the perioperative prognostic risk of breast cancer.

3. Use according to claim 2, wherein the compound is ###0002### The product is intended for the assessment of prognostic risk in breast cancer patients who have received sevoflurane anesthesia, based on the upregulation of KYAT1 protein expression levels or the hydroxylation level of proline at position 352 induced by sevoflurane anesthesia exposure.

4. The use according to claim 2, wherein the compound is ###0002### The prognostic risk refers to the risk of recurrence or metastasis of breast cancer.

5. Use according to any one of claims 1 to 4, wherein the compound is ###0002### The application includes comparing the expression level of KYAT1 protein and / or the hydroxylation level of proline at position 352 in a subject's sample with a reference value. When the expression level or modification level is higher than the reference value, it indicates that the subject has breast cancer or has a high risk of breast cancer recurrence or metastasis.

6. Use according to claim 5, wherein The samples are selected from blood, serum, plasma, or tumor tissue samples.

7. A kit for the auxiliary diagnosis or prognostic prediction of breast cancer, characterized in that, It contains reagents that can specifically detect the level of hydroxylation modification of KYAT1 protein and / or its 352nd proline residue.

8. The kit according to claim 7, characterized in that, The kit also contains reagents for detecting kynurenine levels.

9. The use of the kit according to claim 7 or 8 in the preparation of products for the auxiliary diagnosis of breast cancer or for assessing its perioperative prognostic risk.

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