Endogenous hydrogen sulfide mediated PGK1 sulfhydrylation modification for promoting proliferation and metastasis of breast cancer

Abstract

The invention relates to promotion of breast cancer proliferation and metastasis by endogenous hydrogen sulfide mediated PGK1 sulfhydrylation modification, belongs to the technical field of breast cancer proliferation and metastasis, and aims to solve the problem that partial patients suffer from local relapse or distant metastasis due to drug resistance caused by new mutation of tumor cells. The method comprises the following steps: real-time fluorescent quantitative PCR (Polymerase Chain Reaction), Western blot, H2S content detection of a tissue sample, H2S content detection of a cell sample, Biotin switch assay, cell culture, a plasmid related experiment, a CCK-8 experiment, a plate cloning experiment, a cell scratch experiment, a Transwell migration experiment, a Transwell invasion experiment, glucose level detection, lactic acid level detection, ATP (Adenosine Triphosphate) level detection and determination of an extracellular acidification rate and an oxygen consumption rate. The method comprises the following steps: construction of a mouse breast cancer model, hematoxylin-eosin staining, immunohistochemical staining and statistical analysis, and endogenous H2S regulates glycolysis of breast cancer cells by mediating PGK1 sulfhydrylation modification so as to promote proliferation and metastasis of tumors.

Description

Technical Field

[0001] The present invention relates to the technical field of breast cancer proliferation and metastasis, specifically, endogenous hydrogen sulfide mediates the S-thiolation modification of PGK1 to promote breast cancer proliferation and metastasis. Background Art

[0002] Breast cancer is one of the most common cancers globally. In 2020, breast cancer in women has become the leading cause of cancer incidence worldwide.

[0003] Despite great progress in the treatment of breast cancer, which has evolved from surgical treatment to radiotherapy, chemotherapy, endocrine therapy, targeted therapy, and immunotherapy. Most breast cancer patients respond effectively to treatment initially, but over time, tumor cells acquire new mutations, leading to drug resistance, and some patients experience local recurrence or distant metastasis, resulting in a decline in survival rate. Therefore, exploring new treatment methods and therapeutic targets has become a key research direction in the future.

[0004] In response to the above problems, it is proposed that endogenous hydrogen sulfide mediates the S-thiolation modification of PGK1 to promote breast cancer proliferation and metastasis. Summary of the Invention

[0005] The purpose of the present invention is to provide a method in which endogenous hydrogen sulfide mediates the S-thiolation modification of PGK1 to promote breast cancer proliferation and metastasis. By using this method, the problems in the above background, such as tumor cells acquiring new mutations, resulting in drug resistance, and some patients experiencing local recurrence or distant metastasis, leading to a decline in survival rate, are solved.

[0006] To achieve the above object, the present invention provides the following technical solution: Endogenous hydrogen sulfide mediates the S-thiolation modification of PGK1 to promote breast cancer proliferation and metastasis, including the following steps: S1: Real-time fluorescence quantitative PCR; S2: Western blot; S3: Detection of H2S content in tissue samples; S4: Detection of H2S content in cell samples; S5: Biotin switch assay; S6: Cell culture; S7: Plasmid-related experiments; S8: CCK-8 assay; S9: Colony formation assay; S10: Cell scratch assay; S20: Transwell migration assay; S30: Transwell invasion assay; S40: Glucose level detection; S50: Lactate level detection; S60: ATP level detection; S70: Determination of extracellular acidification rate and oxygen consumption rate; S80: Construction of mouse breast cancer model; S90: Hematoxylin-eosin staining; S100: Immunohistochemical staining; S200: Statistical analysis.

[0007] Furthermore, the specific steps of the real-time fluorescence quantitative PCR described in S1 are as follows: RNA is extracted by the Trizol method. 50 mg of tissue is added to 1 mL of Trizol, ground and centrifuged. The supernatant is taken, 200 μL of chloroform is added, mixed well and centrifuged. The aqueous phase is collected, 500 μL of isopropanol is added, and precipitated at -20 °C for 30 min. After centrifugation, it is washed with ethanol 3 times. After air drying, it is dissolved in 20 μL of enzyme-free water and the concentration is measured; RNA reverse transcription: After thawing the reagent, a 20 μL reaction system is prepared, shaken and mixed well, reacted at 15 min → 85 °C, and the product is collected by centrifugation; PCR is detected on the machine; The reaction mixture is added to an 8-well strip tube, DNA template is added, centrifuged instantaneously and mixed well, the program is set, and the qPCR instrument is started for detection; The specific steps of the Western blot described in S2 are as follows: Protein extraction: After the tissue is thawed, add RIPA lysis buffer according to 100 mg:1 mL, ground and centrifuged to take the supernatant; After cell lysis, scrape it, centrifuge to take the supernatant, and store it after boiling after BCA quantification; Electrophoresis, gel preparation, pouring of separating gel and stacking gel are carried out; Loading: 80 V for 30 min → 120 V for 2 h; Transfer membrane: After the PVDF membrane is activated with methanol, transfer the membrane at a constant current of 200 - 300 mA for 1 - 2 h; Blocking, at room temperature for 2 h; Primary antibody: Shake at 4 °C overnight; Secondary antibody: at room temperature for 2 h; ECL luminescence development, and the gray value is analyzed by ImageJ; The specific steps of the detection of H2S content in the tissue sample described in S3 are as follows: Prepare a 1 mmol standard product, measure the OD value at 665 nm with an enzyme-labeled instrument to draw a standard curve; The tissue sample is homogenized with normal saline at a ratio of 1:9, centrifuged to take the supernatant, and the content (μmol / gprot) is calculated according to the formula (Δ665 - b) / a × f / Cpr.

[0008] Furthermore, the specific steps of the detection of H2S content in the cell sample described in S4 are as follows: The cell sample is ultrasonically broken with the extraction solution, centrifuged to take the supernatant, and the content (nmol / 10 4 cells) is calculated according to the standard curve y = 0.0044x and the formula 340.9 × ΔA / number of cells; All centrifugations are at 4 °C and 12,000 rpm; The specific steps of the Biotin switch assay described in S5 are as follows: collect cell or tissue samples, wash with PBS and then lyse, ultrasonicate and centrifuge to obtain the supernatant; after BCA quantification, take 500 μg of protein, block free thiol groups with MMTS, precipitate with acetone and label the thiol sites with Biotin-HPDP; after washing with acetone, bind with streptavidin agarose beads at 4°C overnight, wash with NaCl buffer 5 times the next day, and finally add loading buffer to boil and denature to detect the thiol modification level; The cell culture described in S6 is as follows: resuscitate the cells and subculture them, change the medium regularly to remove metabolic waste, freeze them for backup, and count them using the formula: number of cells / mL = (total number of four grids / 4) × 10 4 × dilution multiple; sterile operation was performed throughout the process, and the culture conditions were maintained at 37°C and 5% CO2.

[0009] Furthermore, the plasmid-related experiments described in S7 were specifically performed as follows: after constructing the PGK1 wild-type / mutant plasmid, it was amplified in LB medium, extracted using the alkaline lysis method, and finally transfected into breast cancer cells using Lipo8000™, and the samples were collected after 48 h; The specific steps of the CCK-8 experiment described in S8 are as follows: 3000 cells / well were inoculated into a 96-well plate, cultured at 37°C until adhered to the wall, and then replaced with drug-containing culture medium. 10% CCK-8 reagent was added at 0 / 24 / 48 h in the dark, and the absorbance at 450 nm was measured after incubation for 2 h.

[0010] Furthermore, the plate cloning experiment described in S9 has the following specific steps: after digesting breast cancer cells, 1000-3000 cells / well were inoculated into a six-well plate, cultured for 7-14 days until visible clones were formed, fixed with 4% paraformaldehyde for 30 minutes, stained with 0.1% crystal violet for 20 minutes, washed with PBS, photographed and counted using ImageJ; The cell scratch experiment described in S10 has the following specific steps: the breast cancer cell suspension is plated and cultured overnight until confluence, scratched with a pipette tip and then washed with PBS, the drug-containing / serum-free culture medium is replaced, and photos are taken and recorded at 0 / 24 / 48h. The scratch area is analyzed by ImageJ and the migration rate is calculated.

[0011] Furthermore, the Transwell migration assay described in S20 has the following specific steps: Transwell migration assay steps: MCF-7 cells were starved and then a suspension was prepared. 200 μL of serum-free culture medium was added to the upper chamber, and 600 μL of culture medium containing 20% ​​FBS was added to the lower chamber. After culturing for 24 hours, the cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet for 20 minutes. The cells were photographed under a microscope and the number of migrated cells was counted using ImageJ.

[0012] Furthermore, the specific steps of the Transwell invasion assay described in S30 are as follows: MCF-7 cells were starved 24 h in advance, Matrigel was pre-cooled at 4 °C, 50 μL of Matrigel was plated in the upper chamber, and after hydration for 30 min, the cell suspension was inoculated. 600 μL of medium containing 20% FBS was added to the lower chamber. After culturing for 48 h, the cells were fixed and stained, and then photographed and counted under a microscope; The specific steps of the glucose level detection described in S40 are as follows: The cell supernatant was collected, a standard curve was established with a glucose standard from 0 to 2000 mg / dL. 20 μL of the sample was mixed with 170 μL of the detection reagent, heated at 95 °C for 8 min and then cooled at 4 °C, and the OD value was measured at 630 nm to calculate the concentration.

[0013] Furthermore, the specific steps of the lactate level detection described in S50 are as follows: The enzyme working solution and the color reagent were prepared. 1 mL of the prepared enzyme working solution and 200 μL of the color reagent were added to each EP tube containing the sample to be tested, and the mixture was inverted up and down to mix evenly. Then it was placed in a water bath at 37 °C for 10 minutes. After the above reaction ended, the absorbance value at 530 nm was detected using an enzyme-linked immunosorbent assay (ELISA) reader. The lactate content was obtained according to the following calculation method: Lactate content (mmol / L) = (A 测定 - A 空白） / (A 标准 - A 空白 )× standard concentration of the standard product × dilution factor; The specific steps of the ATP level detection described in S60 are as follows: After the sample was treated with the lysis buffer and centrifuged, the supernatant was taken. The ATP standard was diluted with the lysis buffer, and the ATP detection working solution was prepared at a ratio of 1:9. 100 μL of the working solution was added to a 96-well plate and allowed to stand at room temperature for 3 - 5 minutes. The RLU value was measured and a standard curve was plotted to calculate the ATP concentration; The specific steps of the determination of extracellular acidification rate and oxygen consumption rate described in S70 are as follows: The cells were plated on an XF 96-well plate, 180 μL of the hydration solution was added to the lower layer, and the plate was hydrated overnight in a 37 °C CO2-free incubator. At the same time, the drug was prepared and the pH of the Seahorse XF medium was adjusted to 7.4. The next day, the medium was preheated in a water bath. After washing the cells, 175 μL of the medium was added to each well, and the cells were cultured in a 37 °C CO2-free incubator for 1 hour. The drug was diluted, and 25 μL was added to the upper layer of each well. After detection for 30 minutes, the plate was replaced with a cell plate for continued detection.

[0014] Furthermore, the construction of the mouse breast cancer model described in S80 is specifically as follows: Construct a mammary in-situ tumor model. Inject the MDA-MB-231-LUC cell suspension into the mice. After the tumor grows to 100 mm³, randomly divide them into a PBS group (0.01 mL / g), a PAG (40 mg / kg) group, and a PAG + NaHS (10 mg / kg) reversal group, and observe the tumor changes; Construct a mammary lung metastasis model. After injecting the same cell suspension, group them according to the fluorescence intensity (PBS group, PAG group, PAG + NaHS reversal group) after tumor formation, and monitor the metastasis situation; The hematoxylin-eosin staining described in S90 is specifically as follows: After the tumor and lung tissues are fixed for 24 hours, wash them with PBS, dehydrate them with gradient alcohol, make them transparent with xylene, infiltrate them with paraffin and embed them, and then section them; After the sections are baked at 65 °C, remove the wax with xylene, hydrate them with gradient alcohol, stain them with hematoxylin, differentiate them with acid alcohol, blue them, stain them with eosin, and then dehydrate and mount them; The immunohistochemical staining described in S100 is specifically as follows: After dewaxing the sections, repair the antigens by high-pressure with preheated EDTA repair solution and cool them; Dry the sections, add H2O2 to block endogenous peroxidase, and block them with goat serum for 15 minutes; Drop the primary antibody and incubate overnight at 4 °C; The next day, rewarm at 37 °C for 30 minutes, wash with PBS, then drop the secondary antibody and incubate at 37 °C for 30 minutes, and wash with PBS again; Observe by DAB color development, counterstain with hematoxylin, and then dehydrate and mount.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: It is verified that H2S produced by the metabolism of CBS and CSE promotes the malignant biological behavior and glycolysis of breast cancer cells; Endogenous H2S induces the thiolation modification of PGK1 at the Cys108 and Cys316 sites; The thiolation-modified PGK1 promotes the glycolysis and malignant biological behavior of breast cancer cells; H2S promotes the growth and metastasis of tumors in vivo, providing a new idea and target for the treatment of breast cancer. Description of the Drawings

[0016] Figure 1 It is the pLVX-C-FLAG-mCMV-ZsGreen-IRES-Puro map of the present invention; Figure 2 It is the pLVX-hPGK1-Flag-ZsGreen-Puro map of the present invention; Figure 3 It is the schematic diagram of the detection of the expression of CBS and CSE and the H2S level of the present invention; Figure 4 It is the schematic diagram of the determination of the optimal concentration of the H2S synthase inhibitor of the present invention; Figure 5 It is the schematic diagram of reducing the H2S content in breast cancer cells by inhibiting the H2S synthase of the present invention; Figure 6 Schematic diagram of the inhibition of H2S synthase of the present invention on the proliferation of breast cancer cells; Figure 7 Schematic diagram of the inhibition of H2S synthase of the present invention on the migration and invasion of breast cancer cells; Figure 8 Schematic diagram of the inhibition of H2S synthase of the present invention on the EMT of breast cancer cells; Figure 9 Schematic diagram of the inhibition of H2S synthase of the present invention on increasing the glucose content in the supernatant of breast cancer cells; Figure 10 Schematic diagram of the changes in the lactate and ATP contents of breast cancer cells after the inhibition of H2S synthase of the present invention; Figure 11 Schematic diagram of the endogenous H2S sulfhydrylation modification of PGK1 of the present invention; Figure 12 Schematic diagram of the sulfhydrylation modification site of PGK1 of the present invention; Figure 13 Schematic diagram of DTT blocking the sulfhydrylation modification of PGK1 of the present invention; Figure 14 Schematic diagram of blocking the sulfhydrylation modification of PGK1 to reduce the glycolysis of breast cancer cells of the present invention; Figure 15 Schematic diagram of blocking the sulfhydrylation modification of PGK1 to inhibit the malignant biological behavior of breast cancer cells of the present invention; Figure 16 Schematic diagram of H2S promoting the growth and metastasis of breast tumors in mice of the present invention; Figure 17 Schematic diagram of the endogenous H2S mediating the sulfhydrylation modification of PGK1 to promote the glycolysis of breast cancer cells, and further promoting the proliferation and metastasis of breast cancer cells of the present invention; Figure 18 Schematic diagram of the overall process of the present invention; Figure 19 Schematic diagram of the specific steps of real-time fluorescence quantitative PCR of the present invention; Figure 20 Schematic diagram of the specific steps of Western blot of the present invention; Figure 21 Schematic diagram of the specific steps for detecting the H2S content in tissue samples of the present invention; Figure 22 Schematic diagram of the specific steps for detecting the H2S content in cell samples of the present invention; Figure 23 Schematic diagram of the specific steps of Biotin switch assay of the present invention; Figure 24Schematic diagram of the specific steps of the plasmid-related experiments of the present invention; Figure 25 Schematic diagram of the specific steps for measuring the extracellular acidification rate and oxygen consumption rate of the present invention; Figure 26 Schematic diagram of the specific steps for constructing a mouse breast cancer model of the present invention. Detailed implementation manners

[0017] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0018] In order to further clarify the occurrence and development mechanism of breast cancer and provide new therapeutic reference targets for clinical practice, as Figures 1 - 26 shown, the following preferred technical solutions are provided: Real-time fluorescence quantitative PCR, including the following steps: Extract RNA by the Trizol method. Add 50 mg of tissue to 1 mL of Trizol, grind and centrifuge (4 °C, 12,000 rpm, 15 min). Take the supernatant, add 200 μL of chloroform, mix well and centrifuge. Collect the aqueous phase, add 500 μL of isopropanol, precipitate at -20 °C for 30 min, wash 3 times with ethanol after centrifugation, air-dry and dissolve with 20 μL of enzyme-free water and measure the concentration (perform the whole process at 4 °C without enzyme operation). RNA reverse transcription: After thawing the reagent, prepare a 20 μL reaction system, shake well, react at 15 min → 85 °C (in a reverse transcription instrument), and centrifuge to collect the product (perform the whole process on ice).

[0019] Western blot, including the following steps: Protein extraction: After thawing the tissue, add RIPA lysis buffer (containing protease inhibitor) according to 100 mg: 1 mL, grind and centrifuge (4 °C, 12,000 rpm, 30 min) to take the supernatant. After cell lysis, scrape and centrifuge (under the same conditions) to take the supernatant, perform BCA quantification and boil for storage. Electrophoresis: Gel preparation: Pour the separating gel / concentrating gel. Loading: 80 V for 30 min → 120 V for 2 h (until the 10 kDa marker reaches the bottom). Transfer: After activating the PVDF membrane with methanol, transfer at a constant current of 200 - 300 mA for 1 - 2 h. Blocking: At room temperature for 2 h. Primary antibody: Incubate overnight on a shaker at 4 °C. Secondary antibody: At room temperature for 2 h (wash 3 × 10 min with TBST after each antibody incubation). Perform ECL chemiluminescence imaging and analyze the gray value with ImageJ (key parameters: perform all centrifugations at 4 °C; remove air bubbles during transfer; optimize the antibody dilution ratio according to the instructions).

[0020] Detection of H2S content in tissue samples includes the following steps: Prepare a 1 mmol standard, measure the OD value at 665 nm with an enzyme-linked immunosorbent assay (ELISA) reader to plot a standard curve; Homogenize the tissue sample with normal saline at a ratio of 1:9, centrifuge, and take the supernatant. Calculate the content (μmol / gprot) according to the formula (Δ665 - b) / a × f / Cpr. After ultrasonic disruption of the cell sample with an extraction solution, centrifuge and take the supernatant. Calculate the content (nmol / 10 4 cells) according to the standard curve y = 0.0044x and the formula 340.9 × ΔA / number of cells. All centrifugations are performed at 4°C and 12,000 rpm.

[0021] Biotin switch assay includes the following steps: Collect cell or tissue samples, wash them with phosphate-buffered saline (PBS) and then lyse (use RIPA buffer for cells and grind tissues), perform ultrasonic disruption, centrifuge, and take the supernatant; After BCA quantification, take 500 μg of protein, block free thiol groups with methyl methanethiosulfonate (MMTS) (50°C for 30 min), precipitate with acetone, and label the thiolated sites with Biotin-HPDP (incubate for 4 h); After washing with acetone, bind to streptavidin agarose beads at 4°C overnight, wash 5 times with NaCl buffer the next day, and finally add loading buffer, boil to denature, and detect the level of thiolation modification. (Key parameters: All centrifugation steps are performed at 4°C; MMTS blocking is at 50°C; Biotin-HPDP labeling is at room temperature for 4 h).

[0022] Cell culture includes the following steps: After resuscitating the cells, passage them (isolate and re-inoculate during the logarithmic growth phase), change the medium regularly to remove metabolic wastes, freeze and store backups, and use them after counting (formula: number of cells / mL = (total number of four large grids / 4) × 10 4 × dilution factor). Perform aseptic operation throughout the process and maintain the culture conditions at 37°C and 5% CO2. (Note: The passage density is adjusted according to cell type; the cryopreservation solution contains 10% dimethyl sulfoxide (DMSO)).

[0023] Plasmid-related experiments include the following steps: After constructing wild-type / mutant PGK1 plasmids, amplify them in LB medium (containing ampicillin), extract them using the alkaline lysis method (treat with P1 / P2 / P3 solutions and purify with a CP3 column), and finally transfect them into breast cancer cells with Lipo8000™ (in a six-well plate, 2.5 μg of plasmid + 4 μL of transfection reagent), and collect samples 48 h later. Key parameters: Amplify the bacterial solution on a shaker at 37°C and 120 rpm for 14 - 16 h; Centrifuge at 12,000 rpm for plasmid extraction; Change the medium 4 - 6 h after transfection.

[0024] The flat-plate cloning experiment includes the following steps: After digesting breast cancer cells, inoculate a six-well plate at a density of 1000 - 3000 cells / well, culture for 7 - 14 days until visible clone clusters are formed, fix with 4% paraformaldehyde for 30 minutes, stain with 0.1% crystal violet for 20 minutes (protected from light), wash with PBS, take pictures, and count using ImageJ. (Key points: The inoculation density is adjusted according to cell type; wash thoroughly after staining).

[0025] The cell scratch experiment includes the following steps: Spread the breast cancer cell suspension on the plate and culture overnight until confluent, scratch with a pipette tip, wash with PBS, replace with drug-containing / serum-free medium, take pictures at 0 / 24 / 48 h, analyze the scratch area using ImageJ, and calculate the migration rate (migration rate = (area at 0 h - area at t hours) / area at 0 h × 100%). Key points: The scratch width is consistent; fix the observation site.

[0026] The Transwell migration experiment includes the following steps: After starvation treatment of MCF-7 cells, prepare a cell suspension. Add 200 μL of serum-free medium (containing 1.5×10 5 cells) to the upper chamber, add 600 μL of medium containing 20% FBS to the lower chamber, culture for 24 hours, fix with 4% paraformaldehyde, stain with 0.1% crystal violet for 20 minutes, take pictures under a microscope, and count the number of migrated cells using ImageJ. (Key parameters: Inoculate 3×10 4 cells / well for MDA-MB-231 cells; wipe off the non-migrated cells in the upper chamber with a cotton swab after staining).

[0027] The Transwell invasion experiment includes the following steps: Starve MCF-7 cells 24 h in advance, pre-cool Matrigel (diluted 1:8 with DMEM) at 4°C. Spread 50 μL of Matrigel on the upper chamber (gel at 37°C for 2 h), hydrate for 30 min, then inoculate the cell suspension (200 μL / well). Add 600 μL of medium containing 20% FBS to the lower chamber, culture for 48 h, then fix and stain (4% paraformaldehyde + 0.1% crystal violet), and take pictures and count under a microscope. (Key points: Keep Matrigel at 4°C during operation; avoid moving the chamber after inoculation).

[0028] The glucose level detection includes the following steps: Collect the cell supernatant (12,000 g, 5 min), establish a standard curve using a glucose standard from 0 - 2000 mg / dL. Mix 20 μL of the sample with 170 μL of the detection reagent (total volume 190 μL), heat at 95°C for 8 min, then cool at 4°C, measure the OD value at 630 nm, and calculate the concentration.

[0029] Lactic acid level detection, including the following steps: Prepare an enzyme working solution and a color reagent. Add 1 mL of the prepared enzyme working solution and 200 μL of the color reagent to each group of EP tubes containing the sample to be tested. Invert the tubes up and down to mix well, and place them in a 37°C water bath for 10 minutes. After the above reaction ends, use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance value at 530 nm. Obtain the lactic acid content according to the following calculation method: Lactic acid content (mmol / L) = (A 测定 -A 空白） / (A 标准 -A 空白 ) × standard product concentration × dilution factor.

[0030] ATP level detection, including the following steps: After the sample is treated with a lysis solution and centrifuged, take the supernatant. Dilute the ATP standard product with the lysis solution, and prepare an ATP detection working solution at a ratio of 1:9. Add 100 μL of the working solution to a 96-well plate, and let it stand at room temperature for 3 - 5 minutes. Measure the relative light unit (RLU) value and draw a standard curve to calculate the ATP concentration.

[0031] Measurement of extracellular acidification rate and oxygen consumption rate, including the following steps: Seed the cells on an XF 96-well plate, add 180 μL of hydration solution to the lower layer, and incubate overnight in a 37°C CO2-free incubator for hydration. At the same time, prepare the drug and adjust the pH of the Seahorse XF medium to 7.4. The next day, preheat the medium in a water bath, wash the cells, and add 175 μL of medium to each well. Incubate at 37°C in a CO2-free environment for 1 hour. Dilute the drug, add 25 μL to the upper layer of each well, and after 30 minutes of detection, replace it with a cell plate for continued detection.

[0032] Construction of a mouse breast cancer model, including the following steps: Construct an orthotopic tumor model. Inject the MDA-MB-231-LUC cell suspension into the mice. After the tumor grows to 100 mm³, randomly divide the mice into a PBS group (0.01 mL / g), a PAG (40 mg / kg) group, and a PAG + NaHS (10 mg / kg) reversal group, and observe the tumor changes. Construct a breast cancer lung metastasis model. After injecting the same cell suspension, group the mice according to the fluorescence intensity (PBS group, PAG group, PAG + NaHS reversal group) after tumor formation, and monitor the metastasis situation.

[0033] Hematoxylin-eosin (HE) staining, including the following steps: Fix the tumor and lung tissues for 24 hours, wash them with PBS, dehydrate them with gradient alcohol, clear them with xylene, infiltrate them with paraffin and embed them, and then section them. After baking the sections at 65°C, dewax them with xylene, hydrate them with gradient alcohol, stain them with hematoxylin, differentiate them with acid alcohol, blue them, stain them with eosin, dehydrate them, and mount them.

[0034] Immunohistochemical staining includes the following steps: After dewaxing the sections, antigen retrieval is performed by high-pressure using preheated EDTA retrieval solution and then cooled; the sections are dried, H2O2 is added to block endogenous peroxidase, and goat serum is used for blocking for 15 minutes; primary antibodies against Ki-67 (1:500), E-cadherin (1:400), and Vimentin (1:500) are respectively added and incubated overnight at 4°C; the next day, the sections are rewarmed at 37°C for 30 minutes, washed with PBS, secondary antibody is added and incubated at 37°C for 30 minutes, and then washed with PBS again; DAB color development is observed, counterstained with hematoxylin, dehydrated, and sealed.

[0035] Statistical analysis was performed using SPSS 26.0 statistical software, and graphing was done using Graphpad Prism 9.5. Each experiment was independent and repeated 3 times, followed by normal distribution and homogeneity of variance tests, and the results were expressed as mean ± standard deviation. The comparison between two independent samples was analyzed using the t-test. ns indicates P >0.05, * indicates P <0.05, ** indicates P <0.01, *** indicates P <0.001, when P <0.05, it indicates that the difference is statistically significant.

[0036] The results of qRT-PCR, Western blot, and immunohistochemistry showed that the mRNA and protein expressions of CBS and CSE in breast cancer tissues were significantly higher than those in adjacent tissues ( Figure 3 A - C). Detection by methylene blue method confirmed that the H2S level in tumor tissues was also significantly increased ( Figure 3 D).

[0037] Figure 3 In A, it represents the mRNA expression of CBS and CSE detected by qRT-PCR in tissue samples; B - C represent the protein expressions of CBS and CSE detected by Western blot in tissue samples; D represents the H2S level detected by methylene blue method in tissues.

[0038] As Figure 4 shown, H2S is produced by the metabolism of CBS and CSE. Regulating their expression and activity can change the H2S level and affect the tumor microenvironment and progression. In this study, AOAA (CBS inhibitor) and PAG (CSE inhibitor) were used to inhibit the enzyme activity to evaluate their effects on the H2S content in breast cancer cells.

[0039] The IC50 values of AOAA and PAG were determined by cytotoxicity experiments to determine the optimal inhibitory concentration: 30 mM PAG and 900 μM AOAA were used for MDA-MB-231 cells, and 20 mM PAG and 600 μM AOAA were used for MCF-7 cells (Figure 4 A - D).

[0040] Figure 4 In it, A represents the identification of the concentration of PAG drug in MDA - MB - 231 cells; B represents the identification of the concentration of PAG drug in MCF - 7 cells; C represents the identification of the concentration of AOAA drug in MDA - MB - 231 cells; D represents the identification of the concentration of AOAA drug in MCF - 7 cells.

[0041] As Figure 5 shown, the methylene blue method detection showed that in MDA - MB - 231 and MCF - 7 cells, the treatment with AOAA (CBS inhibitor) and PAG (CSE inhibitor) significantly reduced the intracellular H2S level ( Figure 5 A - D), confirming that the regulation of H2S synthase activity generates H2S metabolism.

[0042] Figure 5 In it, A represents the change in the content of H2S in MDA - MB - 231 cells after adding PAG, n = 6; B represents the change in the content of H2S in MCF - 7 cells after adding PAG, n = 3; C represents the change in the content of H2S in MDA - MB - 231 cells after adding AOAA, n = 3; D represents the change in the content of H2S in MCF - 7 cells after adding AOAA, n = 3.

[0043] As Figure 6 shown, in order to further explore the effect of endogenous H2S on the proliferation ability of breast cancer cells, the CCK - 8 and plate cloning experiments were used to detect the proliferation ability of breast cancer cells after inhibiting H2S synthase. The results showed that compared with the untreated group, the proliferation and clone formation numbers of breast cancer cells in the PAG and AOAA inhibitor groups were significantly reduced ( Figure 6 A - H). It is suggested that inhibiting the activity of H2S synthase can reduce the proliferation ability of breast cancer cells.

[0044] Figure 6 In it, A - D represent the changes in the proliferation and clone formation abilities of MDA - MB - 231 and MCF - 7 cells treated with PAG detected by the CCK - 8 and plate cloning experiments, n = 3; E - H represent the changes in the proliferation and clone formation abilities of MDA - MB - 231 and MCF - 7 cells treated with AOAA detected by the CCK - 8 and plate cloning experiments, n = 3.

[0045] As Figures 7 - 8 shown, in order to further explore the effect of endogenous H2S on the migration and invasion abilities of breast cancer cells, the scratch assay and Transwell migration assay were used to detect the changes in the migration ability of breast cancer cells after adding PAG and AOAA drug inhibitors. It was found that the degree of scratch healing in the PAG and AOAA drug treatment groups was significantly lower than that in the untreated group (Figure 7 (A-B), the experiment showed that the number of migrating cells in the PAG and AOAA inhibitor groups was significantly less than that in the untreated group ( Figure 7 (C-F). Similarly, the results of the Transwell invasion assay showed that PAG and AOAA could significantly inhibit the invasion ability of MDA-MB-231 and MCF-7 cells compared with the untreated group ( Figure 7 (C-F). EMT is a key factor leading to tumor metastasis. To further investigate the effect of H2S synthase inhibitors on EMT in breast cancer cells, the expression of key EMT proteins was detected. The results showed that PAG and AOAA could increase the expression of E-cadherin protein and decrease the expression of N-cadherin and Vimentin proteins in breast cancer cells ( Figure 8 (A-B). Collectively, the above results indicate that inhibiting the endogenous H2S level in breast cancer cells weakens the migration, invasion, and EMT of breast cancer cells.

[0046] Figure 7 In A, the wound healing of breast cancer cells treated with PAG was detected by scratch assay, scale bar: 200 μm, n = 3; in B, the wound healing of breast cancer cells treated with AOAA was detected by scratch assay, scale bar: 200 μm, n = 3; in C and E, the migration and invasion of breast cancer cells treated with PAG were detected by Transwell assay, scale bar: 100 μm, n = 3; in D and F, the migration and invasion of breast cancer cells treated with AOAA were detected by Transwell assay, scale bar: 100 μm, n = 3.

[0047] Figure 8 In A-H, the expression of EMT-related proteins in breast cancer cells treated with PAG and AOAA for 48 h was detected by Western blot, n = 3.

[0048] As Figure 9 shown, based on the mechanism that H2S can enhance the glycolysis of tumor cells, the change in the glucose content in the supernatant of breast cancer cells after treatment with PAG and AOAA was first detected using a glucose kit. The results showed that the glucose content in the supernatant of breast cancer cells in the inhibitor group was significantly increased ( Figure 9 (A-D). It can be judged that inhibiting H2S synthase can reduce glucose consumption.

[0049] Figure 9 In A-B, the change in the glucose content in the cell supernatant after treatment with PAG was shown, n = 5; in C-D, the change in the glucose content in the supernatant of breast cancer cells after treatment with AOAA was shown, n = 5.

[0050] As Figure 10As shown in the figure, we further explored the changes in lactate and ATP in breast cancer cells after inhibiting H2S synthase. It was detected that the lactate content in the supernatant of breast cancer cells treated with the inhibitor decreased ( Figure 10 A-D). Similarly, using a kit to detect the intracellular ATP content, it was found that the intracellular ATP content of breast cancer cells in the inhibitor group decreased significantly ( Figure 10 E-H). This indicates that inhibiting H2S synthase can reduce lactate secretion and ATP production in breast cancer cells.

[0051] Figure 10 In A-D, it represents the change in lactate content in the cell supernatant after treatment with PAG and AOAA, n = 5; in E-H, it represents the change in intracellular ATP content after treatment with PAG and AOAA, n = 3.

[0052] As Figure 11 shown, H2S can cause protein thiolation modification to change its activity. From the above results, it is known that H2S plays an important role in the glycolysis process of breast cancer cells by affecting glycolysis-related fluxes. First, the Biotin-switch experiment was used to detect the thiolation modification level of PGK1, and it was found that it could undergo thiolation modification, and the thiolation modification level was higher in breast tumor tissues ( Figure 11 A-B), which showed a correlation with its endogenous hydrogen sulfide level. Next, we also found that after using PAG and AOAA to inhibit the activity of H2S synthase, the thiolation modification level of PGK1 decreased ( Figure 11 C-J). From the above description, it is known that endogenous H2S can cause PGK1 to undergo thiolation modification.

[0053] Figure 11 In A-B, it represents the detection of PGK1 thiolation modification in tissue samples by the Biotin-switch experiment; in C-F, it represents the change in PGK1 thiolation modification in breast cancer cells under the action of PAG, n = 3; in G-J, it represents the change in PGK1 thiolation modification in breast cancer cells under the action of AOAA, n = 3.

[0054] As Figure 12 shown, to further explore the thiolation modification site of PGK1, the 7 cysteine residues in the PGK1 peptide chain were mutated to alanine, and point mutation plasmids were constructed, named C50A, C99A, C108A, C316A, C367A, C379A and C380A respectively. The wild-type plasmid and the above point mutation plasmids were transfected into PGK1 KOIn MDA-MB-231 cells, Biotin-switch detected the thiol modification level of PGK1. The results showed that compared with the wild-type group, the thiol modification levels of C50A, C99A, C367A, C379A, and C380A did not change significantly, while the thiol modification levels of C108A and C316A were significantly reduced ( Figure 12 AB). This indicates that PGK1 undergoes thiol modification at Cys108 and Cys316.

[0055] Figure 12 AB in the figure represents the thiol modification of PGK1 after point mutation detected by Biotin-switch, n=4.

[0056] like Figure 13 As shown in the figure, in order to better explore the effect of PGK1 thiol modification on glycolysis of breast cancer cells, the thiol modification blocker DTT was used. It was found that compared with the control group, the PGK1 thiol level in the DTT group was weakened. After the addition of NaHS, the effect of DTT on PGK1 thiol modification was reversed ( Figure 13 AD). This indicates that DTT blocked the endogenous H2S-mediated PGK1 thiolation modification.

[0057] Figure 13 AD in the figure indicates the changes of PGK1 thiol modification after the action of DTT and NaHS on breast cancer cells detected by Biotin-switch experiment, n=3.

[0058] like Figure 14 As shown in the figure, in order to further explore the specific role of PGK1 thiol modification in the glycolysis process of breast cancer cells, DTT was added to block the thiol modification of PGK1, and the changes in the glucose content in the cell supernatant and the generation of lactate and ATP were detected. The results showed that compared with the control group, the addition of DTT significantly increased the glucose in the cell supernatant, significantly reduced the lactate, and significantly decreased the intracellular ATP level ( Figure 14 AC). The Seahorse experiment was used to detect the changes in the extracellular acidification rate and oxygen consumption rate of breast cancer cells after DTT blocked the thiol modification of PGK1, and further determine the effect of blocking the thiol modification of PGK1 on the glycolysis of breast cancer cells. It was found that compared with the untreated group, after DTT blocked the thiol modification of PGK1, the extracellular acidification rate of breast cancer cells decreased and the oxygen consumption rate increased, while NaHS reversed the above phenomenon ( Figure 14 GJ). The above results suggest that endogenous H2S promotes glycolysis in breast cancer cells by mediating the thiol modification of PGK1.

[0059] Figure 14A-B in it represents the change in the glucose content in the cell supernatant of breast cancer cells under the action of DTT, n = 5; C-D represents the change in the lactic acid content in the cell supernatant of breast cancer cells under the action of DTT, n = 5; E-F represents the change in the intracellular ATP content of breast cancer cells under the action of DTT, n = 3; G-H represents the change in the extracellular acidification rate and oxygen consumption rate of MDA-MB-231 cells, n = 6; I-J represents the change in the extracellular acidification rate and oxygen consumption rate of MCF-7 cells, n = 5.

[0060] As Figure 15 shown, through research, it was found that H2S promoted the proliferation, migration, invasion and epithelial-mesenchymal transition of breast cancer cells, and regulated the glycolysis of breast cancer cells by mediating the thiolation modification of PGK1. First, DTT was used to block the thiolation modification of PGK1 in MDA-MB-231 cells and MCF-7 cells, and the change in the proliferation ability of breast cancer cells was detected. As Figure 15 shown in A, the number of colony formations of breast cancer cells decreased significantly after DTT blocked the thiolation modification of PGK1. Next, the scratch assay and Transwell migration assay were used to detect the migration of breast cancer cells after eliminating the thiolation modification of PGK1, and it was found that the degree of scratch healing in the DTT group was significantly less than that in the control group ( Figure 15 B); the results of the Transwell migration assay showed that DTT treatment significantly inhibited the migration of breast cancer cells ( Figure 15 C-D). In addition, the Transwell invasion assay also obtained consistent results, and DTT significantly inhibited the invasion of breast cancer cells ( Figure 15 C-D). The expression of EMT marker proteins was detected by Western blot, and it was found that after blocking the thiolation modification, the expression of E-cadherin protein increased, while the expression of N-cadherin and Vimentin proteins decreased ( Figure 15 E). The above results indicate that endogenous H2S promotes the malignant biological behavior of breast cancer cells by mediating the thiolation modification of PGK1.

[0061] Figure 15 A in it represents the number of colony formations of breast cancer cells detected by the plate colony assay after adding DTT, n = 3; B represents the change in the scratch healing area of breast cancer cells detected by the scratch assay after DTT treatment, scale bar: 200 μm, n = 3; C-D represents the migration and invasion of breast cancer cells detected by the Transwell assay after DTT treatment, scale bar: 100 μm, n = 3; E represents the expression of EMT-related proteins detected by Western blot after DTT treatment of breast cancer cells, n = 3.

[0062] As Figure 16As shown, to further investigate the effect of H2S on breast tumor growth in vivo, an NCG mouse xenograft tumor model was constructed using MDA-MB-231 cells. As Figure 16 shown in A, the tumor volume in the PAG group was smaller than that in the PBS group, while the tumor volumes in the PAG and NaHS groups were larger than that in the PAG group ( Figure 16 B - C). Immunohistochemical results showed that the expression of Ki-67 protein decreased in the PAG group, while the expression of Ki-67 protein increased after the combined use of PAG and NaHS ( Figure 16 D). The above results indicate that H2S promotes the growth of transplanted tumors in mice. The level of H2S in the body of mice in the PAG group was significantly lower than that in the PBS group, while NaHS reversed the regulation of H2S by PAG ( Figure 16 E). By studying the changes in the level of PGK1 thiolation modification in mice, it was found that the level of thiolation modification in the PAG group was significantly lower than that in the PBS group, while the level of PGK1 thiolation modification in the PAG + NaHS group increased ( Figure 16 F - G). The above results indicate that H2S promotes breast cancer proliferation through PGK1 thiolation modification.

[0063] To explore the effect of endogenous H2S on tumor metastasis in mice, a mouse breast cancer lung metastasis model was constructed. The fluorescence intensity of tumor cells was observed by in vivo imaging technology. As Figure 16 shown in H, the fluorescence signal in the PAG group was weaker than that in the PBS group, and the fluorescence signal in the combined drug group of PAG and NaHS was enhanced. Next, HE staining of lung tissues showed that there were obvious lung metastasis lesions in the PBS group and the combined drug group of PAG and NaHS, and the number of lung metastasis nodules was large, while the number of lung metastasis nodules in the PAG group was significantly reduced ( Figure 16 I - J). To further study the role of H2S in lung metastasis, immunohistochemical staining was performed on the lung tissues of mice to detect the expression of EMT-related proteins. Compared with the control group, the expression of E-cadherin protein in the lung tissues of mice in the PAG group increased significantly, and the expression of Vimentin protein decreased. This phenomenon was reversed after adding NaHS to PAG ( Figure 16 K). The above results indicate that H2S promotes breast cancer metastasis in mice. In summary, H2S can promote the growth and metastasis of xenograft tumors.

[0064] Figure 16In A, the tumor growth of mice was detected by in vivo imaging technology (n = 4); in B, the excised solid tumors were shown (n = 4); in C, the curve of tumor volume change was presented; in D, the expression of Ki-67 protein in the mice was detected by immunohistochemical staining, scale bar: 100 μm; in E, the change of H2S level in the tumor tissues of mice was detected by methylene blue method, n = 4; in F-G, the change of PGK1 sulfhydryl modification in the tumor tissues of mice was detected by Biotinswitch assay, n = 4; in H, the lung metastasis of mice was detected by in vivo imaging technology; in I-J, the HE staining images of lung tissues and the quantification of lung metastasis nodules were shown, scale bar: 200 μm, n = 6; in K, the expression of EMT-related proteins in the lung tissues of mice was detected by immunohistochemistry, scale bar: 50 μm.

[0065] As Figure 17 shown, the following conclusions can be drawn: endogenous H2S regulates the glycolysis of breast cancer cells by mediating PGK1 sulfhydryl modification, thereby promoting tumor proliferation and metastasis.

[0066] It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device.

[0067] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Endogenous hydrogen sulfide mediates the S-thiolation modification of PGK1 to promote the proliferation and metastasis of breast cancer, which is characterized in that: It includes the following steps: S1: Real-time fluorescence quantitative PCR; S2: Western blot; S3: Detection of H2S content in tissue samples; S4: Detection of H2S content in cell samples; S5: Biotin switch assay; S6: Cell culture; S7: Plasmid-related experiments; S8: CCK-8 assay; S9: Plate cloning assay; S10: Cell scratch assay; S11: Transwell migration assay; S12: Transwell invasion assay; S13: Glucose level detection; S14: Lactate level detection; S15: ATP level detection; S16: Determination of extracellular acidification rate and oxygen consumption rate; S17: Construction of mouse breast cancer model; S18: Hematoxylin-eosin staining; S19: Immunohistochemical staining; S20: Statistical analysis.

2. Endogenous hydrogen sulfide mediates the thiolation modification of PGK1 to promote the proliferation and metastasis of breast cancer, characterized in that: For the real-time fluorescence quantitative PCR described in S1, the specific steps are as follows: RNA is extracted by the Trizol method. 50 mg of tissue is added to 1 mL of Trizol, ground and centrifuged. The supernatant is taken, 200 μL of chloroform is added, mixed well and centrifuged. The aqueous phase is collected, 500 μL of isopropanol is added, and precipitated at -20 °C for 30 min. After centrifugation, it is washed with ethanol 3 times. After air drying, it is dissolved in 20 μL of enzyme-free water and the concentration is measured; RNA reverse transcription: After thawing the reagent, a 20 μL reaction system is prepared, shaken and mixed well, reacted at 15 min → 85 °C, and the product is collected by centrifugation (the whole process is performed on ice); qPCR detection: The reaction mixture is added to an 8-strip tube, DNA template is added, centrifuged briefly and mixed well, the program is set, and the qPCR instrument is started for detection; For the Western blot described in S2, the specific steps are as follows: Protein extraction: After the tissue is thawed, add RIPA lysis buffer according to 100 mg:1 mL, ground and centrifuged to take the supernatant. After the cells are lysed, scrape and centrifuge to take the supernatant. After BCA quantification, it is boiled and stored; Electrophoresis, gel preparation, pouring of separating gel and stacking gel are carried out; Loading: 80 V for 30 min → 120 V for 2 h; Transfer: After the PVDF membrane is activated with methanol, transfer at a constant current of 200 - 300 mA for 1 - 2 h; Blocking, at room temperature for 2 h; Primary antibody: Overnight on a shaker at 4 °C; Secondary antibody: At room temperature for 2 h; ECL luminescence development, and the gray value is analyzed by ImageJ; For the detection of H2S content in tissue samples described in S3: Prepare a 1 mmol standard product, measure the OD value at 665 nm with an enzyme-labeled instrument to draw a standard curve; The tissue sample is homogenized with normal saline at a ratio of 1:9, centrifuged to take the supernatant, and the content (μmol / gprot) is calculated according to the formula (Δ665 - b) / a × f / Cpr; In S4, after the cell sample is ultrasonically disrupted with the extraction solution, the supernatant is taken by centrifugation, and the content is calculated according to the standard curve y = 0.0044x and the formula 340.9×ΔA / cell number (nmol / 10 4 cells); all centrifugations are carried out at 4 °C and 12,000 rpm; The biotin switch assay operation process described in S5 is as follows: collect cell or tissue samples, wash with PBS and then lyse, ultrasonicate and centrifuge to obtain the supernatant; after BCA quantification, take 500 μg of protein, block free thiol groups with MMTS, precipitate with acetone and label the thiol sites with biotin-HPDP; after washing with acetone, bind with streptavidin agarose beads at 4°C overnight, wash with NaCl buffer 5 times the next day, and finally add loading buffer to boil and denature to detect the thiol modification level; Cell culture operation procedure described in S6: Passage the cells after resuscitation, change the medium regularly to remove metabolic waste, freeze and backup, and use the formula after counting: Cells / mL = (Total number of four large grids / 4) × 10 4 × Dilution factor; Perform aseptic operation throughout the process and maintain the culture conditions of 37°C and 5% CO2.

3. The endogenous hydrogen sulfide mediates the thiolation modification of PGK1 to promote the proliferation and metastasis of breast cancer according to claim 2, characterized in that: The specific steps of the plasmid-related experiments described in S7 are as follows: after constructing the PGK1 wild-type / mutant plasmid, it was amplified in LB medium, extracted by alkaline lysis method, and finally transfected into breast cancer cells with Lipo8000™, and the samples were collected after 48 hours; The CCK-8 experimental procedure described in S8 was as follows: 3000 cells / well were inoculated into a 96-well plate, cultured at 37°C until adhered to the wall, and then replaced with drug-containing medium. 10% CCK-8 reagent was added at 0 / 24 / 48 h in the dark, and the absorbance at 450 nm was measured after incubation for 2 h.

4. The endogenous hydrogen sulfide mediates the thiolation modification of PGK1 to promote the proliferation and metastasis of breast cancer according to claim 3, characterized in that: The specific steps of the plate cloning experiment described in S9 are as follows: after digestion of breast cancer cells, 1000-3000 cells / well were seeded in a six-well plate, cultured for 7-14 days until visible clones were formed, fixed with 4% paraformaldehyde for 30 minutes, stained with 0.1% crystal violet for 20 minutes, washed with PBS, photographed and counted with ImageJ; The cell scratch experiment described in S10 has the following specific steps: the breast cancer cell suspension is plated and cultured overnight until confluence, scratched with a pipette tip and then washed with PBS, the drug-containing / serum-free culture medium is replaced, and photos are taken and recorded at 0 / 24 / 48h. The scratch area is analyzed by ImageJ and the migration rate is calculated.

5. The endogenous hydrogen sulfide mediates the thiolation modification of PGK1 to promote the proliferation and metastasis of breast cancer according to claim 4, characterized in that: The Transwell migration assay described in S20 has the following specific steps: Transwell migration assay steps: After starvation treatment of MCF-7 cells, a suspension was prepared, 200 μL of serum-free culture medium was added to the upper chamber, and 600 μL of culture medium containing 20% ​​FBS was added to the lower chamber. After culturing for 24 hours, the cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet for 20 minutes. The cells were photographed under a microscope and the number of migrated cells was counted using ImageJ.

6. The endogenous hydrogen sulfide mediates the thiolation modification of PGK1 to promote the proliferation and metastasis of breast cancer according to claim 5, characterized in that: The specific steps of the Transwell invasion assay described in S30 are as follows: MCF-7 cells were starved 24 h in advance, Matrigel was precooled at 4°C, 50 μL of Matrigel was spread in the upper chamber, the cell suspension was inoculated after hydration for 30 min, 600 μL of medium containing 20% ​​FBS was added to the lower chamber, and the cells were fixed and stained after 48 h of culture, and photographed and counted under a microscope; The glucose level detection described in S40 has the following specific steps: collecting cell supernatant (12,000 g, 5 min), establishing a standard curve with glucose standard, mixing 20 μL sample with 170 μL detection reagent, heating at 95°C for 8 min and then cooling at 4°C, measuring OD value at 630 nm and calculating concentration.

7. The promotion of breast cancer proliferation and metastasis mediated by H2S through the sulfhydrylation of PGK1 according to claim 6, wherein: The lactic acid level detection described in S50 is specifically carried out as follows: Prepare an enzyme working solution and a color reagent. Add 1 mL of the prepared enzyme working solution and 200 μL of the color reagent to each EP tube containing the sample to be tested, mix well by inverting up and down, react in a 37 °C water bath for 10 minutes. After the reaction, use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance value at 530 nm. Calculate the lactic acid content according to the following method: Lactic acid content (mmol / L) = (A 测定 -A 空白） / (A 标准 -A 空白 ) × standard concentration × dilution factor; The ATP level detection described in S60 is carried out as follows: After the sample is treated with lysis buffer and centrifuged, the supernatant is taken. The ATP standard is diluted with the lysis buffer, and the ATP detection working solution is prepared at a ratio of 1:

9. Add 100 μL of the working solution to a 96-well plate, let it stand at room temperature for 3 - 5 minutes, measure the RLU value and draw a standard curve to calculate the ATP concentration; The determination of extracellular acidification rate and oxygen consumption rate described in S70 is carried out as follows: Seed the cells in an XF 96-well plate, add 180 μL of hydration solution to the lower layer, and incubate overnight in a 37 °C CO2-free incubator. At the same time, prepare the drug and adjust the pH of the Seahorse XF medium to 7.4; The next day, preheat the medium in a water bath, wash the cells, and add 175 μL of medium to each well, and culture in a 37 °C CO2-free environment for 1 hour; Dilute the drug, add 25 μL to the upper layer of the plate for each well, and replace it with a cell plate for continuous detection after 30 minutes of detection.

8. Endogenous hydrogen sulfide mediates the thiolation modification of PGK1 to promote the proliferation and metastasis of breast cancer, characterized in that: The construction of the mouse breast cancer model described in S80 is carried out as follows: Construct an orthotopic tumor model by inoculating MDA-MB-231-LUC cells into mice. After the tumor grows to 100 mm³, randomly divide them into a PBS group (0.01 mL / g), a PAG (40 mg / kg) group, and a PAG + NaHS (10 mg / kg) reversal group, and observe the tumor changes; Construct a breast-lung metastasis model. After injecting the same cell suspension into the tail vein, group them according to the fluorescence intensity (PBS group, PAG group, PAG + NaHS reversal group) after tumor formation, and monitor the metastasis situation; The hematoxylin-eosin staining described in S90 is carried out as follows: After the tumor and lung tissues are fixed for 24 hours, wash them with PBS, dehydrate them with gradient ethanol, make them transparent with xylene, infiltrate them with paraffin and embed them, and then section them; After the sections are baked at 65 °C, remove the wax with xylene, hydrate them with gradient ethanol, stain them with hematoxylin, differentiate them with acid alcohol, blue them back, stain them with eosin, dehydrate them and mount the slides; The immunohistochemical staining described in S100 is carried out as follows: After de-waxing the sections, repair the antigens by high-pressure with preheated EDTA repair solution and cool them; Wipe the sections dry, add hydrogen peroxide to block endogenous peroxidase, and block with goat serum for 15 minutes; Dropwise add the primary antibodies of Ki-67 (1:500), E-cadherin (1:400), and Vimentin (1:500) respectively, and incubate overnight at 4 °C; The next day, rewarm at 37 °C for 30 minutes, wash with PBS, then add the secondary antibody and incubate at 37 °C for 30 minutes, and wash with PBS; Develop color with DAB, counterstain with hematoxylin, dehydrate and mount the slides.

9. The endogenous hydrogen sulfide mediates the thiolation modification of PGK1 to promote the proliferation and metastasis of breast cancer, characterized in that: The statistical analysis described in S200 is carried out as follows: Each experiment is independent and repeated 3 times, and then tested for normal distribution and homogeneity of variance. It is expressed as mean ± standard deviation, and the comparison of two independent samples is analyzed by t-test.

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