Application of CD36-mediated sodium palmitate to promotion of CML drug resistance through metabolic reprogramming

Abstract

The invention discloses application of CD36-mediated sodium palmitate to promotion of CML drug resistance through metabolic reprogramming, and relates to the technical field of biological medicine. The medicine for treating the CML is screened by utilizing the CD36-EHHADH metabolic axis, and the specific method comprises the following steps: co-culturing a candidate medicine and a CML cell line, detecting the influence of the medicine on the key molecular expression and cell proliferation of the CD36-EHHADH metabolic axis, and screening out the medicine with the treatment potential. According to the method for screening the medicine for treating the CML by utilizing the CD36-EHHADH metabolic axis, the candidate medicine and the CML cell line are co-cultured, the influence of the medicine on key molecule expression and cell proliferation is detected, and the medicine with treatment potential can be rapidly screened out; according to the method, blind screening is avoided, the efficiency and success rate of new drug research and development are improved, and more effective therapeutic drug choices are expected to be provided for CML patients.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of CD36-mediated sodium palmitate to promote CML drug resistance through metabolic reprogramming. Background Technology

[0002] Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm (MPN) characterized by the abnormal expansion of immature hematopoietic stem cells (HSCs) in the bone marrow, leading to elevated levels of myeloid cells in the peripheral blood. CML accounts for approximately 15% of adult leukemias, and its incidence is higher in men. The median age of diagnosis in Western countries is approximately 60 years, but it is younger in Asians. Due to the introduction of tyrosine kinase inhibitors (TKIs), the age-adjusted mortality rate has significantly decreased from 0.85 / 100,000 in 1998 to 0.31 / 100,000 in 2020, and the current 10-year survival rate is 60%–90%.

[0003] Detection of BCR-ABL1 is essential for the diagnosis of CML. The BCR-ABL1 gene produces the BCR-ABL1 transcript, which plays a crucial role in the lateral progression of CML. This gene possesses tyrosine kinase (TK) activity, playing a vital role in activating downstream signaling pathways that inhibit apoptosis and promote CML cell proliferation. In 2001, the US FDA designated imatinib (IM) as a tyrosine kinase inhibitor, making it a first-line treatment for CML. IM competitively inhibits the ATP site of the BCR-ABL1 protein by blocking its TK activity and preventing substrate phosphorylation, thereby blocking signal transduction that leads to cell proliferation. With continued IM treatment, the growth of differentiated cells and leukemia progenitor cells can be controlled. IM has shown good efficacy in treatment, but 25% of patients develop drug resistance, and the disease relapses. Mutations in the BCR-ABL kinase domain are one of the most common mechanisms leading to imatinib resistance.

[0004] The protective role of the bone marrow microenvironment (BMM) for leukemia cells is crucial, but adipocytes, as an important component of the BMM, are often overlooked. Traditionally, bone marrow adipocytes (BMAds) were considered inert cells, merely used to fill excess space in the bone marrow cavity and store energy. However, increasing research indicates that the hyperlipidemic environment induced by BMDAs can promote abnormal proliferation and chemotherapy resistance in leukemia cells, and the role of BMDAs varies among different types of leukemia. In acute myeloid leukemia (AML), BMDAs can release large amounts of free fatty acids (FFA) through lipolysis, promoting excessive FFA uptake by leukemia cells and generating a large amount of energy needed for tumor cell growth and proliferation. In acute lymphoblastic leukemia (ALL), BMDAs promote the secretion of inflammatory cytokines and inhibit the cytotoxicity of the chemotherapeutic drug daunorubicin. The exact mechanisms of action of BMDAs in chronic myeloid leukemia (CML) remain to be explored.

[0005] The fatty acid receptor CD36 (Cluster-differentiated 36) is an integrated transmembrane protein widely distributed in various tissues, exhibiting a high affinity for the uptake of long-chain fatty acids. It participates in various normal and pathobiological processes, including angiogenesis, immune inflammation, atherosclerosis, and phagocytosis. CD36 plays a crucial role in the development of various metabolic diseases, such as cardiovascular disease, non-alcoholic fatty liver disease, and diabetes. In AML, leukemia cells promote chemotherapy resistance by overexpressing CD36; treatment of AML transplanted mice with CD36 blocking antibodies significantly prolongs survival and reduces tumor cell metastasis. In CLL, STAT3 has been shown to activate CD36 by binding to the CD36 promoter, promoting fatty acid uptake. In CML, clinical studies have shown that obesity plays a significant role in the risk of chronic myeloid leukemia progression, suggesting that a hyperlipidemic state may be associated with poor prognosis in CML.

[0006] Fatty acids ingested from the outside are mainly converted into ATP and reduced equivalent NADPH through fatty acid oxidation via mitochondria, thus participating in cellular energy supply.

[0007] In hematologic malignancies, literature reports that leukemia-associated fatty acid cells (LSCs) in relapsed AML patients rely more on the fatty acid oxidation (FAO) pathway to meet their metabolic needs than on glycolysis. CPT1, a key rate-limiting enzyme for FAO in mitochondria, is widely expressed in AML, CLL, and multiple myeloma. The CPT1-specific inhibitor ST1326 can significantly inhibit its enzyme activity, reducing fatty acid oxidation in leukemia cells, thereby inhibiting tumor cell growth and promoting apoptosis. In CML, it is speculated that fatty acid oxidation occurs via the peroxisome pathway, with EHHADH as a key enzyme.

[0008] This invention primarily elucidates the mechanism of action of PA in CML-TKI resistance through the CD36-EHHADH lipid metabolism axis using clinical samples, cellular and molecular biology levels. Summary of the Invention

[0009] The purpose of this invention is to address the shortcomings of existing technologies by proposing an application of CD36-mediated sodium palmitate to promote CML drug resistance through metabolic reprogramming.

[0010] In order to achieve the above object, the present invention adopts the following technical solutions:

[0011] An application of CD36-mediated sodium palmitate to promote CML drug resistance through metabolic reprogramming was conducted. The CD36-EHHADH metabolic axis was used to screen for drugs treating CML. The specific method is as follows:

[0012] Candidate drugs were co-cultured with CML cell lines to examine their effects on the expression of key molecules in the CD36-EHHADH metabolic axis and cell proliferation, thereby screening for drugs with therapeutic potential.

[0013] Preferably, the CML cell line includes the K562R cell line, and the candidate drug includes small molecule inhibitors and traditional Chinese medicine extracts.

[0014] Preferably, in the method, when detecting the effect of the drug on cell proliferation, a cell proliferation detection method is used, including the CCK-8 assay and the MTT assay.

[0015] Preferably, in the method, when culturing the CML cell line, RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin is used.

[0016] An application of CD36-mediated sodium palmitate promoting CML drug resistance through metabolic reprogramming involves using a CML diagnostic kit based on the CD36-EHHADH metabolic axis to assist in the diagnosis of CML. The kit contains antibodies or nucleic acid detection probes that can specifically recognize CD36 and EHHADH to detect the expression levels of corresponding proteins or genes in CML patient samples.

[0017] Preferably, the antibody includes an anti-human antigen CD36 antibody and an anti-human antigen EHHADH antibody, and the nucleic acid detection probe is capable of specifically binding to specific sequences of the CD36 and EHHADH genes.

[0018] An application of CD36-mediated sodium palmitate promoting CML drug resistance through metabolic reprogramming, the application including: elucidating the molecular mechanism of CML drug resistance and overcoming drug resistance in CML cells;

[0019] The specific method for revealing the molecular mechanism of CML resistance is as follows: differential gene expression analysis is performed on CML drug-resistant cell lines and drug-sensitive cell lines to detect the expression differences of CD36, EHHADH, PPARγ and fatty acid metabolism-related genes.

[0020] Preferred method: The specific way to overcome the drug resistance of CML cells is to downregulate the expression or activity of CD36 and EHHADH through siRNA, small molecule inhibitors or gene editing technology, so as to make CML cells sensitive to TKIs again.

[0021] Preferred method: The method for revealing the molecular mechanism of CML drug resistance is based on bioinformatics analysis and in vitro detection of CD36-EHHADH metabolic axis expression in CML, and in vitro experimental analysis of the effect of PA on CML drug resistance through CD36-EHHADH lipid metabolism axis. Specifically, in the bioinformatics analysis and in vitro detection of CD36-EHHADH metabolic axis expression in CML, flow cytometry analysis of PPARγ and EHHADH antibody expression levels in patient samples includes the following steps:

[0022] S1: First, prepare the necessary test tubes and label them with the antibodies you want to add.

[0023] S2: First, add 5 μL of CD34-PC5 antibody to each tube;

[0024] S3: Then add the corresponding antibodies according to the markings on the tube. It must be a combination of FITC and PE labeled antibodies.

[0025] S4: Add 100 μl of bone marrow specimen to each tube, shake to mix, and incubate at room temperature in the dark for 20-30 minutes before testing.

[0026] Preferably, the in vitro experimental analysis of the effect of PA on CML resistance via the CD36-EHHADH lipid metabolism axis includes the following steps:

[0027] S1: Cell culture, cell passage, cell resuscitation, cell cryopreservation;

[0028] S2: Preparation of IM solution and sodium palmitate solution;

[0029] S3: siRNA transfection, overexpression plasmid transfection;

[0030] S4: RT-PCR, total RNA extraction, cDNA generation, Cq value determination;

[0031] S5: Western Blot; extraction of total protein, determination of protein concentration using the Bradford method, gel electrophoresis; detection of ATP production content;

[0032] S6: Statistical analysis.

[0033] The beneficial effects of this invention are as follows:

[0034] 1. This invention utilizes the CD36-EHHADH metabolic axis to screen drugs for the treatment of CML. Candidate drugs are co-cultured with CML cell lines, and the effects of the drugs on the expression of key molecules and cell proliferation are detected. This method can quickly screen out drugs with therapeutic potential. This method avoids blind screening, improves the efficiency and success rate of new drug development, and is expected to provide more effective treatment options for CML patients.

[0035] 2. This invention reverses drug resistance in CML cells by regulating the CD36-EHHADH metabolic axis, such as by downregulating the expression or activity of key molecules through small interfering RNA, small molecule inhibitors, or gene editing technology. This can re-sensitize CML cells to TKIs and effectively overcome the drug resistance problem of CML cells. This is of great significance for improving the treatment effect of CML patients and helps to prolong their survival.

[0036] 3. This invention utilizes a diagnostic kit based on the CD36-EHHADH metabolic axis to specifically detect the expression levels of CD36 and EHHADH proteins or genes in CML patient samples, providing a more accurate and reliable basis for CML diagnosis. The kit contains specific antibodies or nucleic acid detection probes, as well as supporting equipment and reagents, which can effectively improve diagnostic efficiency and accuracy, and help to detect CML patients early and carry out timely treatment. Attached Figure Description

[0037] Figure 1 This is a schematic diagram illustrating the distribution and changes of adipocytes in the bone marrow microenvironment under different clinical treatment conditions of CML in bone marrow biopsy analysis according to the present invention;

[0038] Figure 2 This is a schematic diagram of the flow cytometry analysis of CD36 expression according to the present invention;

[0039] Figure 3 This is a schematic diagram illustrating the effect of PA on cell apoptosis according to the present invention;

[0040] Figure 4This is a schematic diagram illustrating the expression of the CD36-EHHADH metabolic axis in CML using bioinformatics combined with clinical data analysis according to the present invention.

[0041] Figure 5 This is a schematic diagram showing the expression levels of CD36 and EHHADH in cells according to the present invention.

[0042] Figure 6 This is a schematic diagram of the in vitro cell experiment results of the present invention. Detailed Implementation

[0043] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0044] Example 1:

[0045] An application of CD36-mediated sodium palmitate (PA) to promote CML drug resistance through metabolic reprogramming includes: exploring the role of PA in the bone marrow microenvironment in CML drug-resistant patients, bioinformatics analysis and in vitro detection of CD36-EHHADH metabolic axis expression in CML, in vitro experimental analysis of the effect of PA on CML drug resistance through CD36-EHHADH lipid metabolic axis, and taking appropriate measures to achieve or optimize CML treatment.

[0046] The study investigated the role of PA in the bone marrow microenvironment in patients with drug-resistant chronic myeloid leukemia. The specific experimental methods are as follows:

[0047] 1.1 Material Preparation

[0048] 1.2 Test Methods

[0049] 1.2.1 HE staining to observe adipocytes in bone marrow

[0050] 1) Obtain bone marrow fluid by bone marrow aspiration, spread it on a glass slide to form a thin film, and shake or fan it dry in the air. Fix with fixative for more than 10 minutes, wash with distilled water for 2 minutes, replace with fresh distilled water, and wash again for 2 minutes.

[0051] 2) Stain with hematoxylin staining solution for 10 minutes (the time can be adjusted according to the staining results and requirements).

[0052] 3) Rinse with tap water to remove excess staining solution for about 10 minutes.

[0053] 4) Rinse again with distilled water (for a few seconds).

[0054] 5) Stain with eosin staining solution for 2 minutes.

[0055] 6) Wash twice with 70% ethanol and observe under a microscope. The cell nucleus appears blue, while the cytoplasm appears pink or red.

[0056] 1.2.2 Processing of bone marrow supernatant specimens before mass spectrometry.

[0057] 1) Add 225 μL of methanol to the EP tube, then add 225 μL of sample plasma and vortex at maximum speed for 10 seconds.

[0058] 2) Add an appropriate internal standard and mix well.

[0059] 3) Add 750 μL LMTBE (methyl tert-butyl ether), vortex at maximum speed for 10 s, and let stand at room temperature for 30 min.

[0060] 4) Add 188 μL of water for mass spectrometry and vortex for 20 s.

[0061] 5) Let it sit at room temperature for 10 minutes.

[0062] 6) Centrifuge at 4 degrees Celsius and 15,000 rpm for 15 minutes. Take 700 μL of the supernatant (the sample is divided into three layers, from top to bottom: lipid phase, aqueous phase, and solid residue) into a new EP tube.

[0063] 7) Use a nitrogen blower to dry the supernatant.

[0064] 8) Add 50 μL of reconstitution solution to recover lipids. The reconstitution solution consists of dichloromethane and methanol in a 1:1 volume ratio.

[0065] 9) Vortex for 10 seconds, then centrifuge at 14000g (4℃) for 10 minutes.

[0066] 10) Aspirate the supernatant into the sample tube for testing.

[0067] 1.2.3 Determination of the appropriate concentration of PA by CCK8 method

[0068] Cell growth rate was detected using the CellCounting Kit-8 assay. K562R CML cells, supplemented with imatinib (IM) at a concentration of 5 μmol / L, were seeded in 96-well plates. A control group was prepared without IM or PA. The seeding density was 3500 cells / well. Sodium palmitate (PA) concentrations of 0, 80, 160, and 320 μmol / L were added to the cell suspension, and the cells were cultured for 0, 24, 48, and 72 hours. Subsequently, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated for another 4 hours. Before measurement, the plates were shaken to avoid cell overlap affecting absorbance at 450 nm. The experiment was repeated at least three times to ensure the reliability of the results.

[0069] 1.2.4 Apoptosis Detection

[0070] K562R cells were cultured in a medium containing 160 μmol / L LPA and 1M 5 μmol / L PA for 48 hours. The control group received no PA. Cells were then collected, centrifuged at 1500 rpm for 5 min, washed three times with PBS, and then added to a 1×10⁶ cell buffer solution at a 1:3 ratio. 100 μL of the buffer solution was added to a flow cytometry tube, followed by 5 μL of FITC reagent. The cells were incubated at room temperature in the dark for 15 min, then 10 μL of LPI reagent was added. After mixing, 300 μL of PBS was added, and the assay was performed within 1 hour. Early apoptotic cells showed positive staining for annexin-V and negative for propidium iodide, while late apoptotic cells showed positive staining for both annexin-V and propidium iodide.

[0071] 1.2.5 Flow cytometry detection of CD36 expression in bone marrow serum

[0072] 1) Take 100 μL from each bone marrow blood sample and place it in a 2 ml EP tube. Prepare three additional EP tubes containing 100 μL of sample each as a negative control, a CD38APC positive control, and a CD34APC positive control, respectively.

[0073] 2) Add no antibody or add the corresponding single antibody to the control tube as indicated. Add 2uLCD38APC and 2uLCD34APC to each of the remaining 100uL of whole blood and incubate in the dark for 30 min.

[0074] 3) After incubation, add 500 μL of red blood cell lysis buffer to each tube, gently mix with a pipette, and let stand in the dark for 5 minutes.

[0075] 4) After lysis, centrifuge the sample at 1000 rcf for 10 min at 18-25℃, discard the supernatant, wash once with PBS, and centrifuge again.

[0076] 5) Discard the supernatant and resuspend in 600ul PBS. Store in the dark until ready for use.

[0077] 6) Before the sample is tested, adjust the voltage of the flow cytometer using a blank control sample tube.

[0078] Fluorescence compensation was then performed using two single-antibody labeled control sample tubes in the PE and APC fluorescence channels. After adjusting the appropriate parameters, the samples were tested, with 10,000 cells counted for each sample.

[0079] 7) Using the forward scattering and side scattering values, delineate the area containing white blood cells and avoid the red blood cell debris area.

[0080] 8) Further, scatter plots were created in the leukocyte region using CD38 APC and CD34 APC values ​​to separate monocytes;

[0081] 9) FlowJo software was used to analyze CD36 expression in monocytes.

[0082] result

[0083] Bone marrow fat cell staining

[0084] like Figure 1 As shown, hematoxylin-eosin (HE) staining of specimens is performed. Hematoxylin is an alkaline staining solution, primarily staining the chromatin in the cell nucleus and nucleic acids in the cytoplasm a purplish-blue color; eosin is an acidic dye, primarily staining the components in the cytoplasm and extracellular matrix red. Neutral fats appear red, and cell nuclei appear blue. The staining results are observed under a microscope.

[0085] like Figure 2 A clinical analysis of flow cytometry results from bone marrow cells of 87 CML patients at various stages and 73 healthy donors revealed that, compared with hematopoietic stem cells from healthy individuals, the expression level of CD36 in CML leukemia stem cells was significantly increased, reaching as high as 90% with an average of around 40%, while the average CD36 expression in healthy donors was around 20%. Furthermore, a higher proportion of CD36+ cells generally indicates a worse prognosis. Figure 2 In patients who respond well to TKIs, the proportion of CD36+ cells decreases rapidly after treatment. However, in CML patients with kinase domain mutations or who relapse after drug withdrawal, the CD36+ cell count remains at a high level.

[0086] like Figure 3 As shown, the CCK-8 experiment was conducted at PA concentrations of 40, 80, 160, and 320 μM. The most significant effect was observed at 160 μM, and the addition of PA significantly reduced apoptosis.

[0087] In flow cytometry apoptosis experiments, the cell mortality rate was 3.28% after adding PA and 10.56% after adding IM.

[0088] This further confirms that high concentrations of PA can promote CML cells' resistance to the killing effect of TKIs.

[0089] The bioinformatics analysis and in vitro detection of CD36-EHHADH metabolic axis expression in CML were performed using the following specific experimental methods:

[0090] 1.1 Material Preparation

[0091] 1.2. Methods

[0092] 1.2.1 Bioinformatics Analysis

[0093] We searched the TCGA (The Cancer Genome Atlas) database for fatty acid metabolism-related cells K562R and K562, and used the "Limma(3.40.2)" software package in R to investigate differential expression. Adjusted p-values ​​were analyzed in TCGA to correct for false positives. "Adjusted p < 0.05 and log2FC > 1 or log2FC < -1" was defined as differential expression criteria to identify differentially expressed genes. We focused on analyzing the expression of key genes ACSS2 and CPT1A in the endogenous fatty acid synthesis pathway and the fatty acid mitochondrial FAO pathway, as well as the expression of the exogenous fatty acid uptake molecule CD36 and the peroxisome metabolic enzyme gene EHHADH. GO and KEGG analyses were performed on CD36-related pathways.

[0094] 1.2.2 Flow cytometry analysis of PPARγ and EHHADH antibody expression levels in patient specimens

[0095] 1) First, prepare the necessary test tubes and label them with the antibodies you want to add.

[0096] 2) First, add 5 μL of CD34-PC5 antibody to each tube.

[0097] 3) Then, add the corresponding antibodies according to the markings on the tube. It must be a combination of FITC and PE labeled antibodies.

[0098] 4) Add 100 μl of bone marrow specimen to each tube, shake to mix, and incubate at room temperature in the dark for 20-30 minutes before testing.

[0099] like Figure 4 Bioinformatics analysis of differentially expressed genes related to fatty acid metabolism in K562R and K562 cells revealed that, compared to K562 cells, K562R cells showed significantly decreased expression of key genes ACSS2 and CPT1A in the endogenous fatty acid synthesis pathway and the fatty acid mitochondrial FAO pathway, while the expression of exogenous fatty acid uptake molecule CD36 and the important peroxisome metabolic enzyme gene EHHADH was significantly increased. This indicates that CML drug-resistant cells do not function through increased fatty acid synthesis and the fatty acid mitochondrial FAO pathway, but are closely related to the exogenous fatty acid uptake-peroxisome pathway. GO and KEGG analyses revealed that CD36 is closely related to multiple key signaling pathways, suggesting that the CD36-EHHADH lipid metabolism axis may be involved in important drug resistance-related signaling pathways. Subsequently, we also preliminarily examined the expression of EHHADH and PPARγ in patient samples, finding that the protein levels of EHHADH and PPARγ were significantly increased in leukemia stem cells from CML drug-resistant patients.

[0100] Among them, the in vitro experiments analyzed the effect of PA on CML resistance through the CD36-EHHADH lipid metabolism axis. The specific experimental methods are as follows:

[0101] 1.1 Material Preparation

[0102] 1.2 Experimental Methods

[0103] 1.2.1 Cell Culture

[0104] K562 and K562R cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin, and placed in an incubator containing 5% CO2 at 37°C.

[0105] 1.2.2 Cell passage

[0106] Wipe the surface of the biosafety cabinet with 75% alcohol. Place the necessary consumables and pipettes into the biosafety cabinet and sterilize them with UV irradiation for 30 minutes. Simultaneously, remove the prepared 10% FBS 1640 culture medium and PBS solution from the 37°C freezer and allow them to reach room temperature. Next, remove the cell culture flasks from the 37°C incubator containing 5% CO2 and observe the cell growth under a microscope. Then, spray the cell culture flasks, PBS solution, and 10% FBS 1640 culture medium with 75% alcohol for sterilization and place them back into the biosafety cabinet for further processing, ensuring aseptic operation throughout the entire process. Transfer the cell suspension from the cell culture flask into a 15ml centrifuge tube, carefully pipetting to mix thoroughly. Centrifuge at 800 rpm for 5 minutes. Discard the supernatant after centrifugation, add 3ml of PBS solution for washing, mix well, centrifuge at 800 rpm for 5 minutes, discard the supernatant, then add the supernatant to bring the volume to 8ml. Gently pipet to mix thoroughly, then transfer to a new cell culture flask. Write the cell name and treatment time on the flask for easy observation later. Finally, place it in a 37℃ 5% CO2 incubator for continued culture.

[0107] 1.2.3 Cell resuscitation

[0108] Preheat the water bath to 37°C. Locate the cells to be cryopreserved from the -80°C freezer and quickly transfer them to the preheated water bath, shaking continuously until the cell culture medium is liquid. The subsequent working environment is a biosafety cabinet that has been wiped with 75% alcohol and irradiated with UV light for 30 minutes. All procedures involving entry and exit from the biosafety cabinet are disinfected with 75% alcohol. Aspirate the cell suspension from the cryopreservation tubes, mix well, and transfer to a 15ml centrifuge tube. Add 2ml of 10% FBS 1640 medium, mix well, centrifuge at 800 rpm for 5 minutes, discard the supernatant, and bring the volume to 8ml with fresh medium. Gently mix and transfer to a cell culture flask, then incubate at 37°C with 5% CO2.

[0109] 1.2.4 Cell cryopreservation

[0110] Cryopreserved cells were collected by centrifugation at 800 rpm for 5 minutes, and 1 ml of serum-free cryopreservation solution was added and gently mixed. The cell type and operation time should be labeled on the cryopreservation tube. The cells were placed in a programmed cooling box and stored at -80°C overnight, and then transferred to a cell cryopreservation box for preservation.

[0111] 1.2.5 Preparation of IM solution and sodium palmitate solution

[0112] Dissolve one tablet of imatinib in 5 ml of DMSO solution to prepare a 5 nmol / L IM solution, and store it protected from light.

[0113] Preparation of PA solution: Mix the two solutions (BSA:PA) at a volume ratio of 2:1 at 60°C and store at 4°C to prepare a PA solution with a concentration of 25 mMMPa.

[0114] 1.2.6 The siRNA and overexpression plasmid were provided by Shanghai Jikai Gene.

[0115] 1.2.6.1 siRNA transfection

[0116] Collect 2 × 10⁵ K562R cells and resuspend each cell in 2 ml of complete RPMI-1640 medium. Add 40 μl of virus and incubate at 37°C for 12 h, not exceeding 16 h. On the second day, collect cells from each well into EP tubes, centrifuge at 2000 rpm for 2 min, discard the supernatant and replace with complete culture medium, gently mix and continue incubation. Observe cell condition on the third and fourth days to determine if medium replacement is necessary. After 72 h of infection, observe infection efficiency and observe green fluorescence using a fluorescence microscope.

[0117] 1.2.6.2 Transfection with overexpression plasmids

[0118] Collect 2×10⁵ K562 and K562R cells respectively, and resuspend them in 2 ml of complete RPMI-1640 medium. Add 40 μl of virus and incubate at 37°C for 12 h, not exceeding 16 h. On the second day, collect the cells from each well into EP tubes, centrifuge at 2000 rpm for 2 min, discard the supernatant and replace with complete culture medium, gently mix and continue incubation. Observe the cell condition on the third and fourth days to see if medium replacement is necessary. After 72 h of infection, observe the infection efficiency and observe green fluorescence using a fluorescence microscope.

[0119] 1.2.7 RT-PCR

[0120] 1.2.7.1 Total RNA Extraction

[0121] Follow the kit instructions. If the extracted RNA is not used, store it at -70°C.

[0122] 1.2.7.2cDNA generation

[0123] RNA concentration was measured using a Thermo Fisher Scientific instrument; the total concentration was 1000 ng / μl, and the amount of RNA added was calculated. A 20 μl solution was used for subsequent operations. Amplification conditions: 37℃ for 15 min; 85℃ for 5 s.

[0124] 1.2.7.3 Cq value determination

[0125] PCR reagent components: 10 μl of 2×SYBR, 7.2 μl of distilled water, 0.4 μl of primer R, 0.4 μl of primer F, and 2 μl of cDNA;

[0126] Configure the qPCR reaction system according to the table above, then add 2 μL of cDNA, for a total reaction volume of 20 μL.

[0127] Amplification conditions: 95℃ pre-denaturation for 30 seconds, 95℃ denaturation for 5 seconds, 60℃ annealing for 30 seconds, for 41 cycles.

[0128] Data processing and plotting;

[0129] Use GraphpadPrism8 to plot bar charts of human CD36 and EHHADH gene mRNA expression levels.

[0130] 1.2.8 Western Blot

[0131] 1.2.8.1 Extraction of total protein

[0132] Cells were collected by centrifugation at 1000 rpm for 5 min, washed with ice-cold PBS, and the supernatant was discarded. Depending on the imatinib concentration, 200 μL, 160 μL, 120 μL, and 80 μL of protein lysis buffer were added, along with pmsf (protease inhibitor) at a 1:100 ratio. The mixture was then placed on ice and shaken every 10 min to ensure complete lysis. After 30 minutes of lysis, EP tubes were placed in a pre-chilled 4°C centrifuge and centrifuged at 12000 rpm for 10 min. The supernatant was collected and labeled with the date, the amount of lysis buffer added, and the cell name. This supernatant contains the extracted total protein. Unused supernatant was stored at -20°C.

[0133] 1.2.8.2 Bradford method for protein concentration determination

[0134] First, configure the protein standard curve.

[0135] Prepare standard protein at concentrations of 0, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, and 5 mg / ml according to the table below. Shake well after each dilution to ensure thorough mixing.

[0136] The item number, diluent volume, standard volume, and final concentration are as follows:

[0137] A: 20 μL lddH2O + 0 μL BSA

[0138] B: 19 μl dH2O + 1 μl BSA

[0139] C: 18 μl dH2O + 2 μl BSA

[0140] D: 17 μl dH2O + 3 μl BSA

[0141] E: 16 μl dH2O + 4 μl BSA

[0142] F: 15 μl dH2O + 5 μl BSA

[0143] Add 20 μL of sample to each well of a 96-well plate, from tubes A to F. Repeat this process once more for the standard curve. Add 1 μL of the protein sample to 19 μL of water to each well of the 96-well plate, repeating this process three times. Finally, add 180 μL of LG250 staining solution to all wells, and then measure the absorbance at wavelength A595 using a microplate reader. The protein concentration in the sample is then predicted based on the standard curve and the amount of sample added.

[0144] 1.2.8.3 Gel electrophoresis

[0145] Sample preparation: Use a 12.5% ​​concentration gel from Yaxin, and prepare the gel according to the instructions. Place the electrophoresis rack into the electrophoresis tank, fill the inner tank with electrophoresis buffer, and add an appropriate amount of electrophoresis buffer to the outer tank. Slowly remove the comb and add the sample. For protein samples, calculate the loading volume based on 20 μg of protein. Boil at 100℃ for 10 minutes, then cool to 4℃ before use. Store any unused sample at -20℃. Load 4 μL of sample onto the left side of the Maker and 2 μL onto the right side to distinguish the samples.

[0146] Electrophoresis: Run at a constant voltage of 80V for 30 minutes to allow the stacking gel and separating gel to separate, then switch to 120V for 70 minutes. Continue until the bromophenol blue reaches the bottom, then remove the upper stacking gel for transfer.

[0147] Transfer molding: Activate the PVDF membrane by soaking it in methanol for 30 seconds. Pre-cool the transfer solution and soak filter paper and black foam in it. Following the principle of black plate to gel and transparent plate to PVDF membrane, use a sandwich method: foam, filter paper, gel, PVDF membrane, filter paper, foam. A large amount of heat will be generated during the transfer process; add ice to the outside of the tank, and place ice blocks and pre-cooled transfer solution inside the tank.

[0148] Transformation conditions: 110V, 70min.

[0149] Sealing: After the membrane has cooled down, remove the PVDF membrane and seal it at room temperature for 20 minutes.

[0150] Washing: Wash three times with 1×TBST, 5 minutes each time.

[0151] Primary antibody incubation: Place the band containing the target protein molecular weight into the prepared antibody and incubate overnight at 4°C on a shaker.

[0152] Secondary antibody incubation: Wash three times with 1×TBST, 10 min each time. Immerse the PVDF membrane in the prepared secondary antibody and incubate at room temperature for one hour. Wash the membrane three times with 1×TBST, 10 min each time. The internal control does not require incubation with the secondary antibody. Recover the primary and secondary antibodies and store them in a -20°C freezer. Discard the secondary antibody after three uses. Immerse the bands in TBST.

[0153] Exposure: Absorb the moisture on the membrane with absorbent paper, place it on an exposure plate, and evenly drop a 1:1 mixture of ultrasensitive exposure solution onto it. Expose the membrane using a gel imaging analysis system to obtain bands.

[0154] 1.2.9 Detection of ATP production content

[0155] First, collect cells or bacteria into centrifuge tubes, discard the supernatant, and follow the ratio of bacteria or cells (10⁴) to extraction liquid volume (mL) of 1000:1 (it is recommended to add 1 mL of extraction liquid for 5 million bacteria or cells). Sonicate the cells (ice bath, 200W power, 2s sonication, 1s pause, total time 1 min), and centrifuge at 10000g 4℃ for 10 min. Transfer the supernatant to another EP tube, add 500 μL of chloroform, shake thoroughly to mix, and centrifuge at 10000g 4℃ for 3 min. Collect the supernatant and place it on ice for analysis. Preheat the UV spectrophotometer for at least 30 min, adjust the wavelength to 340 nm, and zero the instrument with distilled water. For standard solution dilution: Take 100 μL of 10 μmol / mL ATP standard solution, add 1.5 mL of distilled water, mix thoroughly, and prepare a 0.625 μmol / mL standard solution for use immediately.

[0156] ATP content (μmol / 10⁶ cell) = ΔA determination ÷ (ΔA standard ÷ C standard) × V extraction ÷ 5 = 0.125 × ΔA determination ÷ ΔA standard

[0157] C standard: standard solution concentration, 0.625 μmol / mL; V extraction: volume of added extraction solution, 1 mL; V serum (plasma): serum (plasma) volume, 0.1 mL.

[0158] 1.2.10 Statistical Analysis

[0159] All data are presented as mean ± standard deviation (SD) of three independent experiments. GraphPad was used.

[0160] Prism 9 software is used to create line graphs or corresponding bar charts. Student's t-test is used for pairwise comparisons.

[0161] P < 0.05 is considered statistically significant.

[0162] Western blotting confirmed that in ( Figure 5 In K562 cells, CD36 levels decreased with increasing drug administration time and concentration, while in K562R cells, CD36 levels remained stable without decreasing with increasing drug administration time and concentration. EHHADH levels decreased with time in K562 cells but increased with time in K562R cells. Figure 5 The concentration at which IM is most sensitive was determined to be 5 μmol / L.

[0163] In vitro cell experiments confirmed that TKIs-induced CML resistant cells K562R highly expressed CD36, and the CD36 expression level was not affected by Bcr-Abl tyrosine kinase activity. Figure 6 K562R, compared to K562, exhibits a significantly enhanced ability to uptake PA. Figure 6 Knocking down CD36 expression not only reduces the uptake of PA by K562R cells (…); Figure 6 It can also restore sensitivity to TKIs. Figure 6 Therefore, the above results suggest that the fatty acid transporter CD36 may be a key pathway for CML drug-resistant cells to over-take PA and enter the cells.

[0164] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. An application of CD36-mediated sodium palmitate promoting CML drug resistance through metabolic reprogramming, characterized in that, The CD36-EHHADH metabolic axis was used to screen for drugs to treat CML. The specific method is as follows: Candidate drugs were co-cultured with CML cell lines to examine their effects on the expression of key molecules in the CD36-EHHADH metabolic axis and cell proliferation, thereby screening for drugs with therapeutic potential.

2. The application of CD36-mediated sodium palmitate promoting CML resistance through metabolic reprogramming according to claim 1, characterized in that, The CML cell line includes the K562R cell line, and the candidate drugs include small molecule inhibitors and traditional Chinese medicine extracts.

3. The application of CD36-mediated sodium palmitate promoting CML resistance through metabolic reprogramming according to claim 1, characterized in that, In the method described, when detecting the effect of the drug on cell proliferation, cell proliferation detection methods are used, including the CCK-8 assay and the MTT assay.

4. The application of CD36-mediated sodium palmitate promoting CML resistance through metabolic reprogramming according to claim 1, characterized in that, In the method described, when culturing the CML cell line, RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin is used.

5. An application of CD36-mediated sodium palmitate promoting CML drug resistance through metabolic reprogramming, characterized in that, A CML diagnostic kit based on the CD36-EHHADH metabolic axis is used to assist in the diagnosis of CML. The kit contains antibodies or nucleic acid detection probes that can specifically recognize CD36 and EHHADH to detect the expression levels of corresponding proteins or genes in CML patient samples.

6. The application of CD36-mediated sodium palmitate promoting CML resistance through metabolic reprogramming according to claim 5, characterized in that, The antibodies include anti-human antigen CD36 antibody and anti-human antigen EHHADH antibody, and the nucleic acid detection probe can specifically bind to specific sequences of the CD36 and EHHADH genes.

7. An application of CD36-mediated sodium palmitate promoting CML drug resistance through metabolic reprogramming, characterized in that, The applications include: revealing the molecular mechanisms of drug resistance in CML and overcoming drug resistance in CML cells; The specific method for revealing the molecular mechanism of CML resistance is as follows: differential gene expression analysis is performed on CML drug-resistant cell lines and drug-sensitive cell lines to detect the expression differences of CD36, EHHADH, PPARγ and fatty acid metabolism-related genes.

8. The application of CD36-mediated sodium palmitate promoting CML resistance through metabolic reprogramming according to claim 7, characterized in that, The specific method to overcome drug resistance in CML cells is to downregulate the expression or activity of CD36 and EHHADH through siRNA, small molecule inhibitors, or gene editing techniques, thereby making CML cells sensitive to TKIs again.

9. The application of CD36-mediated sodium palmitate promoting CML resistance through metabolic reprogramming according to claim 7, characterized in that, The study elucidates the molecular mechanism of CML drug resistance based on bioinformatics analysis and in vitro detection of CD36-EHHADH metabolic axis expression in CML, and in vitro experimental analysis of the effect of PA on CML drug resistance through CD36-EHHADH lipid metabolism axis. Specifically, the bioinformatics analysis and in vitro detection of CD36-EHHADH metabolic axis expression in CML include flow cytometry analysis of PPARγ and EHHADH antibody expression levels in patient samples, comprising the following steps: S1: First, prepare the necessary test tubes and label them with the antibodies you want to add. S2: First, add 5 μL of CD34-PC5 antibody to each tube; S3: Then add the corresponding antibodies according to the markings on the tube. It must be a combination of FITC and PE labeled antibodies. S4: Add 100 μl of bone marrow specimen to each tube, shake to mix, and incubate at room temperature in the dark for 20-30 minutes before testing.

10. The application of CD36-mediated sodium palmitate promoting CML resistance through metabolic reprogramming according to claim 7, characterized in that, The in vitro experimental analysis of the effect of PA on CML resistance via the CD36-EHHADH lipid metabolism axis included the following steps: S1: Cell culture, cell passage, cell resuscitation, cell cryopreservation; S2: Preparation of IM solution and sodium palmitate solution; S3: siRNA transfection, overexpression plasmid transfection; S4: RT-PCR, total RNA extraction, cDNA generation, Cq value determination; S5: Western Blot; extraction of total protein, determination of protein concentration using the Bradford method, gel electrophoresis; detection of ATP production content; S6: Statistical analysis.

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