Use of ABCG1 phosphorylation sites as tumor biomarkers

Abstract

The present invention relates to the use of reagents and / or devices for detecting human ABCG1 phosphorylation sites and kits for use in preparing reagents for tumor risk assessment, tumor diagnosis, or tumor prognosis evaluation, and to the use of reagents and kits for inhibiting human ABCG1 phosphorylation sites in preparing drugs for tumor prevention and / or treatment. The human ABCG1 phosphorylation sites are selected from one or more of Y97, T99, S120, S125, T272, S277, S441, and Y655. Phosphoantigen peptides and antibodies of the human ABCG1 protein, as well as uses and kits thereof, are also disclosed. The use of any one or more of the ABCG1 phosphorylation sites has important clinical significance in tumor risk assessment, diagnosis, treatment, and prognosis evaluation.

Description

[Technical Field]

[0001] The present invention belongs to the technical field of molecular diagnostic medicine, and specifically relates to the use of ABCG1 phosphorylation sites as biomarkers in the diagnosis and prognosis evaluation of tumors. [Background technology]

[0002] The ABC (ATP-binding cassette) transporter superfamily is a type of membrane protein, and ABCG1 belongs to one of the ABCG subfamily members. Initial studies suggest that ABCG1 is primarily responsible for the transport of cholesterol and phospholipids in macrophages and may also regulate lipid balance in some cells.

[0003] In a recent study of tumor-associated ABCG1, Roundhill et al. (Non-Patent Document 1) found that ABCG1 was the only gene upregulated in the self-renewing, drug-resistant cell line HOS-EC50.SR. Targeting ABCG1 may eliminate drug-resistant, self-renewing cells (OST-CSCs) and improve survival outcomes in osteosarcoma patients. ABCG1 is crucial for the survival of malignant glioma cells, and targeting ABCG1 may be beneficial for the treatment of malignant gliomas (Non-Patent Document 2). ABCG1 deletion may inhibit tumor growth by regulating intratumoral macrophages. ABCG1 may also be involved in the induction of cisplatin resistance and tumor metastasis after chemotherapy in lung adenocarcinoma cells. Molecules such as ABCG1 are more highly expressed in normal CD34+ / CD38- bone marrow cells than in acute myeloid leukemia CD34+ / CD38- cells, resulting in differences in cholesterol metabolism. Liver X receptor (LXR) inhibitors can inhibit differentiation and induce apoptosis in breast cancer MCF-7 cells, and LXR activation significantly promotes ABCG1 expression and cellular cholesterol efflux. Saracatinib upregulates ABCG1 and reduces the antitumor effect of oxaliplatin. Li Hongtao et al. (Non-Patent Document 3) discovered that miR-519 may regulate breast cancer sensitivity to gemcitabine by targeting ABCG1.

[0004] Regarding tumor metastasis, it has been confirmed that ABCG1, together with molecules such as Slug, may mediate metastasis and cisplatin resistance in lung adenocarcinoma (Non-Patent Document 4). Related research on ovarian cancer has shown that normal ovarian epithelial cells treated with progesterone can upregulate molecules such as ABCG1, thereby regulating the balance of cholesterol and lipids within the cells, which may be of interest in the prevention and treatment of ovarian cancer (Non-Patent Document 5). ABCG1 is highly expressed in high-grade serous ovarian cancer and may be closely related to survival (Non-Patent Document 6). Scutellaria baicalensis extract (SB) can inhibit molecules including ABCG1 and promote the killing effect of platinum-based drugs on ovarian cancer cells (Non-Patent Document 7). Recent research has found that the combination of progesterone-calcitriol and platinum-based drugs can inhibit drug efflux by attenuating the expression of ABCG1 and other molecules, thereby promoting platinum-based killing of ovarian cancer cells (Non-Patent Document 8). ABCG1 is found to be closely associated with tumor initiation, metastasis and drug resistance.

[0005] Among ABC transporters, members of the ABCA, ABCB, and ABCC subfamilies are reported to be phosphorylated to regulate their function, while among the ABCD subfamily members, only ABCD1 and ABCD3 are tyrosine phosphorylated. Among the ABCG subfamily members, the only molecule currently known to contain the correct phosphorylation site is ABCG2 (T362). According to a series of studies by Nagelin et al., ABCG1 in macrophages is degraded by 12 / 15-lipoxygenase via JNK2- and p38-mediated N-terminal serine phosphorylation, resulting in cholesterol accumulation in macrophages and atherosclerosis (Non-Patent Document 9). The authors constructed S27A, S28A, S43A, S45A, S80A, and S85A mutants using the ABCG1 isoform (ORF638), and all of these mutants could be degraded by 12 / 15LO. They also constructed S65A, S70A, S119A, S141A, and S168A mutants and found that these mutants were resistant to 12 / 15LO-induced ABCG1 phosphorylation and degradation. This suggests that these serine sites in the N-terminus of ABCG1 may be phosphorylated to mediate ABCG1 degradation (Non-Patent Document 10), but the specific functional phosphorylation sites have not been identified. Ogura et al. discovered that ABCG1 degradation may be associated with the ubiquitin proteasome (Non-Patent Document 11), but no in-depth, clear reports have been found. Gelissen et al. used a protein kinase A (PKA) inhibitor to investigate the related functions of cholesterol regulation and ABCG1 protein stability for predicted potential phosphorylation sites in the two ABCG1 isoforms, ABCG1(+12) and ABCG1(-12). They found that S389 in ABCG1(+12) is crucial for regulating cholesterol efflux and protein stability, and that it can be regulated and phosphorylated by PKA inhibitors.The S377 site corresponding to ABCG1(-12) was not regulated by PKA inhibitors, suggesting that S377 may not be phosphorylated in ABCG1(-12) (Non-Patent Document 12). The specific phosphorylation site of ABCG1 was not conclusive in this study. Watanabe recently discovered that after ABCG1 is phosphorylated by protein kinase C (PKC), it can increase cholesterol efflux by promoting ABCG1 stability and inhibiting its degradation (Non-Patent Document 13). However, the specific ABCG1 site phosphorylated by PKC is unknown.

[0006] In summary, it is of great importance to explore and identify the specific phosphorylation sites of ABCG1 and use them in tumor risk assessment, diagnosis, treatment, and prognosis evaluation. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] EARoundhill et al., Cancer Lett, 453 (2019) 142-157 [Non-patent document 2] YHChen et al., Oncotarget, 7(2016)23416-23424 [Non-patent document 3] Li Hongtao et al., Chinese Journal of Experimental Surgery, 37(2020)721-723 [Non-patent document 4] J. Zhan et al., Theranostics, 9 (2019) 2084-2099 [Non-Patent Document 5] A. Paucarmayta et al., Biomedicines, 8(2020) [Non-patent document 6] CB Wilcox et al., BMC Cancer, 7 (2007) 223 [Non-Patent Document 7] K. Elsnerova et al., Oncology reports, 35 (2016) 2159-2170 [Non-patent document 8] I. Hussain et al., Journal of cellular biochemistry, 119 (2018) 7515-7524 [Non-Patent Document 9] MH Nagelin et al., Arteriosclerosis, thrombosis, and vascular biology, 28 (2008) 1811-1819. [Non-Patent Document 10] MHNagelin et al., The Journal of biological chemistry, 284(2009)31303-31314 [Non-Patent Document 11] M. Ogura et al., Arteriosclerosis, thrombosis, and vascular biology, 31 (2011) 1980-1987 [Non-Patent Document 12] ICGelissen et al., Journal of lipid research, 53 (2012) 2133-2140 [Non-Patent Document 13] T. Watanabe et al., Journal of biochemistry, 166 (2019) 309-315 Summary of the Invention

[0008] To solve the above problems, the present invention discovers and validates the use of ABCG1 phosphorylation sites and antibodies thereof in tumor diagnosis, treatment and prognosis. Specifically, the present invention is as follows:

[0009] A first aspect of the present invention provides use of a reagent and / or device for detecting human ABCG1 phosphorylation sites in the preparation of a reagent for assessing the risk of tumor development, diagnosing tumors or assessing tumor prognosis.

[0010] In one embodiment, the ABCG1 phosphorylation site is selected from Y97, T99, S120, S125, T272, S277, S441, and Y655.

[0011] In some embodiments, the tumor is selected from osteosarcoma, glioblastoma, lung cancer, leukemia, breast cancer, ovarian cancer, cervical cancer, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, bladder cancer, colon cancer, and prostate cancer.

[0012] A second aspect of the present invention provides the use of an agent for inhibiting human ABCG1 phosphorylation sites in the preparation of a medicament for the prevention and / or treatment of tumors.

[0013] In one embodiment, the ABCG1 phosphorylation site is selected from Y97, T99, S120, S125, T272, S277, S441, and Y655.

[0014] In some embodiments, the tumor is selected from osteosarcoma, glioblastoma, lung cancer, leukemia, breast cancer, ovarian cancer, cervical cancer, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, bladder cancer, colon cancer, and prostate cancer.

[0015] A third aspect of the present invention provides a phosphoantigen peptide of human ABCG1 protein, wherein the antigen peptide is selected from the amino acid sequences shown in any one of SEQ ID NOs: 1 to 8.

[0016] A fourth aspect of the present invention provides an antibody specific to a phosphorylation site of human ABCG1 protein, the antibody being prepared by immunizing an animal with a phosphoantigen peptide of ABCG1 protein described in the third aspect of the present invention.

[0017] A fifth aspect of the present invention provides use of a phosphoantigen peptide of human ABCG1 protein according to the third aspect of the present invention in the preparation of a formulation for detecting phosphorylation sites of ABCG1 protein.

[0018] A sixth aspect of the present invention provides use of the antibody specific to the phosphorylation site of human ABCG1 protein according to the fourth aspect of the present invention in the preparation of a formulation for detecting the phosphorylation site of ABCG1 protein.

[0019] In one embodiment, the phosphorylation site of the ABCG1 protein is selected from Y97, T99, S120, S125, T272, S277, S441, and Y655.

[0020] A seventh aspect of the present invention provides a kit for assessing the risk of tumor development, or for diagnosing, preventing, treating, or evaluating the prognosis of a tumor, the kit comprising a reagent for detecting the human ABCG1 phosphorylation site described in the first aspect of the present invention, an agent for inhibiting / promoting the human ABCG1 phosphorylation site described in the second aspect of the present invention, or an antibody specific to the phosphorylation site of the human ABCG1 protein described in the fourth aspect of the present invention. [Effects of the Invention]

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] The present invention has confirmed that at least eight amino acid sites in the ABCG1 protein, Y97, T99, S120, S125, T272, S277, S441, and Y655, are phosphorylated and expressed. The use of any one or more of these sites has important clinical significance in the risk assessment, diagnosis, treatment, and prognosis evaluation of tumors, such as ovarian cancer, osteosarcoma, glioblastoma, lung cancer, leukemia, breast cancer, cervical cancer, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, bladder cancer, colon cancer, and prostate cancer.

[0023] The present invention further provides a phosphoantigen peptide of human ABCG1 protein, which can be used to prepare an antibody specific to the phosphorylation site of human ABCG1 protein, and the obtained antibody can be used for detecting and functionally studying the phosphorylation site of ABCG1 protein. [Brief explanation of the drawings]

[0024] Other features, objects and advantages of the present invention will become more apparent from a reading of the following detailed description of non-limiting embodiments, which is given with reference to the drawings, in which:

[0025] [Figure 1] ABCG1 phosphorylation sites detected by mass spectrometry. Of these, only one or both of S120 and S125 may be phosphorylated, and only one or both of T272 and S277 may be phosphorylated. [Figure 2] This is a secondary mass spectrum for detecting ABCG1 phosphorylation sites. [Figure 3] Antibody detection of ABCG1 phosphorylation. "*" indicates that the appearance of non-target bands in the GST-ABCG1 sample detected with the phospho-Ser antibody may be due to the presence of small amounts of other serine-phosphorylated proteins in the purified product. The GST antibody was from CST #2622S, and the pSer / pThr / pTyr antibodies were from Abcam, #ab9332 / ab9337 / ab10321. [Figure 4] Detection of ABCG1 phosphorylation in ovarian cancer cell lines SKOV3 and HEYA8. The primary antibody used was an antibody against the ABCG1-specific phosphorylation site. The control antigen was a phosphorylated polypeptide fragment conjugated to BSA, and GAPDH was used as a loading control for the cell samples. The antibody was from Cell Signaling, catalog number #5174. [Figure 5] This immunohistochemistry (IHC) assay was used to detect ABCG1 phosphorylation in ovarian cancer tissues. The primary antibody used was an antibody against specific ABCG1 phosphorylation sites, the secondary antibody was goat anti-rabbit IgG conjugated to HRP, and the coloring agent was 3,3'-diaminobenzidine (DAB). [Figure 6] IHC (immunohistochemistry) detection of ABCG1 phosphorylation in lung, ovarian, and breast cancer tissues and surrounding tissues, and the correlation between phosphorylation and overall survival of the corresponding tumor patients. [Figure 7] IHC (immunohistochemistry) detection of ABCG1 phosphorylation in lung, ovarian, and breast cancer tissues and surrounding tissues, and the correlation between phosphorylation and overall survival of the corresponding tumor patients. [Figure 8]IHC (immunohistochemistry) detection of ABCG1 phosphorylation in lung, ovarian, and breast cancer tissues and surrounding tissues, and the correlation between phosphorylation and overall survival of the corresponding tumor patients. [Figure 9] IHC (immunohistochemistry) detection of ABCG1 phosphorylation in lung, ovarian, and breast cancer tissues and surrounding tissues, and the correlation between phosphorylation and overall survival of the corresponding tumor patients. [Figure 10] IHC (immunohistochemistry) detection of ABCG1 phosphorylation in lung, ovarian, and breast cancer tissues and surrounding tissues, and the correlation between phosphorylation and overall survival of the corresponding tumor patients. [Figure 11] IHC (immunohistochemistry) detection of ABCG1 phosphorylation in lung, ovarian, and breast cancer tissues and surrounding tissues, and the correlation between phosphorylation and overall survival of the corresponding tumor patients. [Figure 12] IHC (immunohistochemistry) detection of ABCG1 phosphorylation in lung, ovarian, and breast cancer tissues and surrounding tissues, and the correlation between phosphorylation and overall survival of the corresponding tumor patients. [Figure 13] IHC (immunohistochemistry) detection of ABCG1 phosphorylation in lung, ovarian, and breast cancer tissues and surrounding tissues, and the correlation between phosphorylation and overall survival of the corresponding tumor patients. [Figure 14] IHC (immunohistochemistry) detection of ABCG1 phosphorylation in lung, ovarian, and breast cancer tissues and surrounding tissues, and the correlation between phosphorylation and overall survival of the corresponding tumor patients. [Figure 15] IHC (immunohistochemistry) detection of ABCG1 phosphorylation in lung, ovarian, and breast cancer tissues and surrounding tissues, and the correlation between phosphorylation and overall survival of the corresponding tumor patients. [Figure 16] IHC (immunohistochemistry) detection of ABCG1 phosphorylation in lung, ovarian, and breast cancer tissues and surrounding tissues, and the correlation between phosphorylation and overall survival of the corresponding tumor patients. [Figure 17]IHC (immunohistochemistry) detection of ABCG1 phosphorylation in lung, ovarian, and breast cancer tissues and surrounding tissues, and the correlation between phosphorylation and overall survival of the corresponding tumor patients. DETAILED DESCRIPTION OF THE INVENTION

[0026] In order to clarify the objectives, technical solutions and advantages of the embodiments of the present invention, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the drawings of the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, but not all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by those skilled in the art without any creative work fall within the protection scope of the present invention.

[0027] [Example 1] Experimental materials and methods 1.1 Eukaryotic GST-pulldown experiments Cells in a 10 cm dish were harvested, lysed, and protein concentration was measured. 500 μg of protein was taken and the cell lysate was added to the lysate to a final volume of 1 mL. 20 μL of Glutathione Sepharose 4B was added to the lysate. The mixture was incubated for 2 h on a shaker at 4°C, then centrifuged at 500 g for 5 min in a 4°C centrifuge. The supernatant was aspirated, and 1 mL of cell lysate was added. The mixture was then inverted and washed, and centrifuged at 500 g for 5 min in a 4°C centrifuge. This washing and centrifugation process was repeated twice. 20–40 μL of 2x SDS loading buffer was added to the precipitate, and the sample was boiled in a metal bath at 100°C for 2–4 min. The supernatant was aspirated for SDS-PAGE analysis, and the ABCG1 molecular weight fragment was excised.

[0028] 1.2 Mass spectrometry detection of ABCG1 protein phosphorylation The gel excised from the GST-pulldown product was sent directly to Zhongke New Life Sciences for protein modification mass spectrometry analysis. The basic steps were as follows:

[0029] The protein analysis mass spectrometer was a Thermo Q Exactive Plus Orbitrap LC-MS / MS. Peptide fragments were quality-controlled filtered with a 1% FDR and 1 unique peptide.

[0030] Tandem mass spectra were analyzed using Mascot. A database search was performed using trypsin enzyme digestion. Search parameters were a fragment ion mass tolerance of 0.05 Da, a parent ion mass tolerance of 7 ppm, and a maximum number of miscuts of 4.

[0031] Fixed modification: Carbamidomethylation (urine methylation) 57.02; Variable modifications: Oxidation (M, oxidation) 15.99, Phosphorylation (STY, phosphorylation) 79.97, Acetylation (Protein N-term, acetylation): 42.01. The present invention focused on phosphorylation modifications and finally found the corresponding secondary mass spectrum from the phosphorylated peptide fragments.

[0032] 1.3 Preparation of ABCG1 phosphoantibody A. Synthesis of peptide fragments: These include phosphorylated and non-phosphorylated peptide fragments, as shown in Table 1.

[0033] [Synthesis of phosphorylated and non-phosphorylated peptide fragments] [Table 1]

[0034] *The Cys amino acid added to the N- or C-terminus of the peptide fragment allows the peptide fragment to be conjugated to other macromolecules such as KLH (Keyhole Limpet Hemocyanin) and BSA (Bovine Serum Albumin) for subsequent use.

[0035] B. Animal Immunization A New Zealand white rabbit (female, >2.5 kg, Shandong Elike Biotechnology Co., Ltd., Animal Experiment Permit Number: SYXK(Lu)20190018) was tied to a platform. The rabbit's buttocks were held down with one hand and the head with the other to prevent movement and expose the rear of the neck as much as possible. The immunization point on the rear of the neck and the surrounding area were disinfected with 75% alcohol. The skin was lifted with one hand, and the immunization needle was held with the other hand. The needle was inserted approximately 0.5 cm toward the thumb of the hand used to lift the skin. A total of five injection points were used. The first immunization was performed using phosphopolypeptide (1 mg / ml) mixed with Freund's complete adjuvant (F5881, SIGMA). The second immunization was performed three times at different sites. The phosphopolypeptide was mixed with Freund's incomplete adjuvant (F5506, SIGMA) at a concentration of 1 mg / ml. Detailed vaccination times and doses are shown in Table 2.

[0036] [Time, dose and schedule of animal immunization] [Table 2]

[0037] C. Detection of antibody titers by ELISA

[0038] c-1 Coating: The required antigen volume and coating solution volume were calculated, and the protein coating concentration was 1-2 μg / ml, and the polypeptide coating concentration was 1-5 μg / ml.

[0039] c-2 Blocking: Take out the coated ELISA plate from the 37℃ incubator or 4℃ refrigerator, shake the coating solution into a water bath, then add 300ul of 5% milk to each well sequentially, cover and place in a 37℃ incubator for 1 hour, or incubate in a 4℃ refrigerator overnight if blocking is performed after 4pm.

[0040] c-3 Washing: The coated and blocked ELISA plates were removed from the 37°C incubator or 4°C refrigerator, the blocking solution was poured into a water bath, and the plates were washed three times with PBST. The ELISA plates were then bagged, organized by item number and sequence number. They were stored at -20°C for up to two months before use and discarded after two months. If the primary antibody needed to be added immediately, it could be added directly and incubated, eliminating the need to store at -20°C in the refrigerator. Primary antibody incubation: Depending on the experimental requirements, 100 μl of primary antibody was added to each well, the wells were covered, and the plates were placed in a 37°C incubator for 60 minutes, after which they were removed. The primary antibody was discarded, washed three times with PBST, and patted dry with an absorbent paper towel.

[0041] c-4 Secondary antibody incubation: Depending on the experimental requirements, add 100 μl of the corresponding secondary antibody to each well, cover, and place in a 37°C incubator to incubate for 30 minutes, then remove. Discard the secondary antibody, wash three times with PBST, and pat dry with an absorbent paper towel.

[0042] c-5 TMB color development: The prepared TMB color development solution was poured into a sample addition tank with clean PE gloves, and 100 μl of the solution was added sequentially to each well of the ELISA plate using a multichannel pipette. The plate was then covered and placed in an incubator for 5 to 10 minutes, and the color development was observed.

[0043] c-6 End: The microplate reader was turned on and preheated for 1 minute, the ELISA plate was removed from the incubator, and 50 μl of 2 M HCl was added to each well of the ELISA plate sequentially.

[0044] c-7 Reading: Turn on the Thermo microplate reader software, select a wavelength of 450-620 nm, wipe the bottom of the ELISA plate with an absorbent towel, place it in the slot of the microplate reader, click start, and read. The relative antibody titer was calculated based on the read value.

[0045] D. Antibody purification

[0046] d-1: A purification column was prepared and fixed to an iron foam plate. The CNBr-Sepharose-activated resin was removed from the 4°C refrigerator and equilibrated at room temperature for half an hour. 0.3 g (to prepare a 1 ml column) was weighed into a 4 ml centrifuge tube and thoroughly dissolved in pre-cooled 1 mM (pH 3.0) hydrochloric acid. The column was then transferred to a purification column, washed with 50 ml of 1 mM hydrochloric acid, and then soaked in 5 ml of 1 mM (pH 3.0) hydrochloric acid for 10 minutes.

[0047] d-2: 5 mg of polypeptide was dissolved in 4 ml of binding buffer, mixed uniformly, transferred to a 4 ml centrifuge tube, and incubated at room temperature for 2 to 3 hours with repeated inversion.

[0048] d-3: The incubated packing material was transferred to a purification column, the flow-through was collected, washed with 10 ml of blocking buffer, and further soaked in blocking buffer at room temperature for 2 hours.

[0049] d-4: Washing was performed three times, alternating with Wash Buffer 3 and Wash Buffer 4, using 5 ml each time.

[0050] d-5: When not in use temporarily, the membrane was washed with 10 ml of PBS and then with 5 ml of 20% ethanol, and then immersed and stored at 4°C.

[0051] d-6: When choosing to use, the freshly prepared purification column was washed with 20 ml of PBS and the pretreated sample to be purified was circulated.

[0052] d-7: The column was passed through for 45 minutes, the flow-through was collected, and the column material was washed with 20 ml of PBS, 10 ml of Wash Buffer 2, and another 10 ml of PBS.

[0053] d-8: Elution was performed with Elution Buffer, and 5 ml of the eluate was dropped into a 15 ml centrifuge tube and neutralized with Neutralization Buffer. After elution of 20 ml of eluate, Coomassie Brilliant Blue was used to detect whether protein had leaked out of the eluate. If protein had leaked out, an additional 10 ml was eluted and detected again. The packing material was washed with 20 ml of Elution Buffer, 10 ml of PBS, and another 10 ml of Storage Buffer. The column was then immersed in three column volumes of Storage Buffer and stored at 4°C.

[0054] d-9: Antibody specificity was detected by the above-mentioned ELISA method, and antibody concentration was detected by the BCA method.

[0055] 1.4 Western Blot Experiment

[0056] Protein extraction: After observing the cell status, cells in the logarithmic growth phase were lysed. The original medium was aspirated and washed twice with pre-chilled PBS. The PBS was then aspirated. The cells to be collected were placed on ice and an appropriate volume of RIPA lysis solution (pre-added with protease inhibitors, phosphatase inhibitors, and PMSF) was added depending on the cell mass to lyse the cells. The cells were scraped from the cell culture dish with a cell scraper and transferred to a 1.5 mL EP tube with a pipette. The lysate was aspirated and dispensed 6–8 times with a 1 mL syringe until it was no longer viscous. The tube was then left on ice for 30 minutes to fully lyse the cells. The tube was then placed in a 4°C centrifuge at 12,000 rpm for 10 minutes. The supernatant (i.e., the dissolved protein) was aspirated and transferred to a new 1.5 mL EP tube.

[0057] Measurement of protein concentration by BCA method: Preparation of a calibration curve: Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of protein standard (concentration 0.5 μg / μL, BSA) to a new 96-well plate, then add 20, 19, 18, 16, 12, 8, 4, and 0 μL of cell lysate to each well to make up to 20 μL, so that the measured concentrations of the protein standard in each well are 0, 25, 50, 100, 200, 300, 400, and 500 μg / mL, respectively. Three duplicate wells are set for each concentration. Take 1 μL of the protein sample to be measured, add 19 μL of the protein lysate to a 96-well plate, and set up three duplicate wells for each sample. Preparation of BCA working solution: Follow the instructions. Therefore, a BCA working solution was prepared with a ratio of solution A:solution B = 50:1, and 200 μL was added to each well. The 96-well plate was placed in a 37°C incubator and incubated for 30 minutes in the dark, ensuring no air bubbles were present in each well. The absorbance value at 562 nm was read using a microplate reader, and a calibration curve was created based on the concentration of the measured protein standard. The concentration of the measured protein sample was then calculated by adding the measured protein sample to 1x loading buffer to achieve the same concentration. After uniform mixing, the sample was placed in a metal bath and boiled at 100°C for 4-8 minutes depending on the sample volume, and then stored in a -80°C refrigerator for long-term storage.

[0058] Electrophoresis: The prepared gel was placed in the electrophoresis tank, electrophoresis buffer was added, and the loading wells were adjusted with a syringe to ensure there were no impurities or air bubbles in the wells. Protein samples and markers were prepared, and the loading amounts in each well were consistent. Electrophoresis was performed at a constant voltage of 80V for the stacking gel and 120V for the separating gel.

[0059] Membrane transfer: 1x membrane transfer solution was prepared and pre-cooled in a 4°C refrigerator. The PVDF membrane was immersed in methanol for 15 s, ddH2O for 2 min, and finally in membrane transfer solution. The membrane transfer clip, sponge, and filter paper were prepared and thoroughly washed with ddH2O. The glass plate was removed and thoroughly rinsed with ddH2O. The gel was then removed and immersed in membrane transfer solution. The membrane transfer clip was placed with the black side facing down, followed by the sponge, filter paper, adhesive, PVDF membrane, filter paper, and sponge. Air bubbles were removed, the membrane transfer clip was clamped, and the membrane was placed in the membrane transfer tank. The membrane was then transferred for approximately 2 h at a constant current of 200 mA, depending on the molecular weight of the target protein.

[0060] Blocking: The membrane was removed from the membrane transfer tank, placed in the prepared 5% non-fat dry milk, and slowly shaken on a horizontal shaker for blocking at room temperature for 2 hours.

[0061] Antibody incubation: Remove the membrane from the blocking solution and rinse twice with 1x TBST. Primary antibody incubation: Place the membrane in the prepared primary antibody and completely immerse it. Place on a horizontal shaker and gently shake. Incubate overnight at 4°C or at room temperature for 2 hours. After incubation, transfer to a membrane washing box, rinse twice with 1x TBST, and wash three times at 10 min each time. Secondary antibody incubation: Depending on the type of primary antibody, select the corresponding HRP-conjugated secondary antibody and prepare it according to the manufacturer's instructions. Place the membrane in the blocking solution and completely immerse it. Place on a horizontal shaker and gently incubate at room temperature for 2 hours. After incubation, transfer to a membrane washing box, rinse twice with 1x TBST, and wash three times at 15 min each time.

[0062] Color development by HRP luminescence method: According to the instructions for the luminescence solution, solution A and solution B were mixed in an equal ratio of 1:1 to prepare the luminescence solution. In the dark, the membrane was placed on the luminescence plate, an appropriate amount of luminescence solution was added dropwise, and the membrane was placed in a pre-cooled exposure device for exposure, and the analytical results were observed.

[0063] 1.5 Co-immunoprecipitation (CoIP)

[0064] Preparation of cell lysate: Aspirate the medium from a 10 cm cell culture dish, wash once with pre-chilled 1x PBS, aspirate the PBS, add 500 μL of pre-chilled 1x cell lysis solution, and place on ice for 5 minutes. Scrape the cells from the cell culture dish with a cell scraper and transfer them to a 1.5 mL EP tube. Place on ice and repeatedly aspirate and dispense with a 1 mL syringe until the cell lysate is no longer viscous. Centrifuge at 12,000 rpm for 10 minutes at 4°C. Transfer the supernatant to a new 1.5 mL EP tube, and measure the protein concentration.

[0065] Pre-clearance of cell lysate: 500 μg of cell lysate was added to 20 μL of 50% Protein A / G agarose bead slurry, and the mixture was incubated at 4°C for 30 minutes with rotation. Then, the mixture was centrifuged at 12,000 rpm for 10 minutes at 4°C in a centrifuge, and the supernatant was transferred to a new 1.5 mL EP tube.

[0066] Immunoprecipitation reaction: Primary antibodies were added to the above pretreated cell lysates in the appropriate ratio and incubated overnight in a shaker at 4°C. 20 μL of 50% protein A / G agarose bead slurry was added and incubated for 2 hours in a shaker at 4°C. The mixture was then centrifuged at 12,000 rpm for 30 seconds in a centrifuge at 4°C. The supernatant was removed by aspiration, and 500 μL of 1× cell lysate was added. The mixture was mixed repeatedly and washed. This washing and centrifugation process was repeated three times.

[0067] Sample analysis: 20-40 μL of 2×SDS digestion solution was added to the above precipitate, vortexed, centrifuged for 30 s, boiled in a metal bath at 100°C for 2-5 min, and centrifuged at 12,000 rpm for 1 min. The supernatant was aspirated and used for SDS-PAGE detection.

[0068] 1.6 Immunohistochemical analysis (IHC)

[0069] This invention uses ovarian cancer tissue chips purchased from a commercial source (Shanghai Xinchao Biotechnology Co., Ltd.), which can be directly used by following the steps below.

[0070] Deparaffinization: Slides are sequentially placed in xylene, xylene, 100% alcohol, 100% alcohol, 95% alcohol, 90% alcohol, 80% alcohol, and 70% alcohol. Xylene is the primary deparaffinizer, based on the principle that "like dissolves like." Generally, slides are placed in each solution for 10 minutes. If the temperature is high, the deparaffinization time can be reduced by several minutes. Conversely, if the temperature is low, the deparaffinization time must be extended appropriately, typically 12-15 minutes.

[0071] Antigen restoration: After deparaffinization, rinse with clean water for a certain period of time, add 3% H2O2 and soak for 10 minutes to remove endogenous catalase, then discard the H2O2, wash twice with clean water, add citrate buffer, place in a microwave oven and steam for 3 minutes (medium heat), generally boiling is sufficient, cool to room temperature, steam once more, and cool to room temperature. The purpose of steaming is to expose the antigen site.

[0072] Serum blocking: After cooling to room temperature, discard the citrate buffer, rinse twice with water, place the slide in PBS for 5 minutes, rinse twice, wipe off the PBS around the tissue, and immediately add serum to block some nonspecific sites. Place the slide in a 37°C incubator for 30 minutes. Serum was diluted 10-fold (900 μl PBS: 100 μl serum blocking solution).

[0073] Addition of primary antibody: Remove the slide from the incubator, wipe off the serum around the surface and back of the slide with absorbent paper, and add the primary antibody. If a control experiment was performed, add PBS to the control tissue. After adding the primary antibody, store the slide in a refrigerator at 4°C overnight.

[0074] Addition of secondary antibody: The slide glass was removed from the refrigerator, placed in PBS and washed three times for 5 minutes each time. After wiping off the PBS around the tissue, secondary antibody was added and the slide was placed in a 37°C incubator for 30 minutes.

[0075] Addition of SABC: The slides were removed from the incubator and washed three times in PBS for 5 min each. After wiping off the PBS around the tissue, SABC was added and the slides were placed in a 37°C incubator for 30 min. SABC was diluted 100-fold (PBS 990 μl: SABC 10 μl).

[0076] Addition of color developer: Remove the slide from the incubator and wash it in PBS three times for 5 minutes each time. After wiping off the PBS around the tissue, add the color developer. (Preparation of color developer: Add 1 drop of color developer A to 1 ml of water and shake well. Add 1 drop of color developer B and shake well. Add 1 drop of color developer C and shake well.) A: DAB B: H2O2 C: Phosphate buffer.

[0077] Counterstaining: After staining, the slides were rinsed with clean water for a certain period of time and then immersed in hematoxylin for staining. Generally, this was 0.5 minutes for animal tissues and 3-5 minutes for plant tissues.

[0078] Dehydration: After counterstaining, slides were rinsed in water and then sequentially placed in 70% alcohol, 80% alcohol, 90% alcohol, 95% alcohol, 100% alcohol, 100% alcohol, xylene, and xylene. Each slide was immersed for 2 minutes, and finally immersed in xylene before being transferred to a fume hood.

[0079] Sealing: A drop of neutral rubber was placed next to the tissue, then covered with a cover glass, first laying one side flat, then gently lowering the other side to prevent air bubbles from forming. After sealing the cover glass, it was placed in a fume hood to dry. The tissue was observed under a microscope and photographed.

[0080] [Example 2] Experimental results 2.1 Preliminary analysis of ABCG1 phosphorylation sites ABCG1 expressed and purified in ovarian cancer cells (NCBI: NM_016818.3 / NP_058198.2-ABCG1-666aa) was sent to Shanghai Zhongke New Life Biotechnology Co., Ltd. for protein phosphorylation mass spectrometry. Among the six peptide fragments, amino acid sites Y97, T99, S441, and Y655 were preliminarily confirmed in four of the peptide fragments. However, amino acid S120 or S125 in one of the other two peptide fragments may be phosphorylated, potentially simultaneously phosphorylated, as these are located within the ATP-binding domain. Another peptide fragment may be phosphorylated at T272 or S277, potentially simultaneously phosphorylated (Figure 1). Further experiments are required to verify this. Figure 2 shows the secondary mass spectrum of the peptide fragments.

[0081] 2.2 Detection of ABCG1 phosphorylation by antibodies We detected purified ABCG1 using pan-phosphoserine, phosphothreonine, and phosphotyrosine antibodies. We found that these antibodies could detect specific bands corresponding to purified ABCG1 (Figure 3A, arrows). However, we could not clearly detect phosphorylated bands at the same positions in cell lysates expressing GST-ABCG1. We speculated that this was because the expression level of ABCG1, particularly the phosphorylation levels of ABCG1 serine, threonine, or tyrosine, accounted for a low proportion of the total phosphorylated protein in cell lysates in transiently transfected cells (HEK293T). When we detected equivalent amounts of samples with GST antibodies, we found that the GST-ABCG1 levels in cell lysates were 5-10 times lower than those in purified GST-ABCG1 (the band-free areas were used as background, analyzed with Image J software) (Figure 3B). We performed pull-down experiments using phosphoserine, phosphothreonine, or phosphotyrosine antibodies, and further detected the pull-down products using GST antibodies. We found that serine phosphorylation was the highest and tyrosine phosphorylation was the lowest (Figure 3C). This indicates that the order of ABCG1 phosphorylation is serine > threonine > tyrosine. Of course, we cannot rule out the influence of differences in antibody affinity or specificity on our results. These data preliminarily demonstrate that ABCG1 is likely phosphorylated at these amino acids.

[0082] 2.3 Detection of ABCG1 phosphorylation sites in ovarian cancer cells and tissues Eight specific amino acid phosphorylation antibodies were prepared and used to detect ovarian cancer cell lines and ovarian cancer tissues (Figures 4-5). As a result, it was confirmed that at least eight amino acid sites in the ABCG1 protein, Y97, T99, S120, S125, T272, S277, S441, and Y655, were phosphorylated and expressed.

[0083] To further demonstrate the clinical significance of the present invention in tumor risk assessment, diagnosis, treatment, and prognosis, tissue chips were constructed using the T99, S120, T272, and Y655 polyclonal antibodies prepared in the present invention for cancer or cancer-adjacent tissues from patients with lung cancer (lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD) (Figures 6 to 9), ovarian cancer (OV) (Figures 10 to 13), and breast cancer (BRCA) (Figures 14 to 17). Expression levels were detected using immunohistochemistry (IHC), and a biostatistical correlation analysis was performed between the expression levels and overall patient survival.

[0084] Tissue chips constructed with different tumors were purchased from Shanghai Xinchao Biotechnology Co., Ltd., and all cases were Chinese cancer patients.

[0085] [Table 3]

[0086] The follow-up records for all cases included the patient's pathology type and follow-up record, as well as the medical ethics approval number for the human tissue.

[0087] The reagents and methods for immunohistochemical staining were the same as those in 1.6.

[0088] The expression levels of the different markers (T99, S120, T272, and Y655) were calculated as the percentage (%) of tumor cells stained per tissue chip, tumor cell (nuclei)<nuclei、N> , and pulp / membrane<cytoplasm / membrane、C / M> The staining intensity, including colorless (-, 0, no staining), light yellow (+, 1, light yellow), yellow-brown (++, 2, brown yellow), brown (+++, 3, brown), and dark brown (++++, 4, dark brown), is determined and scored under the guidance of a pathologist.

[0089] In Figures 6 to 17, A is a representative image of IHC staining (magnification: 200x), and the blocks are enlarged images of the area (800x).

[0090] In Figures 6 to 17, B shows the analysis of the correlation between the level of expression of each marker in the cell nucleus and cell serosa / membrane (N+C / M) and the survival of all tumor patients. The analysis method was the Kaplan Meier survival curve, and p<0.05 is considered to have biological and statistical significance. Kaplan-Meier survival curves were considered biologically significant at p < 0.05. A is a representative image of IHC staining (magnification 200x), and the block is a magnified region (800x).

[0091] For the above operation method and result determination, please refer to the following literature. Yin H,Wang J,Li H,Yu Y,Wang X,Lu L,Lv C,Chang B,Jin W,Guo W,Ren C,Yang G.Nat Commun.2021 Jul 9;12(1):4230.

[0092] While the basic principles, main features, and advantages of the present invention have been illustrated and described above, it will be apparent to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the present invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, in all respects, the embodiments are to be considered as illustrative and not limiting, and the scope of the present invention is limited not by the above description but by the appended claims, and all changes that come within the meaning and range of equivalents of the claims are therefore intended to be embraced within the present invention. Any reference signs in the claims should not be construed as limiting the scope of the claims.

[0093] Furthermore, although this specification is described according to embodiments, each embodiment does not include only independent technical solutions, and this description of the specification is merely for clarity, and those skilled in the art should understand the specification as a whole, and it should be understood that the technical solutions in each example can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. Use of a reagent and / or device for detecting human ABCG1 phosphorylation sites in the preparation of a reagent for assessing the risk of tumor development, diagnosing tumors, or evaluating tumor prognosis.

2. Use of an agent for inhibiting human ABCG1 phosphorylation sites in the preparation of a medicament for the prevention and / or treatment of tumors, wherein preferably the ABCG1 phosphorylation sites are selected from one or more of Y97, T99, S120, S125, T272, S277, S441, and Y655.

3. The use according to claim 1 or 2, characterized in that the human ABCG1 phosphorylation sites are selected from one or more of Y97, T99, S120, S125, T272, S277, S441, and Y655.

4. The use according to any one of claims 1 to 3, characterized in that the tumor is selected from osteosarcoma, glioblastoma, lung cancer, leukemia, breast cancer, ovarian cancer, cervical cancer, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, bladder cancer, colon cancer, and prostate cancer.

5. A phosphoantigen peptide of human ABCG1 protein, characterized in that the antigen peptide is selected from the amino acid sequences shown in any one of SEQ ID NOs: 1 to 8.

6. An antibody specific to a phosphorylation site of human ABCG1 protein, characterized in that the antibody is prepared by immunizing an animal with a phosphoantigen peptide of ABCG1 protein described in claim 5.

7. Use of the phosphoantigen peptide of human ABCG1 protein according to claim 5 in the preparation of a preparation for detecting phosphorylation sites of ABCG1 protein.

8. Use of an antibody specific to the phosphorylation site of human ABCG1 protein according to claim 6 in the preparation of a preparation for detecting the phosphorylation site of ABCG1 protein.

9. The use according to claim 7 or 8, characterized in that the phosphorylation sites of the ABCG1 protein are selected from one or more of Y97, T99, S120, S125, T272, S277, S441, and Y655.

10. A kit for assessing the risk of tumor development, diagnosis, prevention, treatment, and prognosis evaluation, characterized in that the kit comprises a reagent for detecting the human ABCG1 phosphorylation site described in claim 1, a reagent for inhibiting / promoting the human ABCG1 phosphorylation site described in claim 2, or an antibody specific to the phosphorylation site of the human ABCG1 protein described in claim 6.

Images