New application of sialyltransferase ST3GAL4 as a marker of EGFR-TKI resistance

Abstract

The present invention discloses the application of sialyltransferase ST3GAL4 as a marker for assisting in the assessment of EGFR-TKI resistance, the application of ST3GAL4 in the preparation of a reagent as an EGFR-TKI resistance marker, and the use of ST3GAL4 in the preparation of a kit for assisting in the assessment of EGFR-TKI resistance. Also disclosed is an EGFR-TKI resistance analysis kit, including an ST3GAL4 expression detection reagent. The present invention experimentally confirms that ST3GAL4 is closely related to EGFR-TKI resistance, and ST3GAL4 can be used as a marker for EGFR-TKI resistance. ST3GAL4 detection currently has mature technology, such as ELISA kits for detecting patient blood specimens and IHC for detecting patient tissue wax block specimens. By detecting ST3GAL4, it can be indicated whether the patient is resistant to EGFR-TKI, and timely adjustment of the treatment strategy can be guided.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a new application of sialyltransferase ST3GAL4 as an EGFR-TKI resistance marker and an EGFR-TKI resistance analysis kit. Background Art

[0002] Lung cancer is divided into non-small cell lung cancer and small cell lung cancer, of which non-small cell lung cancer accounts for more than 80%.

[0003] Osimertinib (OSI), as a third-generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), is currently widely used in the first- and second-line treatment of patients with advanced non-small cell lung cancer with EGFR sensitive mutations, with significant efficacy, and is a representative drug of the third-generation EGFR-TKI. Gefitinib (Gefitinib, Iressa) is an oral epidermal growth factor receptor tyrosine kinase (EGFR-TK) inhibitor (a small molecule compound) and a representative drug of the first-generation EGFR-TKI. The first-generation EGFR-TKI also includes erlotinib and icotinib hydrochloride. Afatinib is a potent and irreversible dual inhibitor of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) tyrosine kinase, and is a representative drug of the second-generation EGFR-TKI. The second-generation EGFR-TKI also includes dacomitinib.

[0004] However, EGFR-TKIs such as osimertinib, gefitinib, and afatinib also face the problem of acquired resistance, and the resistance mechanism is complex, which seriously limits their clinical application. Therefore, further exploration of the molecular mechanism and reversal strategy of acquired resistance of EGFR-TKIs such as osimertinib, gefitinib, and afatinib has important scientific significance and clinical application prospects.

[0005] Sialyltransferase ST3GAL4 is an exocytic protein. Currently, there are mature ELISA kits that are economical, reliable and easy to obtain. Summary of the invention

[0006] The inventors of the present invention found through the results of RNAseq differential gene screening of osimertinib-resistant lung cancer cell lines that although osimertinib-resistant lung cancer cell lines and parent cell lines have differences in apoptosis, autophagy and other aspects, it is surprising that the expression of sialyltransferase ST3GAL4 mutation in osimertinib-resistant lung cancer cell lines is abnormally increased, and the difference is very significant. Further Western blot detection confirmed the above findings, and sialyltransferase ST3GAL4 was found to be abnormally increased in serological specimens of osimertinib-resistant patients. By interfering with the expression of ST3GAL4 by siRNA, the resistance level of osimertinib-resistant cell lines was significantly reduced. The inventors further verified through experiments that sialyltransferase ST3GAL4 is closely related to the resistance of the third-generation epidermal growth factor receptor tyrosine kinase inhibitor osimertinib, the first-generation epidermal growth factor receptor tyrosine kinase inhibitors (gefitinib, erlotinib, icotinib), and the second-generation epidermal growth factor receptor tyrosine kinase inhibitors (afatinib, dacomitinib). Based on this, the present invention requests protection of the following technical solutions:

[0007] Application of sialyltransferase ST3GAL4 as a biomarker for auxiliary assessment of EGFR-TKI resistance.

[0008] Application of sialyltransferase ST3GAL4 in the preparation of reagents as EGFR-TKI resistance markers.

[0009] In the above application, the EGFR-TKI includes first, second and third generation EGFR-TKI drugs.

[0010] The present invention also protects the use of sialyltransferase ST3GAL4 in preparing a kit for assisting in evaluating EGFR-TKI resistance.

[0011] The kit assists in evaluating the state of EGFR-TKI resistance based on the content or content change trend of ST3GAL4 in the sample.

[0012] The kit is a protein expression detection kit.

[0013] In the above-mentioned use, the EGFR-TKI includes first-, second- and third-generation EGFR-TKI drugs.

[0014] The present invention also protects an EGFR-TKI resistance analysis kit, comprising a sialyltransferase ST3GAL4 expression detection reagent.

[0015] In the above-mentioned EGFR-TKI resistance analysis kit, the EGFR-TKI includes first-, second- and third-generation EGFR-TKI drugs.

[0016] In the above-mentioned application, or in the above-mentioned use, or in the above-mentioned kit, the first-generation EGFR-TKI drugs include gefitinib, erlotinib, and icotinib, the second-generation EGFR-TKI drugs include afatinib and dacomitinib, and the third-generation EGFR-TKI drugs include osimertinib.

[0017] The beneficial effects of the present invention are as follows: the applicant discovered that the expression of sialyltransferase ST3GAL4 mutation was abnormally elevated in osimertinib-resistant lung cancer cell lines, and subsequent real-time fluorescence quantitative PCR, cell proliferation experiments, clone formation experiments, cell invasion experiments and other experiments further confirmed that ST3GAL4 was closely related to the resistance to EGFR-TKI osimertinib, gefitinib, erlotinib, icotinib, afatinib and dacomitinib, and that ST3GAL4 regulated c-Met overexpression to mediate the resistance to osimertinib, gefitinib, erlotinib, icotinib, afatinib and dacomitinib, revealing for the first time the role of protein glycosylation modification in the resistance to osimertinib, gefitinib, erlotinib, icotinib, afatinib and dacomitinib and its key mechanism. ST3GAL4 can be used as a marker for resistance to EGFR-TKI osimertinib, gefitinib, erlotinib, icotinib, afatinib, and dacomitinib. There are currently mature technologies for ST3GAL4 detection, such as ELISA kits for testing patient blood samples and IHC for testing patient tissue wax block specimens. Testing ST3GAL4 can indicate whether the patient is resistant to EGFR-TKI osimertinib, gefitinib, erlotinib, icotinib, afatinib, and dacomitinib, and guide timely adjustment of treatment strategies, which has important social and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Immunoblotting was used to detect the expression of sialyltransferase ST3GAL4 in osimertinib-sensitive and -resistant cells.

[0019] Figure 2 It is an enzyme-linked immunosorbent assay (ELISA) to detect sialyltransferase ST3GAL4 in patient serological specimens.

[0020] Figure 3 The ST3GAL4 transcription level in PC-9GR and H1975 cells after overexpression of ST3GAL4 was detected by fluorescence quantitative method.

[0021] Figure 4 The MTT assay was used to detect the effect of overexpression of ST3GAL4 on the sensitivity of PC-9GR and H1975 cells to osimertinib.

[0022] Figure 5The MTT experiment was used to detect the effect of ST3GAL4 interference on the sensitivity of osimertinib in osimertinib-resistant cell lines PC-9GROR3 and H1975OR3.

[0023] Figure 6 These are the results of clone formation experiments after overexpression of ST3GAL4 in osimertinib-sensitive cells PC-9GR and H1975.

[0024] Figure 7 The Transwell invasion assay was used to detect the invasion ability of PC-9GR and H1975 cells before and after overexpression of ST3GAL4.

[0025] Figure 8 The CDX model was constructed to detect the efficacy of osimertinib in PC-9GR cells overexpressing ST3GAL4.

[0026] Fig. 9 A CDX model was constructed to detect the efficacy of brigatinib combined with osimertinib on PC-9GROR cells, wherein Figure A is the tumor size, Figure B is the tumor growth curve, Figure C is the average tumor weight, and Figure D is the ST3GAL4 concentration in serum.

[0027] Fig.10 The Transwell invasion assay was used to detect the effects of osimertinib alone, ST3GAL4 small interfering RNA alone, and the combination of the two on the invasive ability of PC-9GROR and H1975OR cells.

[0028] Fig.11 These are the results of the scratch experiment, where Figure A detects the migration ability of PC-9GROR3 cells interfering with siST3GAL4 and PC-9GR cells overexpressing ST3GAL4, and Figure B detects the migration ability of H1975-OR cells treated with osimertinib and interfering with STAGAL4.

[0029] Fig.12 The results of CCK-8 assay for cell proliferation activity after PC-9 cells overexpressing ST3GAL4 were treated with different concentration gradients of gefitinib, erlotinib, icotinib, afatinib, and dacomitinib. Figure A shows the results of gefitinib treatment, Figure B shows the results of erlotinib treatment, Figure C shows the results of icotinib treatment, Figure D shows the results of afatinib treatment, and Figure E shows the results of dacomitinib treatment.

[0030] Fig.13 A clone formation experiment was used to detect the cell proliferation ability of PC-9 cells under the action of gefitinib and afatinib before and after overexpression of ST3GAL4. Figure A shows the result of gefitinib treatment, and Figure B shows the result of afatinib treatment.

[0031] Fig.14The results of Ki67 detection of cell proliferation status after PC-9 cells overexpressing ST3GAL4 were treated with different concentration gradients of erlotinib, icotinib, and dacomitinib.

[0032] Fig.15 This is the result of cell invasion experiment after overexpression of ST3GAL4 in PC-9 cells.

[0033] Fig.16 The results of cell migration assay were used to detect the migration ability of PC-9 cells overexpressing ST3GAL4. DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0035] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0036] The main reagents and consumables used in the examples and their sources:

[0037] Trizol reagent was purchased from Invitrogen. RNA reverse transcription kit, TB Premix Ex Taq fluorescent quantitative PCR kit was purchased from Takara, and DEPC water, DMSO, and PBS solution were purchased from Shanghai Shenggong Biotechnology Co., Ltd.

[0038] Fetal bovine serum was produced by PAN-Seratech. RPMI 1640 medium was produced by Sigma. Penicillin-streptomycin solution was produced by HyClone. EDTA trypsin was produced by Tianjin Haoyang Biological Co., Ltd. Various cell culture dishes, 6-well plates, 12-well plates, and 96-well plates were produced by Corning, USA. CCK-8 was produced by Biomake.

[0039] BCA protein concentration determination kit and PVDF membrane were produced by Millipore. 30% Acr-Bis (29:1), Tris-Hcl (PH8.8 and PH6.8), 10% SDS, 10% ammonium persulfate, TEMED, 5X protein loading buffer, and Western primary antibody diluent were purchased from Beyotime. Western blot glass plates and protein markers were produced by Bio-rad, USA. Skim milk powder was purchased from Biofroxx. Goat anti-rabbit secondary antibody was purchased from Sino Biological. Tissue protein lysis buffer and phosphate inhibitor were purchased from Thermo. TRIS and Glycine were purchased from Solarbio. SDS and Sodium Chloride were purchased from BIOSHARP. Triton X-100 and goat serum were purchased from Zhongshan Jinqiao, Cy3-conjugated Goat anti-Rabbit IgG was purchased from BIOSS, DAPI was purchased from Med Chem Express, and Ki67 was purchased from Boster.

[0040] Human ST3GAL4 ELISA kit was purchased from Shanghai Fanke Industrial Co., Ltd.

[0041] Transwell cell culture chamber was purchased from Merck Millipore, Inc. Matrigel was purchased from Corning, Inc.

[0042] Lung cancer cell lines PC-9GR and H1975: NSCLC cell line PC-9GR was derived from Guangzhou Medical University (China), which contains the T790M mutation of the EGFR gene. H1975 cells were purchased from the American Cell Bank (ATCC, Rockville, MD, USA), which contains the L858R and T790M mutations of the EGFR gene.

[0043] The drug-resistant cells PC-9GROR1, PC-9GROR2, PC-9GROR3 and H1975OR1, H1975OR2, H1975OR3 were all screened and cultured by our laboratory.

[0044] The other reagents, unless otherwise indicated, are conventional reagents in the art and can be obtained commercially.

[0045] Example 1: Immunoblotting (WB) to detect the expression of sialyltransferase ST3GAL4 in drug-resistant cells

[0046] 1. Experimental materials:

[0047] RPMI 1640 medium, fetal bovine serum, penicillin-streptomycin solution, EDTA trypsin, cell culture flasks, cell culture dishes, tissue protein lysis buffer, BCA protein concentration assay kit, 30% Acr-Bis (29:1), Tris-Hcl (PH8.8 and PH6.8), 10% SDS, 10% ammonium persulfate, TEMED, 5× protein loading buffer, ST3GAL4 antibody, GAPDH antibody (internal reference antibody), Western primary antibody diluent, protein marker, skim milk powder, goat anti-rabbit secondary antibody, TRIS, SDS, Glycine, sodium chloride, Tween-20.

[0048] Cell culture medium: RPMI 1640 medium + 10% fetal bovine serum + 0.1% penicillin-streptomycin solution

[0049] 10× electrophoresis buffer: Dissolve 10g SDS, 188g Glycine, and 30.2g Tris in 1L distilled water. Dilute with distilled water to 1× electrophoresis buffer before use.

[0050] 10× electrotransfer buffer: Dissolve 29.8g of Glycine, 58g of Tris, and 3.7g of SDS in 1L of distilled water. Dilute 80mL of 10× electrotransfer buffer to 1× electrotransfer buffer and add 200mL of methanol before use.

[0051] 10×TBST solution: Dissolve 88g of Sodium Chloride and 24.2g of Tris in 1l of distilled water. Dilute with distilled water to 1×TBST solution, and add 0.1% Tween-20 before use.

[0052] 5% skim milk powder blocking solution (50 mL): weigh 2.5 g skim milk powder and dissolve it in 50 mL TBST buffer. Prepare and use it immediately.

[0053] 2. Experimental steps:

[0054] 1. Total Protein Extraction

[0055] (1) Osimertinib-sensitive cells PC-9GR and drug-resistant cells PC-9GROR1, PC-9GROR2, and PC-9GROR3 were inoculated, and H1975 and H1975OR1, H1975OR2, and H1975OR3 were inoculated in 6-well plates and cultured at 37° C. and 5% CO 2 for 48 h before extraction of total protein.

[0056] (2) Discard the culture medium, add 80 μL of tissue protein lysis buffer to each well, place on ice for 30 min, and shake every 10 min to allow the lysis buffer to react fully. Scrape the cells from the culture plate with a cell scraper and transfer them to a 1.5 mL centrifuge tube with a pipette.

[0057] (3) Centrifuge at 12,000 rpm for 20 min at 4°C and transfer the supernatant to another centrifuge tube.

[0058] 2. Protein concentration determination by BCA method

[0059] (1) Follow the instructions of the Bio-Tech BCA kit to perform subsequent operations. First, prepare a standard curve. Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of the protein standard to a 96-well plate, and add PBS to make the final volume of each well 20 μL. Add 5 μL of protein sample and 15 μL of PBS to each well of the sample to be tested and mix them evenly.

[0060] (2) Add 220 μL BCA working solution (solution A:solution B = 1:50) to each well, mix again, and incubate at 37°C for 30 min.

[0061] (3) Place the reacted sample into an ELISA instrument, select a wavelength of 562 nm, detect the OD value of the protein sample, and finally draw a standard curve based on the OD value of the standard. Then, substitute the sample OD value into the standard curve to calculate the protein concentration of the sample.

[0062] 3. Western blot

[0063] (1) Prepare polyacrylamide gel: Install the gel preparation plate and prepare gel according to the instructions of the Bio-Tech SDS-PAGE gel preparation kit. First, prepare 10% separation gel. Add the reagent to the gel preparation plate and seal the gel with isopropanol. After solidification, prepare 5% concentrated gel. After filling the gel, insert the comb and wait for solidification.

[0064] (2) Gel electrophoresis: Load the prepared gel into the electrophoresis device, remove the comb, and add 1×SDS-PAGE electrophoresis solution to the electrophoresis tank. The electrophoresis solution should cover the sample wells. Calculate the sample loading amount based on the protein concentration measured by the BCA method. Add 30μg of protein to each sample well and 8μL of protein marker. After installing the electrophoresis device, turn on the power supply and perform electrophoresis at 110V for 75 minutes.

[0065] (3) Transfer: After the electrophoresis is completed, soak the transfer sponge and filter paper in transfer buffer. Find the target protein band on the PAGE gel according to the marker, cut it off and soak it in transfer buffer. At the same time, cut a PVDF membrane of the same size as the target protein gel. The PVDF needs to be soaked in anhydrous methanol for about 30 seconds. After it is activated, it is also immersed in transfer buffer. In order, overlap the transfer clip (white side), sponge, filter paper, PVDF membrane, PAGE gel, filter paper, sponge, and transfer clip (black side), and make sure to remove all bubbles during the process. Put the transfer clip into the transfer tank, add transfer buffer, put the transfer device on ice to keep it low, turn on the power, and transfer the membrane for 95 minutes at 80V.

[0066] (4) Blocking: Prepare blocking solution (5% skim milk powder) in advance. After the transfer is completed, immerse the PVDF membrane in the blocking solution and block it on a shaker at room temperature for 1 h, or at 4°C overnight.

[0067] (5) Primary antibody incubation: After blocking, wash the PVDF membrane three times with TBST, 8 min each time. Dilute the antibody according to the antibody instructions, add the antibody and incubate at 4°C overnight.

[0068] (6) Secondary antibody incubation: Wash the membrane three times with TBST, 8 min each time. Dilute the secondary antibody according to the antibody instructions. After adding the secondary antibody, incubate on a shaker at room temperature for 1 h.

[0069] (7) Color development: After the antibody incubation, wash with TBST three times, 8 min each time. Add ECL chemiluminescent reagent (solution A:solution B = 1:1) on the membrane and expose it in a chemiluminescent imager.

[0070] 3. Experimental Results

[0071] Test results such as Figure 1 As shown, compared with osimertinib-sensitive cells PC-9GR, the resistant cell lines PC-9GROR1, PC-9GROR2, PC-9GROR3 and H1975OR2, H1975OR3 have enhanced expression of sialyltransferase ST3GAL4. Example 2: Enzyme-linked immunosorbent assay (ELISA) was used to detect the concentration changes of sialyltransferase ST3GAL4 in patient serological specimens before and after osimertinib resistance.

[0072] 1. Experimental Materials

[0073] Human ST3GAL4 ELISA kit, patient serum.

[0074] 2. Experimental steps

[0075] (1) Dilution of standard

[0076] This kit provides one original standard, which should be diluted in a small test tube according to the following table.

[0077] 240pg / ml Standard No. 5 150 μl of original standard is added to 150 μl of standard diluent 120pg / ml Standard No. 4 150 μl of standard No. 5 was added to 150 μl of standard diluent 60pg / ml Standard No. 3 150μl of standard No. 4 was added to 150μl of standard diluent 30pg / ml Standard No. 2 150 μl of standard No. 3 was added to 150 μl of standard diluent 15pg / ml Standard No. 1 150μl of standard No. 2 was added to 150μl of standard diluent

[0078] (2) Sample addition

[0079] Set up blank wells, standard wells, and wells for samples to be tested. Accurately add 50μl of the standard sample to the ELISA coated plate, add 40μl of sample diluent to the wells for samples to be tested, and then add 10μl of the sample to be tested (the final dilution of the sample is 5 times). Add the sample to the bottom of the well of the ELISA plate, try not to touch the well wall, and shake it gently to mix well.

[0080] (3) Incubation

[0081] The plate was sealed with a sealing film and incubated at 37°C for 30 minutes.

[0082] (4) Liquid preparation

[0083] Dilute the 30-fold concentrated washing solution with 30-fold distilled water for later use

[0084] (5) Washing

[0085] Carefully peel off the sealing film, discard the liquid, shake dry, fill each well with washing solution, let it stand for 30 seconds and then discard, repeat this 5 times and pat dry.

[0086] (6) Adding enzyme

[0087] Add 50 μl of enzyme labeling reagent to each well, except for the blank well.

[0088] (7) Incubation

[0089] Same operation as step (3)

[0090] (8) Washing

[0091] Same operation as step (5)

[0092] (9) Color rendering

[0093] First add 50 μl of color developer A to each well, then add 50 μl of color developer B, gently shake to mix, and develop the color at 37°C in the dark for 10 minutes.

[0094] (10) Termination

[0095] Add 50 μl of stop solution to each well to terminate the reaction (the blue color immediately turns yellow)

[0096] (11) Determination

[0097] Use the blank well as zero setting, and measure the absorbance (OD value) of each well in sequence at a wavelength of 450 nm. The measurement should be performed within 15 minutes after adding the stop solution.

[0098] (12) Calculation

[0099] Draw a standard curve on the coordinate paper with the concentration of the standard as the horizontal axis and the OD value as the vertical axis. Find the corresponding concentration based on the OD value of the sample using the standard curve; then multiply it by the dilution factor; or use the concentration of the standard and the OD value to calculate the linear regression method of the standard curve, substitute the OD value of the sample into the equation, calculate the sample concentration, and then multiply it by the dilution factor to get the actual concentration of the sample.

[0100] 3. Experimental Results

[0101] The experimental results are as follows Figure 2 As shown, compared with the ST3GAL4 concentration in the patient's serum when the patient was sensitive to osimertinib, the sialyltransferase ST3GAL4 in the serum samples after osimertinib resistance was abnormally increased.

[0102] Example 3

[0103] 1. Obtaining PC-9GR stable cell lines and H1975 stable cell lines overexpressing the Human ST3GAL4 gene

[0104] First, PC-9GR and H1975 cells overexpressed ST3GAL4, and the following steps were followed: using lentivirus as a tool, the exogenous gene Human ST3GAL4 (C-terminal GFP tag) was integrated into the PC-9GR cell line and H1975 cell line, respectively, to obtain PC-9GR stable cell lines and H1975 stable cell lines overexpressing the Human ST3GAL4 gene. (Human ST3GAL4 gene information GenBank: BC010645.1).

[0105] (1) Vector construction and extraction

[0106] Synthesize the target gene ST3GAL4 (fused with C-terminal GFP tag) and insert the synthesized sequence into the lentiviral transfer vector (vector name pLV-ST3GAL4, eukaryotic resistance is puromycin, prokaryotic resistance is ampicillin). Pick multiple clones for sequencing identification, select the correct activated strain, transfer to LB medium, and shake the bacteria at 37°C overnight. After overnight culture, use a plasmid DNA large-scale extraction kit to extract plasmid DNA. Sequencing verifies the correctness of the vector sequence.

[0107] (2) Lentivirus preparation

[0108] a. Obtain high-quality plasmids: pLV-ST3GAL4, pVSVG, pSD; revive HEK293T cells into T25 cell culture flasks.

[0109] b. Find the appropriate ratio to mix the three plasmids with the transfection reagent and transfect into HEK293T cells. After 48-72 hours of cell culture, centrifuge and collect the cell supernatant. Filter the cell supernatant with a 0.45μm sterile filter and obtain high-titer lentivirus after ultracentrifugation.

[0110] (3) Lentivirus infection of cells

[0111] a. A preliminary experiment was conducted in a 24-well plate. PC-9GR cells and H1975 cells were infected with gradient MOI (multiplicity of infection) respectively. Puromycin was added after 6 hours of culture. The cells were cultured for 48-72 hours and the optimal MOI was determined by qPCR.

[0112] b. PC-9GR cells and H1975 cells cultured in T25 were infected with the optimal MOI conditions, puromycin was added after 6 hours of culture, and then cultured for another 48-72 hours for subsequent experiments.

[0113] (4) Isolation of monoclonal cell lines

[0114] The cloned cell lines obtained by the serial dilution method are generally serially diluted in 12-24 96-well plates. The serial dilution method is as follows:

[0115] a. Add 200uL of the cell suspension in step 3.2 to well A1, and then dilute it from well A1 to well H1 at a 2-fold ratio. For example, pipette 100uL of cell suspension from well A1 into well B1, and add 100uL of culture medium, mix well, then pipette 100uL of cell suspension into well C1, and add 100uL of culture medium.

[0116] b. After mixing the first column, dilute the samples from the first to the twelfth columns at a 2-fold ratio.

[0117] c. Place the plate in an incubator and avoid moving it. After 3 days, continue to observe the cell growth status and mark the position of the single clone on the plate.

[0118] d. Expand the culture of monoclonal cell lines and add resistance screening pressure - puromycin. Continuously increase the resistance screening pressure (increase the concentration of puromycin) to obtain high-expressing cell lines for later identification.

[0119] (5) Identification of monoclonal cell lines

[0120] a. Identify all the cell lines in the above steps by qPCR and screen out the cell lines with high expression of ST3GAL4 gene.

[0121] b. The expression level of ST3GAL4 protein in each monoclonal cell line was further determined by flow cytometry (FCM), fluorescence observation or immunofluorescence (IF).

[0122] (6) Expand the identified cell lines and store them in aliquots for cryopreservation. Obtain PC-9GR stable cell lines overexpressing the Human ST3GAL4 gene and H1975 stable cell lines PC-9GR OE-1 、PC-9GR OE-2 、PC-9GR OE-3 、H1975 OE-1 、H1975 OE-2 、H1975 OE-3 .

[0123] 2. Fluorescence quantitative detection of expression of ST3GAL4 in PC-9GR and H1975 cells after overexpression

[0124] (1) Trizol reagent was used to extract RNA from PC-9GR and H1975 cells overexpressing ST3GAL4.

[0125] 30×10 5 PC-9GR and H1975 cells overexpressing ST3GAL4 were inoculated into 6-well culture plates and cultured at 37℃ and 5% CO2 for 48h. Add 1ml Trizol to fully homogenize and let stand at room temperature for 5min. Add 0.2ml chloroform, oscillate for 15s, and let stand for 2min. Centrifuge at 4℃, 12000g×15min, and take the supernatant. Add 0.5ml isopropanol, gently mix the liquid in the tube, and let stand at room temperature for 10min. Centrifuge at 4℃, 12000g×10min, and discard the supernatant. Add 1ml 75% ethanol and gently wash the precipitate. 4℃, 7500g×5min, discard the supernatant. Invert to dry, add appropriate amount of DEPC H2O to dissolve (65℃ for dissolution for 10-15min).

[0126] (2) Perform reverse transcription using an RNA reverse transcription kit according to the kit instructions.

[0127] (3) Detect the concentration of DNA by spectrophotometry.

[0128] (4) Fluorescence quantitative PCR amplification reaction system is 20 μL: TB Premix Ex Taq (TAKARA) 12.5 μL, upstream and downstream primers 1 μL each, cDNA template 2 μL, balance ddH2O. Fluorescence quantitative PCR reaction conditions are: 95℃30s; 95℃5s, 60℃30s, a total of 40 cycles. The upstream and downstream primer sequences are:

[0129] ST3GAL4-1S:5'-GCCTCTAAGCTCTTTGGCAACTA-3' (SEQ ID NO.1),

[0130] ST3GAL4-1A: 5'-GATGGAGGAGCTGGTGATGG-3' (SEQ ID NO. 2).

[0131] (5) Pass 2 ΔΔCT Methods Expression levels were calculated.

[0132] The results are as follows Figure 3 As shown, the mRNA level of ST3GAL4 was highly expressed after overexpression in PC-9GR and H1975 cells.

[0133] 3. Cell Proliferation Assay

[0134] Different concentration gradients (10 μM, 1 μM, 10 -1 μM, 10 -2 μM, 10 -3 μM) of osimertinib was used to treat PC-9GR and H1975 cells overexpressing ST3GAL4; CCK-8 was used to detect cell proliferation activity. The specific steps were as follows:

[0135] (1) 100 μL of cell suspension of PC-9GR and H1975 cells overexpressing ST3GAL4 (3000 cells) was added to a 96-well plate and pre-cultured in an incubator (37° C., 5% CO 2 ) for 24 h.

[0136] (2) The culture medium in the wells was discarded, and 200 μL of cell culture medium containing drugs at different concentrations was added thereto, and the cells were incubated in an incubator for 48 h (37° C., 5% CO 2 ).

[0137] (3) Add 10 μL of CCK-8 solution to each well and incubate in an incubator for 1 to 4 h.

[0138] (4) Measure the absorbance at 450 nm using an enzyme-labeled instrument.

[0139] The results are as follows Figure 4 The results showed that after PC-9GR was overexpressed, it was resistant to osimertinib and the cell survival rate was significantly increased; after H1975 cells were overexpressed, H1975 OE-1 Compared with H1975 OE-2 and H1975 OE-3 Even less sensitive.

[0140] IV. MTT assay to detect the effect of knocking out ST3GAL4 with small interfering RNA on the sensitivity of osimertinib in drug-resistant cells

[0141] I. Experimental materials: MTT reagent, ribo FECT TM CPTransfection Kit (transfection kit, brand ribobio)

[0142] II. Experimental steps:

[0143] 1. Knockdown of ST3GAL4 by small interfering RNA

[0144] (1) Inoculation of cells

[0145] 3×10 5 H1975OR3 and PC-9GROR3 cells were inoculated into the culture wells of a 6-well plate.

[0146] (2) Transfection

[0147] For each cell sample, prepare as follows:

[0148] a. Dilute siRNA: Dilute 50 μl siRNA storage solution (v3) with 120 μl Buffer (v2) and shake gently.

[0149] b. Preparation of mixed solution: Add 12 μl Reagent (v4), gently pipette to mix, and incubate at room temperature for 0 to 15 minutes to prepare the transfection complex.

[0150] c. Add the transfection mixture to 1863 μl of complete medium without dual antibody (v1) and shake gently.

[0151] d. Place the culture plate in a CO2 incubator at 37°C for 72 hours.

[0152] 2. MTT assay

[0153] (1) Inoculation of cells

[0154] 100 μL of 3,000 H1975OR3 and PC-9GROR3 cells with ST3GAL4 knockout were added to each well. After incubation in the incubator for 24 hours, the cells were replaced with 8 μM, 4 μM, 2 μM, 1 μM, 10 -1 μM, 10 -2 μM, 10 -3 μM, 10 -4 μM osimertinib in the culture medium.

[0155] (2) Preparation of MTT solution

[0156] Dissolve MTT Reagent (Component A) with MTT Solvent (Component B) to prepare 5 mg / ml MTT solution. It can be used immediately after preparation, or stored directly at -20℃ away from light, or it can be appropriately divided and stored at -20℃ away from light as needed.

[0157] (3) Add 10 μL MTT solution to each well to make the final concentration of MTT in each well be 0.5 mg / ml.

[0158] (4) After gently mixing, incubate in a 5% CO2, 37°C incubator for 4 hours.

[0159] (5) Carefully aspirate the culture medium from each well to prevent the cell monolayer from rupturing.

[0160] (6) Add 100 μL Formazan Solubilization Solution (Component C) to each well.

[0161] (7) Place the 96-well plate on an oscillator and gently shake it for 10 minutes until the formazan is completely dissolved as observed under an ordinary optical microscope.

[0162] (8) Measure the absorbance at 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0163] III. Experimental results:

[0164] The results are as follows Figure 5 As shown, PC-9GROR3 is the PC-9GR resistant strain, PC-9GROR3-siST3GAL4-1 is PC-9GROR3 plus ST3GAL4 factor, PC-9GROR3-siST3GAL4-3 is PC-9GROR3 plus ST3GAL4 factor, and H1975OR3 is the H1975 resistant strain, H1975OR3-siST3GAL4-2 is H1975OR3 plus ST3GAL4 factor, and H1975OR3-siST3GAL4-3 is H1975OR3 plus ST3GAL4 factor.

[0165] The results showed that PC-9GROR3, PC-9GROR3-siST3GAL4-1, PC-9GROR3-siST3GAL4-3 and H1975OR3, H1975OR3-siST3GAL4-2, H1975OR3siST3GAL4-3 were treated with different concentration gradients of osimertinib, and the cell proliferation activity was detected by CCK-8. The results suggested that after interfering with ST3GAL4 in PC-9GROR3 cells, they were sensitive to osimertinib; after interfering with H1975OR3 cells, they were sensitive to osimertinib.

[0166] 5. Clone Formation Experiment

[0167] The clone formation assay was used to detect the cell proliferation ability of PC-9GR and H1975 cells before and after overexpression of ST3GAL4 under the action of osimertinib.

[0168] NSCLC cells were seeded in 6-well culture plates at a density of 800 cells / well and cultured for 2 weeks. The cells were then washed 3 times with PBS. Subsequently, the colonies were fixed in 4% paraformaldehyde for 30 min and then stained with 0.1% crystal violet for 30 min at room temperature.

[0169] (1) PC-9GR and H1975 cells and their respective cells overexpressing ST3GAL4 were seeded at a density of 800 cells / well in 6-well culture plates and cultured for 2 weeks (37° C., 5% CO 2 ).

[0170] (2) Observe frequently and terminate the culture when clones visible to the naked eye appear in the culture dish.

[0171] (3) Wash the cells three times with PBS.

[0172] (4) Discard the supernatant, fix with 4% paraformaldehyde for 30 min, and stain with GIMSA staining solution for 10-30 min.

[0173] (5) Calculate the clone formation rate: clone formation rate = (number of clones / number of inoculated cells) × 100%.

[0174] (6) Statistical analysis of results.

[0175] The results are as follows Figure 6 As shown, the monoclonal formation ability of PC-9GR and H1975 cells was enhanced after overexpression of ST3GAL4.

[0176] VI. Cell invasion assay

[0177] The cell invasion assay was performed to detect the invasion ability of PC-9GR and H1975 cells before and after overexpression of ST3GAL4. The procedure was as follows:

[0178] (1) A Transwell cell culture chamber with a PET membrane having a pore size of 8 μm was placed in a 24-well culture plate, and the bottom of the Transwell chamber was coated with 50 μL / well of Matrigel and incubated at 37°C overnight to form a gel.

[0179] (2) PC-9GR and H1975 cells and cells before and after overexpression of ST3GAL4 were digested with trypsin and collected. Cell suspensions were prepared in RPMI 1640 culture medium containing 1% fetal bovine serum. The cell density was adjusted to 2×10 5100 μL / well was inoculated into the upper chamber, and 600 μL of culture medium containing 10% fetal bovine serum was added into the lower chamber.

[0180] (3) Incubate at 37°C for 48 h and remove the Transwell.

[0181] (4) Fix with 4% paraformaldehyde for 30 min and stain with crystal violet for 30 min.

[0182] (5) Carefully wipe off the cells on the top of the microporous filter membrane with a cotton swab, clean the cell chamber with running water, and let it dry naturally.

[0183] (6) Count the number of cells in the lower layer of the filter under a light microscope.

[0184] The results are as follows Figure 7 As shown, the cell counting results showed that after PC-9GR and H1975 cells overexpressed ST3GAL4, the number of invasive cells increased and the invasion ability of cells was enhanced.

[0185] VII. Tumor Cell Line Xenograft Model (CDX Model)

[0186] The CDX model was constructed to detect the efficacy of osimertinib in PC-9GR cells overexpressing ST3GAL4:

[0187] (1) Six-week-old female mice were randomly divided into two groups according to each cell type: no treatment group and osimertinib treatment group.

[0188] (2) PC-9GR, PC-9GR-OE ST3GAL4 The cells were 2×10 6 Each cell type was inoculated into the subcutaneous tissue of the right forelimb of nude mice, and 20 nude mice were inoculated with each cell type.

[0189] (3) Tumor volume (length × width) 2 ) / 2, measured twice a week.

[0190] (4) When the tumor reaches 50 mm 3 At 4 pm, the nude mice in the treatment group were intragastrically administered with 5 mg / kg osimertinib.

[0191] (5) After 4 weeks of drug administration, the nude mice were euthanized to terminate the animal experiment and estimate the tumor volume.

[0192] The results are as follows Figure 8 As shown, the volume and weight of tumor growth slowed down after the use of osimertinib.

[0193] Example 4: Construction of CDX model to detect the efficacy of brigatinib combined with osimertinib on PC-9GROR cells

[0194] (1) Six-week-old female mice were randomly divided into four groups according to each cell type: no treatment group, osimertinib treatment group, brigatinib treatment group, and brigatinib combined with osimertinib treatment group.

[0195] (2) PC-9GROR3 cells were grown at 2×10 6 The cells were inoculated subcutaneously into the right forelimb of nude mice, and 40 nude mice were inoculated.

[0196] (3) Tumor volume (length × width) 2 ) / 2, measured twice a week.

[0197] (4) When the tumor reaches 50 mm 3 At the same time, the nude mice in the osimertinib treatment group were gavaged with 5 mg / kg osimertinib, the nude mice in the brigatinib treatment group were gavaged with 35 mg / kg brigatinib, and the nude mice in the brigatinib combined with osimertinib treatment group were gavaged with 5 mg / kg osimertinib and 35 mg / kg brigatinib.

[0198] (5) After 4 weeks of drug administration, the nude mice were euthanized to terminate the animal experiment and estimate the tumor volume.

[0199] The results are as follows Fig. 9 As shown, after the combination of brigatinib and osimertinib, the tumor volume grew the slowest, the tumor weight grew the slowest, and the ST3GAL4 concentration in the serum was the lowest, indicating that the combination of brigatinib and osimertinib can significantly inhibit the growth of osimertinib-resistant cell transplant tumors.

[0200] Example 5: Cell invasion assay to detect the invasive ability of osimertinib and STAGAL4 factor combined on PC-9GROR and H1975OR cells

[0201] (1) A Transwell cell culture chamber with a PET membrane having a pore size of 8 μm was placed in a 24-well culture plate, and the bottom of the Transwell chamber was coated with 50 μL / well of Matrigel and incubated at 37°C overnight to form a gel.

[0202] (2) PC-9GROR3 and H1975OR3 cells treated with osimertinib and ST3GAL4 small interfering RNA were digested with trypsin and collected, and cell suspensions were prepared in RPMI 1640 culture medium containing 1% fetal bovine serum. The cell density was adjusted to 2×10 5 100 μL / well was inoculated into the upper chamber, and 600 μL of culture medium containing 10% fetal bovine serum was added into the lower chamber.

[0203] (3) Incubate at 37°C for 48 h, remove the Transwell, and carefully wipe off the cells on the microporous filter membrane with a cotton swab.

[0204] (7) Fix with 4% paraformaldehyde for 30 min and stain with crystal violet for 30 min.

[0205] The number of cells in the lower layer of the filter membrane was counted under a light microscope.

[0206] The results are as follows Fig.10 As shown, the cell counting results showed that after PC-9GROR3 and H1975OR3 cells were stimulated with osimertinib and STAGAL4 factor, the number of invasive cells increased and the invasion ability of the cells was enhanced.

[0207] Example 6: Scratch test

[0208] The experimental method of the scratch test is:

[0209] (1) 2.5×10 per hole 5 The cells were inoculated into 6-well plates and the supernatant was discarded after culturing for 12 h.

[0210] (2) The experiment includes a control group (normal cells) and experimental groups with different drug effects. The experimental groups are set up according to the needs of the experiment.

[0211] (3) After continuing to culture in the incubator for 48 h, vertical scratches were made with a 10 μL pipette tip, and the culture medium containing 5% fetal bovine serum was replaced. Pictures were taken under a microscope at 0 and 24 h.

[0212] 1. Cell migration assay to detect the migration ability of PC-9GROR3 cells interfering with siST3GAL4 and PC-9GR cells overexpressing ST3GAL4

[0213] (1) 2.5×10 per hole 5 PC-9GR, PC-9GROR3 (two groups), and PC-9GR cells overexpressing ST3GAL4 were inoculated into 6-well plates, and the supernatant was discarded after 12 h of culture.

[0214] (2) PC-9GROR3 was subjected to interference treatment using the siST3GAL4 kit.

[0215] (3) After continuing to culture in the incubator for 48 h, vertical scratches were made with a 10 μL pipette tip, and the culture medium containing 5% fetal bovine serum was replaced. Pictures were taken under a microscope at 0 and 24 h.

[0216] The results are as follows Fig.11 As shown in A, after PC-9GROR3 cells interfered with siST3GAL4, the number of invasive cells decreased and the cell migration ability weakened; after PC-9GR cells overexpressed ST3GAL4, the number of invasive cells increased and the cell migration ability enhanced.

[0217] 2. Cell migration experiment detected the effect of osimertinib treatment and interference of ST3GAL4 on the migration ability of H1975OR3 cells.

[0218] (1) 2.5×10 per hole 5 H1975OR3 cells (four groups) were inoculated into 6-well plates and the supernatant was discarded after culturing for 12 h.

[0219] (2) The three groups of H1975-OR cells were treated with osimertinib, interference according to the siST3GAL4 kit, or a combination of osimertinib and interference according to the siST3GAL4 kit.

[0220] (3) After continuing to culture in the incubator for 48 h, vertical scratches were made with a 10 μL pipette tip, and the culture medium containing 5% fetal bovine serum was replaced. Pictures were taken under a microscope at 0 and 24 h.

[0221] The results are as follows Fig.11 As shown in B, after H1975-OR cells were treated with osimertinib, the number of invasive cells increased and the migration ability of cells was enhanced; when H1975-OR cells were interfered with STAGAL4 and H1975-OR cells were interfered with STAGAL4 and then treated with osimertinib, the number of invasive cells decreased and the migration ability of cells was weakened.

[0222] Example 7: Sialyltransferase ST3GAL4 indicates resistance to gefitinib, erlotinib, icotinib, afatinib and dacomitinib

[0223] 1. Obtaining PC-9 cells overexpressing the Human ST3GAL4 gene

[0224] (A) Vector construction and extraction

[0225] Synthesize the target gene ST3GAL4 and insert the synthetic sequence into the lentiviral transfer vector (vector name pLV-ST3GAL4, eukaryotic resistance is puromycin, prokaryotic resistance is ampicillin). Pick multiple clones for sequencing identification, select the correct activated strain, transfer to LB medium, and shake the bacteria at 37°C overnight. After overnight culture, use a plasmid DNA extraction kit to extract plasmid DNA. The operation steps are carried out according to the instructions of the plasmid DNA extraction kit. The specific steps are as follows:

[0226] (1) Harvest the bacteria cultured overnight and centrifuge at 6000g, 4°C for 15 min.

[0227] (2) Add 10 mL of Buffer P1 to resuspend the bacteria.

[0228] (3) Add 10 mL of Buffer P2, shake the tube vigorously 4-6 times to mix thoroughly, and leave at room temperature for 5 min. If LyseBlue reagent is used, the solution should turn a uniform blue.

[0229] (4) During the incubation period, open the needle port seal and place the filter in a suitable rack.

[0230] (5) Add 10 mL of Buffer P3 in an ice bath to the lysate and shake the tube vigorously 4-6 times to mix. If LyseBlue reagent is added, mix the reagent until no color is left.

[0231] (6) Pour the lysate into the needle-sealed filter and leave it at room temperature for 10 min without inserting the piston. Open the needle-sealed cap and gently squeeze the piston to filter the lysate into a new 50 mL centrifuge tube.

[0232] (7) Add 2.5 mL of Buffer ER, mix thoroughly 10 times, and incubate on ice for 30 min.

[0233] (8) Hang the QIAGEN-tip 500 column on another centrifuge tube, add 10 mL of Buffer QBT, and allow the solution in the tube to drip down by gravity.

[0234] (9) Add the lysate incubated on ice in step 7 to the QIAGEN-tip column and allow it to drip through the resin.

[0235] (10) Wash the QIAGEN-tip column with 2 × 30 mL Buffer QC.

[0236] (11) Elute the DNA with 15 mL of Buffer QN into a new 50 mL centrifuge tube.

[0237] (12) Add 10.5 mL of room temperature isopropanol, mix, centrifuge at 15,000 g, 4 °C for 30 min, and carefully pour off the supernatant.

[0238] (13) Wash the DNA with 5 mL of 70% ethanol, centrifuge at 15,000 g, 4°C for 10 min, and carefully pour off the supernatant.

[0239] (14) Allow to dry for 5-10 minutes and dissolve the DNA in an appropriate amount of Buffer TE.

[0240] (B) Plasmid transfection

[0241] The extracted ST3GAL4 plasmid DNA was transfected into PC-9 cells (human non-small cell lung cancer cells) using the Effectene Transfection Reagent (brand: QIAGEN) transfection kit. The operation steps were carried out according to the kit instructions. The specific steps are as follows:

[0242] (1) One day before transfection, 3x10 5 PC-9 cells were added to the culture medium and cultured overnight (37°C, 5% CO2)

[0243] (2) On the day of transfection, dilute 0.4 μg of plasmid DNA into TE Buffer to a total volume of 100 μL. Add 3.2 μL of enhancer and vortex for 1 second.

[0244] (3) Incubate at room temperature (15-25°C) for 2-5 minutes.

[0245] (4) Add 10 μL Effectence Transfection to the mixture and mix by pipetting up and down for 10 seconds.

[0246] (5) Incubate at room temperature for 5-10 minutes.

[0247] (6) Replace 1.6 mL of fresh culture medium in the 6-well plate.

[0248] (7) Add 600 μL of culture medium to the mixture, pipette up and down twice, and add dropwise to the cells in the 6-well plate. Rotate the 6-well plate to mix well.

[0249] (8) After 2 days of culture, the cells were collected.

[0250] 2. Cell Proliferation Assay

[0251] PC-9 cells overexpressing ST3GAL4 were treated with gefitinib and afatinib at different concentration gradients (10μM, 2μM, 0.4μM, 0.08μM, 0.016μM, 0.0032μM); PC-9 cells overexpressing ST3GAL4 were treated with erlotinib and icotinib at different concentration gradients (1000μM, 100μM, 10 μM, 1μM, 0.1μM, 0.01μM); PC-9 cells overexpressing ST3GAL4 were treated with dacomitinib at different concentration gradients (100μM, 10μM, 1μM, 0.1μM, 0.01μM, 0.001μM). Cell proliferation activity was detected by CCK-8, and the specific steps were as follows:

[0252] (1) Add 100 μL of a cell suspension of PC-9 cells overexpressing ST3GAL4 (3000 cells) to a 96-well plate and pre-culture in an incubator (37° C., 5% CO 2 ) for 24 h.

[0253] (2) The culture medium in the wells was discarded, and 200 μL of cell culture medium containing drugs at different concentrations was added thereto, and the cells were incubated in an incubator for 48 h (37° C., 5% CO 2 ).

[0254] (3) Add 10 μL of CCK-8 solution to each well and incubate in an incubator for 1 to 4 h.

[0255] (4) Measure the absorbance at 450 nm using an enzyme-labeled instrument.

[0256] The results are as follows Fig.12 As shown, the results suggest that after overexpression of ST3GAL4, PC-9 cells are resistant to gefitinib, erlotinib, icotinib, afatinib and dacomitinib, and the cell survival rate is significantly increased.

[0257] 3. Clone Formation Experiment

[0258] The clone formation assay was used to detect the cell proliferation ability of PC-9 cells before and after overexpression of ST3GAL4 under the action of gefitinib and afatinib.

[0259] (1) PC-9 cells and cells overexpressing ST3GAL4 were seeded at a density of 800 cells / well in a 6-well culture plate, treated with gefitinib or afatinib, and cultured for 2 weeks (37° C., 5% CO 2 ).

[0260] (2) Observe frequently and terminate the culture when clones visible to the naked eye appear in the culture dish.

[0261] (3) Wash the cells three times with PBS.

[0262] (4) Discard the supernatant, fix with 4% paraformaldehyde for 30 min, and stain with GIMSA staining solution for 10-30 min.

[0263] (5) Calculate the clone formation rate: clone formation rate = (number of clones / number of inoculated cells) × 100%.

[0264] (6) Statistical analysis of results.

[0265] The results are as follows Fig.13 As shown, the monoclonal formation ability of PC-9 cells was enhanced after overexpression of ST3GAL4.

[0266] 4. Ki67 experiment (cell immunofluorescence method)

[0267] Ki67 assay was used to detect the cell proliferation ability of PC-9 cells before and after overexpression of ST3GAL4 under the action of dacomitinib, erlotinib and icotinib.

[0268] (1) Plating: PC-9 cells and cells overexpressing ST3GAL4 were plated at 3×10 4 The cells were seeded at a density of 100 / well in 6-well culture plates, treated with dacomitinib, erlotinib and icotinib, and cultured for 2 days (37°C, 5% CO2).

[0269] (2) Fixation: Fix the treated sample slices with 4% paraformaldehyde at room temperature for 20 to 30 minutes.

[0270] (3) Washing: Wash 3 times with PBS, 5 minutes each time.

[0271] (4) Permeabilization: Permeabilize the membrane with 0.2% Triton X-100 at room temperature for 20 minutes.

[0272] (5) Washing: Wash 3 times with PBS, 5 minutes each time.

[0273] (6) Blocking: Add an appropriate amount of blocking solution (10% goat serum) and incubate at room temperature in a wet box for 30 to 60 minutes.

[0274] After blocking, gently wipe off the blocking solution around the sample.

[0275] (7) Add primary antibody: dilute Anti-ki67 Rabbit antibody at 1:100 in blocking buffer; add diluted Anti-ki67 Rabbit antibody to each sample and incubate in a humidified chamber at 4°C in the dark overnight.

[0276] (8) Washing: Wash with PBS three times, 5 minutes each time.

[0277] After washing, gently wipe off the blocking solution around the sample.

[0278] (9) Add fluorescently labeled secondary antibody: Add Cy3-conjugated Goat anti-Rabbit IgG (diluted 1:200) to each sample and incubate at room temperature in the dark for more than 2 h.

[0279] (10) Washing: Wash 3 times with PBS, 5 minutes each time.

[0280] (11) Counterstaining: Before taking pictures, counterstain with DAPI (diluted 1:500) for 15 minutes.

[0281] (12) Washing: Wash with PBS three times, 5 minutes each time.

[0282] (13) Detection and analysis were performed using a fluorescence inverted microscope.

[0283] The results are as follows Fig.14 As shown, the results suggest that after overexpression of ST3GAL4, PC-9 cells are resistant to dacomitinib, erlotinib and icotinib, and the cell survival rate is significantly increased.

[0284] 5. Cell invasion assay

[0285] The cell invasion assay was performed to detect the invasion ability of PC-9 cells overexpressing ST3GAL4 before and after treatment with gefitinib or afatinib, according to the following steps:

[0286] (1) PC-9 cells and cells overexpressing ST3GAL4 were seeded at a density of 800 cells / well in a 6-well culture plate, cultured overnight (37° C., 5% CO 2 ), and treated with gefitinib or afatinib for 2 days.

[0287] (2) A Transwell cell culture chamber with a PET membrane having a pore size of 8 μm was placed in a 24-well culture plate, and the bottom of the Transwell chamber was coated with 50 μL / well of Matrigel and incubated at 37°C overnight to form a gel.

[0288] (3) PC-9 cells and cells overexpressing ST3GAL4 before and after treatment with gefitinib or afatinib were digested with trypsin and collected, and cell suspensions were prepared in RPMI 1640 culture medium containing 1% fetal bovine serum. The cell density was adjusted to 2×10 5 100 μL / well was inoculated into the upper chamber, and 600 μL of culture medium containing 10% fetal bovine serum was added into the lower chamber.

[0289] (4) Incubate at 37°C for 48 h and remove the Transwell.

[0290] (5) Fix with 4% paraformaldehyde for 30 min and stain with crystal violet for 30 min.

[0291] (6) Carefully wipe off the cells on the top of the microporous filter membrane with a cotton swab, clean the cell chamber with running water, and let it dry naturally.

[0292] (7) Count the number of cells in the lower layer of the filter membrane under a light microscope.

[0293] The results are as follows Fig.15 As shown, the cell counting results showed that after PC-9 cells overexpressed ST3GAL4, although treated with gefitinib and afatinib, the number of invasive cells increased and the invasion ability of cells was enhanced, indicating that PC-9 cells overexpressing ST3GAL4 were resistant to gefitinib and afatinib.

[0294] 6. Scratch test

[0295] The experimental method of the scratch test is:

[0296] (1) 2.5×10 per hole 5 The cells were inoculated into 6-well plates and the supernatant was discarded after culturing for 12 h.

[0297] (2) The experiment includes a control group (normal cells) and experimental groups with different drug effects. The experimental groups are set up according to the needs of the experiment.

[0298] (3) After continuing to culture in the incubator for 48 h, vertical scratches were made with a 10 μL pipette tip, and the culture medium containing 5% fetal bovine serum was replaced. Pictures were taken under a microscope at 0 and 24 h.

[0299] The cell migration assay was used to detect the migration ability of PC-9 cells overexpressing ST3GAL4. The operation steps are as follows:

[0300] (1) PC-9 cells and cells overexpressing ST3GAL4 were seeded at a density of 800 cells / well in a 6-well culture plate, cultured overnight (37° C., 5% CO 2 ), and treated with gefitinib or afatinib for 2 days.

[0301] (2) 2.5×10 per hole 5 PC-9 cells and PC-9 cells overexpressing ST3GAL4 were inoculated into 6-well plates and the supernatant was discarded after culturing for 12 h.

[0302] (3) After continuing to culture in the incubator for 48 h, vertical scratches were made with a 10 μL pipette tip, and the culture medium containing 5% fetal bovine serum was replaced. Pictures were taken under a microscope at 0 and 24 h.

[0303] The results are as follows Fig.16 As shown, after PC-9 cells overexpressed ST3GAL4, although they were treated with gefitinib and afatinib, the number of invasive cells increased and the migration ability of cells was enhanced, indicating that PC-9 cells overexpressing ST3GAL4 were resistant to gefitinib and afatinib.

[0304] This example confirms through cell proliferation assay, clone formation assay, Ki67 assay, cell invasion assay, and cell migration assay that ST3GAL4 can be used as a marker for resistance to gefitinib, erlotinib, icotinib, afatinib, and dacomitinib. Sequence Listing <110> Chinese People's Liberation Army Special Medical Center <120> New application of sialyltransferase ST3GAL4 as a marker of EGFR-TKI resistance <160> 2 <170> PatentIn version 3.5 <210> 1 <211> twenty three <212> DNA <213> Artificial sequence <400> 1 gcctctaagc tctttggcaa cta 23 <210> 2 <211> 20 <212> DNA <213> Artificial sequence <400> 2 gatggaggag ctggtgatgg 20

Claims

1. Use of a reagent for detecting sialyltransferase ST3GAL4 in the preparation of a kit for assisting in the assessment of EGFR-TKI resistance in non-small cell lung cancer.

2. The use according to claim 1, characterized in that: The kit assists in evaluating the state of EGFR-TKI resistance based on the content or content change trend of ST3GAL4 in the sample.

3. The use according to claim 2, characterized in that: The kit is a protein expression detection kit.

4. The use according to any one of claims 1 to 3, characterized in that: The EGFR-TKI includes first, second and third generation EGFR-TKI drugs.

5. The use according to claim 4, characterized in that: The first-generation EGFR-TKI drugs include gefitinib, erlotinib, and icotinib, the second-generation EGFR-TKI drugs include afatinib and dacomitinib, and the third-generation EGFR-TKI drugs include osimertinib.

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