Application of Kir2.1 as a target in the preparation of reagents or drugs for the treatment, prevention or diagnosis of medulloblastoma

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to the use of Kir2.1 as a target in the preparation of reagents or drugs for treating, preventing or diagnosing medulloblastoma. The present invention has important clinical application value for the diagnosis, treatment and prevention of WNT / SHH medulloblastoma.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to the application of Kir2.1 as a target in the preparation of reagents or drugs for treating, preventing or diagnosing medulloblastoma. Background Art

[0002] Medulloblastoma (MB) is the most common embryonic malignant brain tumor of the central nervous system in children, accounting for about 25% of all childhood intracranial tumors. Currently, the treatment strategy for medulloblastoma is surgery combined with whole brain spinal cord radiotherapy and adjuvant chemotherapy after radiotherapy. Stratified treatment is performed according to risk factors. At present, the five-year recurrence-free survival rate of standard-risk medulloblastoma is about 70%-80%, and the five-year recurrence-free survival rate of high-risk medulloblastoma is about 60% (Sun Xiaofei, Zhen Zijun. Multidisciplinary diagnosis and treatment expert consensus on childhood medulloblastoma (CCCG-MB-2017) [J]. Chinese Journal of Pediatric Hematology and Oncology, 2018, 23(4)).

[0003] However, due to the side effects of radiotherapy, medulloblastoma survivors often suffer from decreased cognitive function, decreased intelligence, growth retardation, endocrine dysfunction, infertility, and the development of second tumors (Sun Xiaofei, Zhen Zijun. Multidisciplinary Expert Consensus on Diagnosis and Treatment of Childhood Medulloblastoma (CCCG-MB-2017) [J]. Chinese Journal of Pediatric Hematology and Oncology, 2018, 23(4)). Summary of the invention

[0004] In view of this, the object of the present invention is to provide the use of inward rectifier potassium ion channel 2.1 as a target in the preparation of an agent or drug for treating, preventing or diagnosing medulloblastoma.

[0005] To achieve the above object, the technical solution of the present invention is:

[0006] The invention discloses an application of inward rectifier potassium channel 2.1 (Kir2.1 for short) as a target in the preparation of reagents or drugs for treating, preventing or diagnosing medulloblastoma (MB for short).

[0007] Furthermore, the reagent or drug refers to a reagent or drug that regulates or detects the expression level of the inward rectifier potassium channel 2.1.

[0008] Furthermore, the drug is a drug that inhibits the activity of inward rectifier potassium ion channel 2.1.

[0009] Furthermore, the medulloblastoma refers to non-WNT / SHH medulloblastoma (ie, medulloblastoma Group 3 and Group 4 subtypes).

[0010] The present invention also aims to protect the use of inhibitors targeting inward rectifier potassium ion channel 2.1 in the preparation of drugs for treating or preventing medulloblastoma.

[0011] Furthermore, the medulloblastoma refers to non-WNT / SHH medulloblastoma.

[0012] The beneficial effects of the present invention are:

[0013] The present invention uses Kir2.1 as a target to prepare a reagent or drug for treating, preventing or diagnosing medulloblastoma, and has important clinical application value for the diagnosis, treatment and prevention of WNT / SHH medulloblastoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The test result diagram of Example 1, wherein a is a representative immunohistochemical image of Kir2.1 in medulloblastoma samples of different subtypes, b is a medulloblastoma sample whose immunohistochemical score shows that Kir2.1 expression in Group 3 / 4 is higher than that in WNT and SHH classification, c is an immunohistochemical image of Kir2.1 in cancer and paracancerous cells, d is a statistical graph of immunohistochemical images of Kir2.1 that is highly expressed in cancer tissues than in paracancerous tissues, e is a statistical graph of Kir2.1 high expression in non-WNT / SHH subtypes in the GEO database, f is a statistical graph of Kir2.1 expression in Group 3 / 4 that is higher than that in WNT and SHH subtypes, and g is a statistical graph of Kir2.1 in different subtypes. Relationship between expression level and patient prognosis, where the expression of Kir2.1 in Group3 / 4 is correlated with patient prognosis, G3 / 4 represents group 3 and group 4 medulloblastoma subgroups defined by clustering algorithm, SHH represents medulloblastoma subgroup characterized by hyperactivation of SHH pathway, WNT represents medulloblastoma subgroup characterized by hyperactivation of WNT pathway, β-catenin represents β-catenin, GAB1 represents growth factor receptor-associated binding protein 1, ns represents no significant difference, expression represents expression level, IDO / Area represents integrated optical density per unit area, Tumor represents tumor, Incisal margin represents resection margin (paired cancer and adjacent tissue), Normal represents adjacent normal tissue, MB represents medulloblastoma, GSE28245 represents a subset of medulloblastoma data in GEO database, with a total of 64 cases, GSE37418 represents a subset of medulloblastoma data in GEO database, with a total of 73 cases, percentage of Kir1.2 high Cases indicates the percentage of cases with high expression of Kir2.1, subgroup indicates the subtype, overall survival percent indicates the overall survival percentage, and days after surgery indicates the number of days after surgery, which is the same as the figure below;

[0015] Figure 2 The detection result diagram of Example 2, wherein a is the result of in vitro migration experiment of Kir2.1 knockdown and overexpression cells, b is the result of in vitro invasion experiment of Kir2.1 cells, c is the result of in vivo imaging and its statistical graph after in situ injection of Kir2.1 overexpression cells into mice, d is the result of in vivo imaging and its statistical graph after in situ injection of Kir2.1 knockdown cells into mice; e is HE staining of in situ tumor of mice, f is the statistical graph of survival of tumor-bearing mice, g is the effect of Kir2.1 knockdown and overexpression on the expression levels of Vimentin, E-cadeherin and Slug in medulloblastoma cells, mock is the control of the knockdown group, shKir2.1 is the knockdown Kir2.1, ctrl is the control of the overexpression group, ov-Kir2.1 is the overexpression Kir2.1, MB428 is the primary medulloblastoma established in this laboratory, MB913 is the primary medulloblastoma photo established in this laboratory Flu is fluorescence intensity, E-Cadherin is epithelial cadherin, Vimentin is vimentin, Slug is zinc finger transcription factor, and β-actin is β-actin;

[0016] Figure 3 is the result diagram of Example 3, wherein a is the first five proteins interacting with Kir2.1 screened out after the immunoprecipitation-mass spectrometry detection experiment, b is the interaction between Kir2.1 and 5 proteins verified by immunoprecipitation, c is the interaction between Kir2.1 and Notch2 in MB428 and MB913 overexpressing cells, Notch2 is the Notch signaling pathway, HMGB1 is the high mobility group protein, HDGF is the hepatocellular carcinoma derived growth factor, RALA is the Ras-related protein Ral-A, TBX2 is the T-box transcription factor, FLAG is a marker, IB shows the target protein, IP purifies the target protein, input is the positive control, IgG is the negative control, elution is the eluent;

[0017] Figure 4: The result diagram of Example 4, wherein a is the co-localization of Kir2.1 and N2ICD, b is the effect of knockdown and overexpression of Kir2.1 on the distribution of N2ECD detected by flow cytometry, c is the effect of knockdown / overexpression of Kir2.1 on the nuclear entry of N2ICD, d and e are the effects of Kir2.1 expression changes on the expression of Notch2 target protein, f is the expression of Notch2 target protein after silencing Notch2 in Kir2.1 overexpressing cells, g is the result of invasion experiment after silencing Notch2, wherein N2ICD is the intracellular region of Notch2, DAPI is 4',6-diamidino-2-phenylindole, which is a fluorescent dye that can strongly bind to DNA, Merge is a combination diagram, Counts is count, cytoplasm is cytoplasm, nuclear is nucleus, LaminB1 is a nuclear internal reference antibody, GAPDH is a membrane internal reference antibody, c-Myc is a Notch2 downstream target gene, and Hes1 is a Notch2 downstream target gene;

[0018] Figure 5 The figures are the results of Example 5; a is the effect of using S2 / S3 inhibitors on the expression of Notch2 in the membrane / nucleus, b is the flow cytometry detection of the effect of GI254023X on the expression of Notch2 on the cell membrane surface, c is the interaction between Kir2.1 and S2 / S3 cleavage enzymes, d is the effect of GI254023X on the invasion of Kir2.1-overexpressing cells, and e is a molecular mechanism model diagram of Kir2.1 promoting the invasion and metastasis of myeloid blasts, wherein GI254023X is an S2 cleavage inhibitor, RO4929579 is an S3 cleavage inhibitor, mNotch2-FL is the full length of Notch2, mN2ICD is the membrane part of Notch2, and nN2ICD is the nuclear part of Notch2.

[0019] Figure 6 The results of Example 6 are shown in Figures 1 and 2; a and b show the expression of Kir2.1 / Notch2 in non-WNT / SHH MB, and c shows the relationship between Kir2.1 / Notch2 expression and prognosis in non-WNT / SHH MB. high / nN2ICD high Isoforms and non-Kir2.1 high / nN2ICD high The relationship between subtypes and clinical prognosis. low For low expression of Kir2.1, nN2ICD low For low expression of nNotch2, Kir2.1 high For high expression of Kir2.1, nN2ICD highFor the highly expressed nNotch2.

[0020] Figure 7 These are the result graphs of Example 7, a is a live imaging graph of tumor-bearing mice showing that blocking the Notch2 signaling pathway inhibits the growth and metastasis of transplanted tumors formed by Kir2.1-overexpressing medulloblastoma cells, b is a statistical graph of a, c is a graph showing that GI254023X significantly inhibits the invasion and metastasis of orthotopic transplanted tumors, and d is a graph showing that GI254023X significantly prolongs the survival time of mice bearing transplanted tumors formed by MB428 cells overexpressing Kir2.1. DETAILED DESCRIPTION

[0021] The examples are provided to better illustrate the content of the present invention, but the content of the present invention is not limited to the examples. Therefore, those skilled in the art can make non-essential improvements and adjustments to the implementation scheme according to the above content of the invention, which still fall within the protection scope of the present invention.

[0022] Example 1

[0023] Through immunohistochemistry and data analysis, the results are as follows Figure 1 shown.

[0024] Immunohistochemical staining

[0025] 1. Sectioning: All paraffin specimens are uniformly numbered and placed in a -20℃ refrigerator for pre-freezing, followed by sectioning, spreading, and air-drying. The thickness of the sections is 3μm.

[0026] 2. Dewaxing: Place the paraffin sections on an iron rack, bake in a 60℃ oven for 45 minutes, and then quickly soak in xylene for 15 minutes. Then soak in another tank of xylene for 15 minutes. Then soak in 100% alcohol for 10 minutes, 95% alcohol for 5 minutes, 85% alcohol for 5 minutes, 75% alcohol for 5 minutes, tap water for 5 minutes, and PBS for 5 minutes.

[0027] 3. Antigen repair: Rinse with PBS 3 times, 5 minutes each time. Place the slices in a pressure cooker filled with repair solution, heat to 100°C, cover with lid, and apply high pressure for 2 minutes and 30 seconds. Then rinse the pressure cooker with tap water to cool it down, take out the rack and put it into the repair box filled with repair solution, and cool it naturally to room temperature.

[0028] 4. Inactivation of endogenous catalase: Take out the slices immersed in the repair solution, rinse with PBS three times, 5 minutes each time, add 3% H2O2, and act at 37℃ for 30 minutes.

[0029] 5. Blocking: Take out the inactivated slices and rinse with PBS 3 times, 5 minutes each time. Take out the slices and put them in a wet box. Add goat blocking serum on the slices and cover the wet box. Incubate in a 37℃ oven for 30 minutes.

[0030] 6. Primary antibody incubation: Gently shake off the blocking serum from the slices, add diluted antibodies to the tissue slices, cover with a wet box lid, and incubate in a 4°C refrigerator overnight.

[0031] 7. Secondary antibody incubation: Take out the wet box and place it in a 37℃ oven to reheat for 15 minutes. Place the slices in the repair box and rinse with PBS 3 times, 5 minutes each time. Then take out the slices and place them in a wet box. Add horseradish peroxidase (3,3'-diaminobenzidine, DAB) labeled secondary antibody to the slices and cover the wet box. Incubate in a 37℃ oven for 30 minutes.

[0032] 8. Color development: Take out the wet box, place the slices in the repair box and soak them in PBS for 3 times, 5 minutes each time. Then take out the slices and place them under a microscope, add DAB color development solution. When the color develops to a suitable degree, immediately put them in water to terminate the reaction.

[0033] 9. Restaining: Rinse with tap water three times, immerse the sections in hematoxylin solution for 20 seconds, then rinse thoroughly with tap water, immerse in hydrochloric acid alcohol differentiation solution for 1 second, take out, and soak in running tap water to turn blue.

[0034] 10. Dehydration, transparency and sealing: Soak the counterstained sections in 75% alcohol for 5 minutes, 85% alcohol for 5 minutes, 95% alcohol for 5 minutes, and 100% alcohol for 10 minutes. Then soak them in xylene for 15 minutes. Then soak them in another jar of xylene for 15 minutes. Take out the sections, put them in a fume hood to air dry, then add 50μL of neutral gum, seal them with a coverslip, remove bubbles in a 37℃ oven, and scan and save.

[0035] Immunohistochemical scoring

[0036] In order to reduce the interference factors of human interpretation, the immunohistochemical scoring method was performed using Image-pro plus 5.0 software.

[0037] The specific operations are as follows:

[0038] 1. Image collection

[0039] All images were collected under the same microscope, the same brightness, the same magnification, and the same software parameter settings. Five images were randomly collected from each sample slice under a clear field of view.

[0040] 2. Software Analysis

[0041] (1) Calibrate the image optical density: Click the Files button to open the image to be calculated. Under the Measure button, click Calibration. In the Intensity window, click New and select the std option density. Change the Incident level to 230 in Options and do not close the window.

[0042] (2) Color selection: Select Count / size in Measure, click Select colors, use HSI mode, set the H value to 0-30, the S value to 0-255, and the I value to 0-230, then click file-save to save the color selection settings. This color selection settings file will be loaded when calculating the score of each image in the future.

[0043] (3) Select measurement parameters: On the Count / size screen, click Measure, select select measurement, and then select the IOD and Area parameters.

[0044] (4) Measurement: Use the irregular tool to draw the measurement area and click the count button. Read the IOD SUM value of the measurement data in the view statistics window as the cumulative optical density value of this photo. Click covertAOI (s) to Obiect(s) under Edit again and click the count button again. Read the Area SUM value of the measurement data in the view statistics window as the area value of this photo.

[0045] (5) Calculation: The relative optical density of each photo is the value of IOD SUM divided by Area SUM. The average of the five relative optical densities is the immunohistochemical score of the specimen.

[0046] Use of GEO Database

[0047] The Gene Expression Omnibus (GEO) database is affiliated with the National Institutes of Health (NCBI) of the United States, which contains the largest and most comprehensive public gene expression data. We searched the GEO database to further verify the expression of Kir2.1 in myeloma. The specific operations are as follows:

[0048] 1. Searching for Datasets

[0049] Go to NCBI's GEO website (www.ncbi.nlm.nih.gov / geo / ), search for Medulloblastoma as the keyword, select GEO Dataset, set the species to Homo sapiens, and set the Attribute to Tissue. You will get a series of results, check them one by one, and find the dataset with Medulloblastoma.

[0050] 2. Download analysis data

[0051] (1) Download data: Go to the GES website and click the Series Matri Files file under Download family to download the original data file. Click Download on the GPL page under Platforms to download the platform information file of the dataset.

[0052] (2) Analyze data: Find the gene number corresponding to KCNJ2 in the platform information file. Find the data in the data file according to the number, which is the expression data of KCNJ2. At the same time, find the patient and his pathological data and copy them into a new Excel spreadsheet. Analyze the difference in KCNJ2 expression between samples in the new spreadsheet.

[0053] Depend on Figure 1 As shown in c and 1d, the expression of Kir2.1 in tumor tissues was significantly higher than that in adjacent adjacent tissues.

[0054] Depend on Figure 1 a, 1b, 1e and 1f show that the expression of Kir2.1 in non-WNT / SHH subtypes is significantly higher than that in WNT and SHH subtypes, and the expression level of Kir2.1 is significantly positively correlated with the molecular subtypes of tumors in 170 patients.

[0055] Depend on Figure 1 gIt can be seen that high expression of Kir2.1 is associated with poor prognosis of MB patients with non-WNT / SHH subtype.

[0056] Example 2

[0057] Invasion migration and Western Blot experiments were performed, and the results were as follows Figure 2 It is knowable.

[0058] Myeloid blast cell culture

[0059] The myeloid blasts used in this experiment were cultured in DMED medium containing 10% FBS, and the cell incubator was maintained at 5% CO2, 37°C, and 95% humidity. When the cell confluence reached 80%-90%, the cells were digested and subcultured with trypsin cell digestion solution. MB428 and MB913 cells were subcultured 1 to 3 times. Half of each cell was frozen at the second generation, and cells from the third to fifth generations were used for experiments.

[0060] Establishment of a medulloblastoma cell line with stable silencing of Kir2.1

[0061] 1. Kir2.1-shRNA interference lentivirus and nonsense sequence control lentivirus were provided by Santa Cruze.

[0062] 2. After rinsing and digesting the myeloid blasts in the logarithmic growth phase with PBS, resuspend the cells in 3 mL of RPMI 1640 medium containing 10% FBS. After counting the cells, 5×10 cells were added to each well. 4 Cells were seeded in 6-well plates and cultured overnight.

[0063] 3. Add the lentivirus to the cell culture wells and change the medium after culturing for 24 hours.

[0064] 4. Continue culturing, and after the cells have expanded to 2 dishes, use 4 μg / mL of puromycin to screen out drug-resistant cells.

[0065] 5. qRT-PCR and Western blot were used to identify the interference efficiency at the mRNA and protein levels respectively for subsequent experiments.

[0066] Establishment of a Myeloid Cell Line Stably Overexpressing Kir2.1

[0067] 1. The Kir2.1 overexpression lentivirus was constructed, sequenced, and the plasmid was drawn and packaged by Shanghai Genema Gene Co., Ltd.

[0068] 2. Take the myeloid blasts in the logarithmic growth phase, rinse and digest with PBS, resuspend the cells in 3mL of DMEM medium containing 10% FBS, count the cells, and add 5×10 cells per well. 4 Cells were seeded in 6-well plates and cultured overnight.

[0069] 3. Add the diluted lentivirus to the cell culture wells and change the medium after 24 hours of culture.

[0070] 4. Continue culturing, and after the cells have expanded to 3 dishes, use 10 μg / mL puromycin to screen out drug-resistant cells.

[0071] 5. qRT-PCR and Western blot were used to identify the overexpression efficiency at the mRNA and protein levels respectively.

[0072] In vitro Matrigel-Transwell invasion assay of medulloblasts

[0073] The Matrigel-Transwell model was used to detect changes in the invasion ability of cells after knockdown and overexpression of Kir2.1. The specific operations are as follows:

[0074] 1. Mix 40 μL of Matrigel and 80 μL of serum-free DMEM medium in a 1.5 mL EP tube and place on ice for later use.

[0075] 2. Apply 10 μL of the mixture evenly onto the 8 μm Transwell chamber membrane in a 24-well plate and incubate in a 37°C incubator until the liquid is completely dry.

[0076] 3. After rinsing with PBS, the cells were digested with trypsin until the cells became round. The cells were resuspended in 2 mL of DMEM medium containing 10% FBS to terminate the reaction and resuspended. The cells were transferred to a 15 mL centrifuge tube and centrifuged at 800 rpm for 5 min. The cells were washed with PBS for 3 times and the cells were counted.

[0077] 4. Take 1×10 5 Each myeloid blast was resuspended in 200 μL DMEM medium without FBS and added to the upper chamber of the Transwell chamber. The chamber was placed in a well plate, and 500 μL DMEM medium containing 10% FBS was added to the lower chamber. The cells were statically cultured in an incubator at 37°C and 5% CO2.

[0078] 5. Take out the Transwell chamber after 18h and 24h respectively, remove the culture medium, and rinse with PBS three times.

[0079] 6. After culturing the 24-well plate in a 37°C cell culture incubator for 24 hours, rinse the chamber twice with PBS and then fix it with 4% paraformaldehyde solution at room temperature for 20 minutes.

[0080] 7. Stain with crystal violet solution, gently wipe off the cells on the membrane with a cotton swab, wash and dry it, place it under a microscope, randomly select 5 fields of view to photograph and count the cells for statistical analysis.

[0081] In vitro migration ability test of myeloid blasts

[0082] 1. Cell plating: Digest the cells to be tested the day before and add 5×10 5 Cells were cultured overnight in an incubator.

[0083] 2. Observe the cells, and when they are fully grown, use a 10μl pipette tip to draw a smooth straight line vertically at the bottom of the well plate.

[0084] 3. Wash the cells twice with PBS, remove excess cells, and add serum-free DMEM medium.

[0085] 4. Place the cells in an incubator and continue culturing. Take them out after 24 hours and take photos to calculate the migration rate of each cell type.

[0086] Western blot experiments

[0087] 1. Sample Processing

[0088] (1) Remove the culture medium from the cell sample, rinse with pre-cooled PBS three times, wash directly with pre-cooled PBS three times, remove the PBS by centrifugation, and leave the cell pellet, which is placed on ice for later use.

[0089] (2) Add an appropriate amount of cell lysis buffer and mix thoroughly. Place on ice and pipette once every 10 minutes for 30 minutes. Centrifuge at 4°C, 14,000 × g for 15 minutes and aspirate the supernatant into a new EP centrifuge tube.

[0090] (3) The BCA protein concentration detection kit was used to determine the sample concentration. An appropriate amount of protein loading buffer was added to adjust all samples to the same concentration. The samples were placed at 100°C for 5 min to fully denature the protein. The samples were cooled to room temperature and centrifuged for later use.

[0091] 2. Electrophoresis

[0092] (1) Use a microsyringe to load the sample. The total protein loaded in each lane is 20 μg. After mixing the protein marker with 1× loading buffer, add an equal volume of sample to the corresponding well.

[0093] (2) Using bromophenol blue as an indicator, perform constant voltage electrophoresis. When the protein sample is in the upper gel, use 80 V. When the protein sample is compressed very finely and reaches the lower gel, adjust the voltage to 120 V. End the electrophoresis when bromophenol blue reaches the bottom of the gel.

[0094] 3. Wet transfer: Take a PVDF membrane of appropriate size, activate it with methanol and transfer it to the transfer buffer. Place them in the order of filter paper, membrane, gel, and filter paper, remove bubbles thoroughly, place all transfer preparations in ice water, and transfer at 200mA constant current for 2 h.

[0095] 4. Blocking: After electroporation, take out the membrane and place it in the prepared blocking solution and block it at room temperature for 2 hours.

[0096] 5. Primary antibody incubation: After blocking, carefully remove the PVDF membrane with tweezers, cut off the excess corners, place it in the prepared primary antibody, and incubate at 4°C overnight.

[0097] 6. Washing and secondary antibody incubation: On the second day, remove the PVDF membrane and wash it 3 times with PBST shaker, 15 minutes each time. Transfer the membrane to the secondary antibody liquid of the corresponding species, incubate it with PBST shaker for 2 hours, and then wash it 3 times with PBST, 15 minutes each time.

[0098] 7. Image acquisition: Use Thermo developer to develop.

[0099] Establishment of orthotopic medulloblastoma transplantation mouse model

[0100] 1. Experimental animals: Male NOD / SCID mice aged 4-6 weeks were purchased from Hunan SLAC Jingda and continued to be housed in the center after inoculation of transplanted tumors under standard pathogen-free conditions.

[0101] 2. Animal anesthesia: NOD / SCID mice were anesthetized by intraperitoneal injection of 0.8% chloral hydrate solution at 50 mg / kg body weight.

[0102] 3. The number of cells inoculated is 5×10 per NOD / SCID mouse. 4 The cells were resuspended in 10 μl serum-free DMEM basal medium; 10 cells in each group.

[0103] 4. Intracranial transplant tumor inoculation method: Select 2mm to the right of the anterior bregma and 0.5mm before the coronal suture as the needle insertion point, disinfect with 75% alcohol, and fully expose the needle insertion site. Use a sterilized 20μl injector for injection. The needle insertion depth is about 1mm-2mm and there is a sense of breakthrough. Slowly push the injection intermittently, and the injection time is 1min; after the injection is completed, the injector stays for 1min and then slowly withdraws; disinfect the needle entry with 75% ethanol solution.

[0104] 5. After the intracranial orthotopic transplanted tumor is inoculated, the growth of the animal is observed once a day until 3 months after the injection. When the animal is found dead, the brain tissue and spinal cord are promptly removed and fixed into sections for observation of tumor formation.

[0105] Hematoxylin-eosin staining (H&E) of the cerebellum and spinal cord of mice with transplanted tumors

[0106] 1. Dehydration, embedding and sectioning: Take out the cerebellum and spinal cord of the transplanted tumor mouse from formalin, mark them and place them in a dehydration box for dehydration. After overnight, take out the specimen and embed it in an embedding machine. Then slice the paraffin block with a thickness of 3μm, spread the slices in warm water, and dry them for storage.

[0107] 2. Dewaxing: Place the slices in a 60℃ oven for 30 minutes, then quickly soak them in xylene for 15 minutes. Then soak them in another jar of xylene for 15 minutes. Then soak them in 100% alcohol for 5 minutes, 95% alcohol for 5 minutes, 85% alcohol for 5 minutes, 75% alcohol for 5 minutes, tap water for 5 minutes, and PBS for 5 minutes.

[0108] 3. Hematoxylin staining: Immerse the sections in hematoxylin staining solution for 5 minutes, rinse with tap water 3 times, and then immerse in hydrochloric acid alcohol solution for 2 seconds. After taking out, rinse thoroughly with tap water, and then immerse the sections in tap water to return to blue for 10 minutes.

[0109] 4. Eosin staining: Immerse the sections in a gradient alcohol solution containing eosin dye, with concentrations ranging from 75%, 85%, 95% and 100% from low to high, and soak in each concentration of alcohol for 5 minutes.

[0110] 5. Transparentization and sealing: Soak the slices in xylene solution twice, 1 min each time, and then air-dry them in a fume hood. Add 50 μl of neutral gum to each slice to seal it.

[0111] 6. Observation: Place the slice on the stage of an optical microscope, observe the histological morphology of the specimen and take photos.

[0112] Depend on Figure 2 As shown in a and 2b, high expression of Kir2.1 can significantly enhance the invasive ability of cells, while knocking down Kir2.1 can reduce the invasive ability of cells. Figure 2 e It can be seen that after Kir2.1 overexpression, mice had larger tumors and more metastatic foci, and their survival time was also shorter; Figure 2 d shows that Kir2.1 overexpression leads to more obvious invasion margins and metastatic foci; Figure 2 f shows that Kir2.1 is involved in the EMT process.

[0113] Example 3

[0114] The results of immunoprecipitation experiments were shown in Figure 3 shown.

[0115] Co-immunoprecipitation

[0116] The immunoprecipitation experiment used the Co-IP kit from Thermo Company. The specific steps are as follows:

[0117] 1. Antibody cross-linking with resin

[0118] (1) Take the cross-linked resin and mix it gently. Pipette 80 μL into the chromatography column, centrifuge at 1000 × g for 1 min, and wash twice with Coupling buffer.

[0119] (2) Mix 10 μg of primary antibody or corresponding IgG, 10 μL of sodium cyanoborohydride and an appropriate volume of coupling buffer to make 200 μL of antibody cross-linking mixture. Seal the bottom of the chromatography column and add the mixture to the chromatography column. Rotate at 4°C for cross-linking for 2-4 hours:

[0120] 2. Cell Collection, Lysis and Pre-clearing

[0121] (1) When the confluence of myeloma cells overexpressing Kir2.1 reached 80%, the medium was removed, the cells were rinsed three times with pre-cooled PBS, and the cells were collected by scraping into a 15 mL centrifuge tube and centrifuged at 1000×g for 5 min. The cells were then transferred to a 1.5 mL EP tube and the excess PBS was removed after centrifugation.

[0122] (2) Add IP lysis buffer containing protease inhibitors to the collected cells, blow the cells thoroughly until the precipitate disappears, and place them on ice for 30 minutes to lyse, blowing every 10 minutes.

[0123] (3) Pipette 80 μL of the control cross-linked resin into the chromatography column, centrifuge at 1000 × g for 1 min, and wash twice with coupling buffer.

[0124] (4) The cell lysate was centrifuged at 16,000 × g for 15 min, and the supernatant was added to the pre-cleared centrifuge column and vortexed at 4°C for 1 h.

[0125] 3. Second cross-linking of antibodies

[0126] (1) Remove the plug at the bottom of the centrifuge column, centrifuge at 1000 × g for 1 min, wash once with coupling buffer, and wash twice with quenching buffer.

[0127] (2) 10 μL of sodium cyanoborohydride was mixed with 200 μL of quenching buffer, added to the centrifuge column, the bottom was sealed, and vortexed at 4°C for 15 min.

[0128] 4. Antibody-protein cross-linking

[0129] (1) Remove the plug at the bottom of the antibody cross-linking centrifuge column, centrifuge at 1000 × g for 1 min, wash twice with coupling buffer, wash six times with wash buffer, and wash twice with lysis buffer, and seal the bottom.

[0130] (2) Remove the plug at the bottom of the protein centrifuge column, centrifuge at 1000 × g for 1 min, collect the effluent, add it to the antibody-crosslinked centrifuge column, and vortex mix at 4°C overnight.

[0131] 5. Protein elution and denaturation

[0132] (1) Remove the plug at the bottom of the centrifuge column, centrifuge at 1000 × g for 1 min, wash 5 times with lysis buffer, 6 times with wash buffer, and 2 times with cell lysis buffer.

[0133] (2) Replace the collection tube, add 200 μL elution buffer, centrifuge at 100×g for 1 min, add 50 μL elution buffer, incubate for 5 min, and centrifuge at 1000×g for 1 min. Add 50 μL of the collection solution to the centrifuge tube and incubate for 5 min, and centrifuge at 1000×g for 1 min.

[0134] (3) Add 50 μL of 5× loading buffer, boil at 100°C for 5 min, immediately put on ice, centrifuge at 12,000 rpm for 2 min, and then perform Western blot experiment.

[0135] Mass spectrometry analysis

[0136] 1. Sample Preparation

[0137] The sample source for mass spectrometry analysis was immunoprecipitation experiment. The centrifuge tube was not eluted but directly washed with Washbuffer and centrifuged at 1000×g for 1 min. The upper and lower openings were sealed and stored on ice.

[0138] 2. Mass Spectrometry Analysis

[0139] The mass spectrometry of this experiment was mainly completed by the Biomedical Analysis and Testing Center of the Army Medical University. The prepared samples were transported on ice to the Biomedical Analysis and Testing Center and the relevant information forms were filled out.

[0140] Depend on Figure 3 It can be seen that Kir2.1 only interacts with Notch2 (such as Figure 3 b and 3c).

[0141] Example 4

[0142] Fluorescence confocal experiment was performed, and the results were as follows Figure 4 shown.

[0143] Cell immunofluorescence

[0144] 1. Take 1×10 5 The cells were seeded in transwell chambers and cultured overnight at 37°C, 5% CO2, rinsed 3 times with PBS, fixed with 4% paraformaldehyde at room temperature for 15 min, and rinsed 3 times with PBS, 5 min each time.

[0145] 2. Incubate with 0.3% Triton X-100 for 15 minutes to permeabilize the cells, and then rinse with PBS three times, 5 minutes each time.

[0146] 3. Block with 10% goat serum at 37°C for 30 min.

[0147] 4. Add the diluted Kir2.1 (1:200 dilution) and Notch2 (1:100 dilution) antibody mixture and incubate overnight at 4°C.

[0148] 5. Rinse with PBS three times, 5 minutes each time, add diluted 647-labeled (1:500) and 488-labeled (1:500) fluorescent secondary antibody mixture, and incubate at 37°C for 30 minutes.

[0149] 6. Rinse with PBS three times, 5 minutes each time, add DAPI to stain nuclei, and incubate at 37°C for 15 minutes.

[0150] 7. Rinse with PBS three times, 5 min each time, seal the slides with anti-fluorescence quenching sealing medium, and store in a light-proof humidified box at 4°C.

[0151] 8. Observe and collect images using laser confocal microscopy

[0152] Flow cytometry

[0153] 1. Digest the cells to be tested with trypsin, centrifuge at 500×g for 5 min, and remove the excess supernatant.

[0154] 2. Resuspend the precipitate with an appropriate amount of PBS without calcium or magnesium.

[0155] 3. Add Notch2-APC and IgG-APC antibodies to the cells to be tested, incubate on ice for 1 hour, and wash with calcium-free and magnesium-free PBS.

[0156] Wash once, centrifuge at 500 g for 5 minutes, and resuspend.

[0157] 4. After resuspension, use BD Aria II flow cytometry instrument for detection.

[0158] 5.1.5.4 Extraction of cytoplasmic and nuclear proteins

[0159] 1. After trypsin digestion of adherent cells, centrifuge at 500×g for 5 min to collect the cells.

[0160] 2. Wash the collected cells twice with PBS.

[0161] 3. Transfer 5×106 cells to a collection tube, centrifuge at 500×g for 2-3 minutes, and remove excess supernatant.

[0162] 4. Add cold CER I to the cell pellet, vigorously resuspend the cell pellet at the highest speed, and place on ice for 10 minutes

[0163] 5. Add pre-chilled CER II to the lysate.

[0164] 6. Vortex the tube vigorously for 5 seconds, place on ice for 1 minute, vortex vigorously at top speed for 5 seconds, and then centrifuge at 16,000 × g for 5 minutes.

[0165] 7. Immediately transfer the cytoplasmic proteins to a new centrifuge tube and set aside.

[0166] 8. Resuspend the cell pellet from the previous step with pre-chilled NER lysis buffer.

[0167] 9. Vortex the cell nuclei pellet vigorously for 15 seconds each time, then place the sample on ice for 10 minutes. Repeat this step four times to fully lyse the cell nuclei and centrifuge at 16,000×g for 10 minutes.

[0168] 10. Immediately transfer the supernatant to a new pre-cooled centrifuge tube, measure the concentration, and prepare for Western-blot.

[0169] Cell membrane protein extraction

[0170] 1. Use a cell scraper to scrape the cells off the surface of the plate and place 5×10 6 Resuspend the cells in growth medium and centrifuge the resulting cell suspension at 300 × g for 5 minutes.

[0171] 2. Wash the cell pellet with 3 mL of cell washing solution and centrifuge at 300×g for 5 minutes.

[0172] 3. Carefully remove and discard the supernatant. Resuspend the cells in 1.5 mL of cell wash buffer and transfer to a 2 mL centrifuge tube. Centrifuge at 300 × g for 5 minutes and discard the supernatant.

[0173] 4. Add 0.75 mL of permeabilization buffer to the cell pellet. Vortex briefly to obtain a homogenous cell suspension. Incubate at 4°C for 10 minutes with constant mixing.

[0174] 5. Centrifuge the permeabilized cells at 16,000 × g for 15 minutes. Carefully remove the supernatant containing cytoplasmic proteins and transfer to a new centrifuge tube.

[0175] 6. Add 0.5 mL of solubilization buffer to the pellet and resuspend by pipetting up and down. Incubate at 4°C for 30 minutes with constant mixing.

[0176] 7. Centrifuge the tube at 16,000 × g for 15 minutes at 4°C. Transfer the supernatant containing soluble membrane proteins and membrane-associated proteins to a new centrifuge tube.

[0177] 8. Proceed with downstream applications. The cytosolic and membrane fractions on ice can be used immediately or aliquoted and stored at -80°C for future use.

[0178] Depend on Figure 4 a As can be seen from the above, after Kir2.1 overexpression, Notch2 increased in the nucleus; Figure 4 b shows that after overexpression of Kir2.1, the amount of N2ECD on the cell membrane surface was significantly reduced compared with the control; while after knocking down Kir2.1, the amount of N2ECD on the cell membrane surface was significantly increased compared with the control; Figure 4 c shows that overexpression of Kir2.1 can significantly increase the amount of N2ICD in the cytoplasm and nucleus, while silencing Kir2.1 significantly reduces the amount of N2ICD in the cytoplasm and nucleus; Figure 4 d shows that knocking down Kir2.1 can significantly reduce the expression of Notch2 target genes, and overexpression of Kir2.1 or Kir2.1-M can significantly increase the expression of these proteins; Figure 4 e It can be seen that as the gradient of Kir2.1 increases, the expression of N2ICD also increases, and the expression of target proteins of the Notch2 signaling pathway also shows a gradient increasing trend; Figure 4 As shown in f and g, the expression of target proteins C-Myc and Hes1 was significantly reduced after overexpression of Kir2.1 and knockdown of Notch2. After silencing Notch2, the ability of Kir2.1 to promote cell invasion was significantly reduced.

[0179] Example 5

[0180] The overexpressed Kir2.1 cells were inhibited by using S2 enzyme cleavage inhibitor G1254023X and S3 enzyme cleavage inhibitor RO4929597, respectively. Figure 5 shown.

[0181] Western blot experiments

[0182] 1. Sample Lysis

[0183] (1) Remove the culture medium from the cell sample, rinse with pre-cooled PBS three times, wash directly with pre-cooled PBS three times, remove the PBS by centrifugation, and leave the cell pellet, which is placed on ice for later use.

[0184] (2) Add an appropriate amount of cell lysis buffer and mix thoroughly. Place on ice and pipette once every 10 minutes for 30 minutes. Centrifuge at 4°C, 14,000 × g for 15 minutes and aspirate the supernatant into a new EP centrifuge tube.

[0185] (3) The BCA protein concentration detection kit was used to determine the sample concentration. An appropriate amount of protein loading buffer was added to adjust all samples to the same concentration. The samples were placed at 100°C for 5 min to fully denature the protein. The samples were cooled to room temperature and centrifuged for later use.

[0186] 2. Electrophoresis detection

[0187] (1) Use a microsyringe to load the sample. The total protein loaded in each lane is 20 μg. After mixing the protein marker with 1× loading buffer, add an equal volume of sample to the corresponding well.

[0188] (2) Using bromophenol blue as an indicator, perform constant voltage electrophoresis. When the protein sample is in the upper gel, use 80 V. When the protein sample is compressed very finely and reaches the lower gel, adjust the voltage to 120 V. End the electrophoresis when bromophenol blue reaches the bottom of the gel.

[0189] 3. Wet transfer: Take a PVDF membrane of appropriate size, activate it with methanol and transfer it to the transfer buffer. Place them in the order of filter paper, membrane, gel, and filter paper, remove bubbles thoroughly, place all transfer preparations in ice water, and transfer at 200mA constant current for 2 h.

[0190] 4. Blocking: After electroporation, take out the membrane and place it in the prepared blocking solution and block it at room temperature for 2 hours.

[0191] 5. Primary antibody incubation: After blocking, carefully remove the PVDF membrane with tweezers, cut off the excess corners, place it in the prepared primary antibody, and incubate at 4°C overnight.

[0192] 6. Washing and secondary antibody incubation: On the second day, remove the PVDF membrane and wash it 3 times with PBST shaker, 15 minutes each time. Transfer the membrane to the secondary antibody liquid of the corresponding species, incubate it with PBST shaker for 2 hours, and then wash it 3 times with PBST, 15 minutes each time.

[0193] 7. Image acquisition: Use Thermo developer to develop.

[0194] Co-immunoprecipitation

[0195] The immunoprecipitation experiment used the Co-IP kit from Thermo Company. The specific steps are as follows:

[0196] 1. Antibody cross-linking with resin

[0197] (1) Take the cross-linked resin and mix it gently. Pipette 80 μL into the chromatography column, centrifuge at 1000 × g for 1 min, and wash twice with Coupling buffer.

[0198] (2) Mix 10 μg of primary antibody or corresponding IgG, 10 μL of sodium cyanoborohydride and an appropriate volume of coupling buffer to make 200 μL of antibody cross-linking mixture. Seal the bottom of the chromatography column and add the mixture to the chromatography column. Rotate at 4°C for cross-linking for 2-4 hours:

[0199] 2. Cell Collection, Lysis and Pre-clearing

[0200] (1) When the confluence of myeloma cells overexpressing Kir2.1 reached 80%, the medium was removed, the cells were rinsed three times with pre-cooled PBS, and the cells were collected by scraping into a 15 mL centrifuge tube and centrifuged at 1000×g for 5 min. The cells were then transferred to a 1.5 mL EP tube and the excess PBS was removed after centrifugation.

[0201] (2) Add IP lysis buffer containing protease inhibitors to the collected cells, blow the cells thoroughly until the precipitate disappears, and place them on ice for 30 minutes to lyse, blowing every 10 minutes.

[0202] (3) Pipette 80 μL of the control cross-linked resin into the chromatography column, centrifuge at 1000 × g for 1 min, and wash twice with coupling buffer.

[0203] (4) The cell lysate was centrifuged at 16,000 × g for 15 min, and the supernatant was added to the pre-cleared centrifuge column and vortexed at 4°C for 1 h.

[0204] 3. Second cross-linking of antibodies

[0205] (1) Remove the plug at the bottom of the centrifuge column, centrifuge at 1000 × g for 1 min, wash once with coupling buffer, and wash twice with quenching buffer.

[0206] (2) 10 μL of sodium cyanoborohydride was mixed with 200 μL of quenching buffer, added to the centrifugal column, the bottom was sealed, and vortexed at 4°C for 15 min.

[0207] 4. Antibody-protein cross-linking

[0208] (1) Remove the plug at the bottom of the antibody cross-linking centrifuge column, centrifuge at 1000 × g for 1 min, wash twice with coupling buffer, wash six times with wash buffer, and wash twice with lysis buffer, and seal the bottom.

[0209] (2) Remove the plug at the bottom of the protein centrifuge column, centrifuge at 1000 × g for 1 min, collect the effluent, add it to the antibody-crosslinked centrifuge column, and vortex mix at 4°C overnight.

[0210] 5. Protein elution and denaturation

[0211] (1) Remove the plug at the bottom of the centrifuge column, centrifuge at 1000 × g for 1 min, wash 5 times with lysis buffer, 6 times with wash buffer, and 2 times with cell lysis buffer.

[0212] (2) Replace the collection tube, add 200 μL elution buffer, centrifuge at 100×g for 1 min, add 50 μL elution buffer, incubate for 5 min, and centrifuge at 1000×g for 1 min. Add 50 μL of the collection solution to the centrifuge tube and incubate for 5 min, and centrifuge at 1000×g for 1 min.

[0213] (3) Add 50 μL of 5× loading buffer, boil at 100°C for 5 min, immediately put on ice, centrifuge at 12,000 rpm for 2 min, and then perform Western blot experiment.

[0214] Flow cytometry

[0215] 1. Digest the cells to be tested with trypsin, centrifuge at 500×g for 5 min, and remove the excess supernatant.

[0216] 2. Resuspend the precipitate with an appropriate amount of PBS without calcium or magnesium.

[0217] 3. Add Notch2-APC and IgG-APC antibodies to the cells to be tested, incubate on ice for 1 hour, and wash with calcium-free and magnesium-free PBS.

[0218] Wash once by centrifugation at 500 g for 5 minutes and resuspend.

[0219] 4. After resuspension, use BD Aria II flow cytometry instrument for detection.

[0220] Depend on Figure 5 a As can be seen from the figure, overexpression of Kir2.1 significantly reduced the expression of full-length Notch2 (mNotch2-FL) and N2ICD (mN2ICD) on the membrane, but increased the expression of N2ICD (nN2ICD) in the nucleus. Treatment of myeloid blasts overexpressing Kir2.1 with GI254023X significantly reduced the effects of Kir2.1, the expression of mNotch2-FL and mN2ICD increased significantly, and the expression of nN2ICD decreased significantly. Figure 5 c shows that Kir2.1 interacts with Adam10; Figure 5 b shows that after overexpression of Kir2.1, Notch2 on the cell membrane surface was significantly reduced. When the inhibitor GI254023X was used in cells overexpressing Kir2.1, this effect disappeared. The expression of Kir2.1 promoted the invasion of myeloid blasts. After treatment with the inhibitor GI254023X, this ability to promote cell invasion was significantly reduced. Figure 5 e It can be seen that Kir2.1 promotes the cleavage of Notch2 by interacting with Adam10, promoting its maturation and incorporation into the nucleus to activate this signaling pathway.

[0221] Example 6

[0222] Immunohistochemical staining of non-WNT / SHH MB tumors showed Figure 6 a; Survival analysis was performed, and the results Figure 6 c and 6d.

[0223] Immunohistochemical staining

[0224] 1. Sectioning: All paraffin specimens are uniformly numbered and placed in a -20℃ refrigerator for pre-freezing, followed by sectioning, spreading, and air-drying. The thickness of the sections is 3μm.

[0225] 2. Dewaxing: Place the paraffin sections on an iron rack, bake in a 60℃ oven for 45 minutes, and then quickly soak in xylene for 15 minutes. Then soak in another tank of xylene for 15 minutes. Then soak in 100% alcohol for 10 minutes, 95% alcohol for 5 minutes, 85% alcohol for 5 minutes, 75% alcohol for 5 minutes, tap water for 5 minutes, and PBS for 5 minutes.

[0226] 3. Antigen repair: Rinse with PBS 3 times, 5 minutes each time. Place the slices in a pressure cooker filled with repair solution, heat to 100°C, cover with lid, and apply high pressure for 2 minutes and 30 seconds. Then rinse the pressure cooker with tap water to cool it down, take out the rack and put it into the repair box filled with repair solution, and cool it naturally to room temperature.

[0227] 4. Inactivation of endogenous catalase: Take out the slices immersed in the repair solution, rinse with PBS three times, 5 minutes each time, add 3% H2O2, and incubate at 37℃ for 30 minutes.

[0228] 5. Blocking: Take out the inactivated slices and rinse with PBS 3 times, 5 minutes each time. Take out the slices and put them in a wet box. Add goat blocking serum on the slices and cover the wet box. Incubate in a 37℃ oven for 30 minutes.

[0229] 6. Primary antibody incubation: Gently shake off the blocking serum from the slices, add diluted antibodies to the tissue slices, cover with a wet box lid, and incubate in a 4°C refrigerator overnight.

[0230] 7. Secondary antibody incubation: Take out the wet box and place it in a 37℃ oven to reheat for 15 minutes. Place the slices in the repair box and rinse with PBS 3 times, 5 minutes each time. Then take out the slices and place them in a wet box. Add horseradish peroxidase (3,3'-diaminobenzidine, DAB) labeled secondary antibody to the slices and cover the wet box. Incubate in a 37℃ oven for 30 minutes.

[0231] 8. Color development: Take out the wet box, place the slices in the repair box and soak them in PBS for 3 times, 5 minutes each time. Then take out the slices and place them under a microscope, add DAB color development solution. When the color develops to a suitable degree, immediately put them in water to terminate the reaction.

[0232] 9. Restaining: Rinse with tap water three times, immerse the sections in hematoxylin solution for 20 seconds, then rinse thoroughly with tap water, immerse in hydrochloric acid alcohol differentiation solution for 1 second, take out, and soak in running tap water to turn blue.

[0233] 10. Dehydration, transparency and sealing: Soak the counterstained sections in 75% alcohol for 5 minutes, 85% alcohol for 5 minutes, 95% alcohol for 5 minutes, and 100% alcohol for 10 minutes. Then soak them in xylene for 15 minutes. Then soak them in another jar of xylene for 15 minutes. Take out the sections, put them in a fume hood to air dry, then add 50μL of neutral gum, seal them with a coverslip, remove bubbles in a 37℃ oven, and scan and save.

[0234] All data were analyzed using SPSS 20.0 statistical software, and the Kaplan-Meier method was used to estimate the relationship between Kir2.1 expression level and overall survival (OS) and progression-free survival (PFS) of patients with medulloblastoma. p < 0.05 was considered statistically significant.

[0235] Depend on Figure 6 aIt can be seen that the expression of Kir2.1 and nN2ICD is not completely consistent. Figure 6 As shown in c and 6d, patients with high expression of both Kir2.1 and nN2ICD had the shortest survival period.

[0236] Example 7

[0237] Kir2.1-overexpressing orthotopic tumor cells and control cells were transplanted into mice and treated with the S2 cleavage inhibitor G1254023X. Figure 7 a; further survival analysis was performed, and the results were as follows Figure 7 As shown in d.

[0238] Establishment of orthotopic medulloblastoma transplantation mouse model

[0239] 1. Experimental animals: Male NOD / SCID mice aged 4-6 weeks were purchased from Hunan SLAC Jingda and continued to be housed in the center after inoculation of transplanted tumors under standard pathogen-free conditions.

[0240] 2. Animal anesthesia: NOD / SCID mice were anesthetized by intraperitoneal injection of 0.8% chloral hydrate solution at 50 mg / kg body weight.

[0241] 3. The number of cells inoculated is 5×10 per NOD / SCID mouse. 4 The cells were resuspended in 10 μl serum-free DMEM basal medium; 10 cells in each group.

[0242] 4. Intracranial transplant tumor inoculation method: Select 2mm to the right of the anterior bregma and 0.5mm before the coronal suture as the needle insertion point, disinfect with 75% alcohol, and fully expose the needle insertion site. Use a sterilized 20μl injector for injection. The needle insertion depth is about 1mm-2mm and there is a sense of breakthrough. Slowly push the injection intermittently, and the injection time is 1min; after the injection is completed, the injector stays for 1min and then slowly withdraws; disinfect the needle entry with 75% ethanol solution.

[0243] 5. NOD / SCID mice were divided into the following groups: control group, control-GI254023X administration group, Kir2.1 overexpression group, and Kir2.1 overexpression group-GI254023X administration group. Administration began 7 days after transplantation of the tumor. The control-GI254023X administration group and the overexpression group-GI254023X administration group were administered GI254023X (GI254023X was dissolved in 0.1M Na2CO3 solution) at a dose of 100 mg / kg / day. The control group and the Kir2.1 overexpression group were given corresponding doses of placebo (0.1M Na2CO3 solution) for 5 consecutive days per week for two weeks.

[0244] 6. Perform in vivo imaging of small animals on the 7th, 14th and 21st days after tumor implantation. Each mouse was intraperitoneally injected with 200 μl of luciferase substrate, and photographed and observed using an IVIS in vivo imaging instrument after anesthesia with isoflurane.

[0245] 7. After NOD-SCID mice developed severe neurological symptoms, they were euthanized and their survival time was calculated.

[0246] Immunohistochemical staining

[0247] 1. Sectioning: All paraffin specimens are uniformly numbered and placed in a -20℃ refrigerator for pre-freezing, followed by sectioning, spreading, and air-drying. The thickness of the sections is 3μm.

[0248] 2. Dewaxing: Place the paraffin sections on an iron rack, bake in a 60℃ oven for 45 minutes, and then quickly soak in xylene for 15 minutes. Then soak in another tank of xylene for 15 minutes. Then soak in 100% alcohol for 10 minutes, 95% alcohol for 5 minutes, 85% alcohol for 5 minutes, 75% alcohol for 5 minutes, tap water for 5 minutes, and PBS for 5 minutes.

[0249] 3. Antigen repair: Rinse with PBS 3 times, 5 minutes each time. Place the slices in a pressure cooker filled with repair solution, heat to 100°C, cover with lid, and apply high pressure for 2 minutes and 30 seconds. Then rinse the pressure cooker with tap water to cool it down, take out the rack and put it into the repair box filled with repair solution, and cool it naturally to room temperature.

[0250] 4. Inactivation of endogenous catalase: Take out the slices immersed in the repair solution, rinse with PBS three times, 5 minutes each time, add 3% H2O2, and incubate at 37℃ for 30 minutes.

[0251] 5. Blocking: Take out the inactivated slices and rinse with PBS 3 times, 5 minutes each time. Take out the slices and put them in a wet box. Add goat blocking serum on the slices and cover the wet box. Incubate in a 37℃ oven for 30 minutes.

[0252] 6. Primary antibody incubation: Gently shake off the blocking serum from the slices, add diluted antibodies to the tissue slices, cover with a wet box lid, and incubate in a 4°C refrigerator overnight.

[0253] 7. Secondary antibody incubation: Take out the wet box and place it in a 37℃ oven to reheat for 15 minutes. Place the slices in the repair box and rinse with PBS 3 times, 5 minutes each time. Then take out the slices and place them in a wet box. Add horseradish peroxidase (3,3'-diaminobenzidine, DAB) labeled secondary antibody to the slices and cover the wet box. Incubate in a 37℃ oven for 30 minutes.

[0254] 8. Color development: Take out the wet box, place the slices in the repair box and soak them in PBS for 3 times, 5 minutes each time. Then take out the slices and place them under a microscope, add DAB color development solution. When the color develops to a suitable degree, immediately put them in water to terminate the reaction.

[0255] 9. Restaining: Rinse with tap water three times, immerse the sections in hematoxylin solution for 20 seconds, then rinse thoroughly with tap water, immerse in hydrochloric acid alcohol differentiation solution for 1 second, take out, and soak in running tap water to turn blue.

[0256] 10. Dehydration, transparency and sealing: Soak the counterstained sections in 75% alcohol for 5 minutes, 85% alcohol for 5 minutes, 95% alcohol for 5 minutes, and 100% alcohol for 10 minutes. Then soak them in xylene for 15 minutes. Then soak them in another jar of xylene for 15 minutes. Take out the sections, put them in a fume hood to air dry, then add 50μL of neutral gum, seal them with a coverslip, remove bubbles in a 37℃ oven, and scan and save.

[0257] All data were analyzed using SPSS 20.0 statistical software, and the Kaplan-Meier method was used to estimate the relationship between Kir2.1 expression level and overall survival (OS) and progression-free survival (PFS) of patients with medulloblastoma. p < 0.05 was considered statistically significant.

[0258] Depend on Figure 7 As shown in a and b, GI254023X treatment can significantly inhibit the growth of transplanted tumors, and the inhibitory effect on transplanted tumors formed by MB428 cells overexpressing Kir2.1 is significantly higher than that on transplanted tumors formed by cells in the control group. Figure 7 c It can be seen that GI254023X treatment can significantly inhibit the nuclear entry of nN2ICD. Figure 7 It can be seen that the survival time of tumor-transplanted mice can be significantly prolonged after being treated with GI254023X.

[0259] In summary, Kir2.1 / Notch2 is an independent prognostic factor for patients with medulloblastoma, and inhibition of the Notch2 signaling pathway can be used as a therapeutic target for patients with Kir2.1high / nN2ICDhigh medulloblastoma.

[0260] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. Targeting inward rectifier potassium channels Use of the inhibitor of 2.1 in the preparation of a drug for treating medulloblastoma, characterized in that: The inhibitor is GI254023X.

2. The use according to claim 1, characterized in that The medulloblastoma refers to non-WNT / SHH medulloblastoma.

Images