A product for preventing and / or treating glioma formation

Abstract

The present invention discloses the application of substances that inhibit the activity and / or expression level of CDYL in the preparation of products for preventing and treating glioma. Experiments have proven that at the cellular level, knockdown of CDYL can inhibit the proliferation ability, migration ability, and invasion ability of U87MG cells; at the animal level, knockdown of CDYL in U87MG cells significantly inhibits the growth of U87MG subcutaneous transplantation or orthotopic glioma; further, intratumoral injection of AAV-shCDYL to interfere with the expression of CDYL in U87MG xenografts significantly inhibits the growth of xenografts. The present invention provides a potential new target for the clinical treatment of glioma, and also provides new ideas for combination treatment regimens and immunotherapy methods in the clinical treatment of glioma. The present invention has potential clinical value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a product for preventing and / or treating glioma formation. Background Art

[0002] Glioma (also known as brain glioma) is the most common primary malignant central nervous system tumor. Glioma grows in an infiltrative manner, and patients have high recurrence and mortality rates. Traditional clinical treatment is mainly surgery, supplemented by radiotherapy and chemotherapy. In recent years, some new treatment methods have emerged, such as molecular therapy and immunotherapy, but they have not significantly prolonged the survival of patients, and there are large differences between individuals, and some even develop drug resistance. The main reason is that the pathogenesis of glioma is far from being elucidated. Therefore, finding key factors or nodes in the occurrence and development of tumors and providing new targets and new measures for clinical treatment has been the direction of this field. Recent studies have found that changes in epigenetic regulatory factors caused by acquired environmental factors are more common in the occurrence and development of gliomas, so it is of great significance to study the epigenetic regulatory mechanism of gliomas.

[0003] The epigenetic factor CDYL (Chromo-domain Y-like) (Gene ID 9425) is a key transcriptional repressor, first cloned by David C. Page's laboratory in 1999. Structurally, CDYL primarily comprises an N-terminal chromatin domain responsible for chromatin binding. This domain binds to the EZH2 region, the catalytic subunit of PRC2 (Polycomb repressive complex 2), and participates in PRC2-mediated trimethylation of histone H3K27 (H3K27me3). Binding to H3K27me3, CDYL forms heterochromatin, exerting transcriptional repression. Its C-terminus contains a coenzyme A domain that binds to C-terminal binding protein (CtBP) and histone deacetylase (HDAC) 1 / 2 to form a transcriptional repressor complex. Furthermore, Shang Yongfeng's research group reported that CDYL can convert crotonyl-CoA to β-hydroxybutyryl-CoA through enzymatic activity, negatively regulating histone crotonylation, further modulating the transcription of downstream target genes, and ultimately participating in the regulation of spermatogenesis. CDYL is not only abundantly expressed in the testis but also in other tissues such as the brain, lymph nodes, liver, and kidneys. In recent years, the function of CDYL in the central nervous system has received increasing attention. CDYL can inhibit the transcription of brain-derived neurotrophic factor (BDNF) by binding to the catalytic subunit of PRC2, EZH2 (Enhancer of zeste homolog 2), thereby limiting the complexity of neuronal dendrites and regulating dendritic development. Furthermore, CDYL can promote neuronal migration. CDYL-deficient mice exhibit reduced neuronal migration, increased cortical neuronal excitability, and increased susceptibility to epilepsy. The role of CDYL in the development and progression of gliomas and its underlying mechanisms remain unclear. Summary of the Invention

[0004] The purpose of the present invention is to prevent and / or treat glioma.

[0005] The present invention first provides for the use of a substance that inhibits the activity and / or expression of CDYL (Gene ID 9425) in the preparation of a product. The product may have at least one of the following functions: C1) preventing gliomas; C2) treating gliomas; C3) inhibiting glioma growth; C4) inhibiting glioma cell proliferation; C5) inhibiting glioma cell migration; or C6) reducing glioma cell invasiveness.

[0006] In the above application, the substance that inhibits CDYL activity and / or expression may be z1) or z2) or z3) or z4):

[0007] z1) nucleic acid molecule A; the nucleotide sequence of nucleic acid molecule A is shown in SEQ ID NO: 1;

[0008] z2) nucleic acid molecule B; the nucleotide sequence of nucleic acid molecule B is shown in SEQ ID NO: 2;

[0009] z3) shRNA synthesized by the shRNA expression system with the nucleic acid molecule A as the target;

[0010] z4) shRNA synthesized by the shRNA expression system with the nucleic acid molecule B as the target.

[0011] The shRNA expression system can be a lentiviral expression system or an adeno-associated virus expression system.

[0012] The z3) can specifically be the CDYL shRNA 2# mentioned in the example, that is, a recombinant lentivirus constructed by Shanghai Jima Pharmaceutical Technology Co., Ltd. with nucleic acid molecule A as the target and LV16 (U6 / Luciferase17&Puro) as the vector.

[0013] The z3) can specifically be the AAV-shCDYL mentioned in the example, that is, a recombinant adeno-associated virus constructed by Beijing Li Keli Biotechnology Co., Ltd. with nucleic acid molecule A as the target and pAAV-Luciferase-shRNA as the vector.

[0014] The z4) can specifically be the CDYL shRNA 3# mentioned in the example, that is, a recombinant lentivirus constructed by Shanghai Jima Pharmaceutical Technology Co., Ltd. with nucleic acid molecule B as the target and LV16 (U6 / Luciferase17&Puro) as the vector.

[0015] In any of the above applications, the glioma may be an astrocytoma.

[0016] The present invention also protects the use of CDYL as a drug target in the preparation of a product. The product may have at least one of the following functions: C1) to C6) : C1) preventing gliomas; C2) treating gliomas; C3) inhibiting glioma growth; C4) inhibiting glioma cell proliferation; C5) inhibiting glioma cell migration; C6) reducing glioma cell invasiveness.

[0017] In the above application, the CDYL as a drug target can be nucleic acid molecule A or nucleic acid molecule B;

[0018] The nucleotide sequence of the nucleic acid molecule A is shown in SEQ ID NO: 1;

[0019] The nucleotide sequence of the nucleic acid molecule B is shown in SEQ ID NO: 2.

[0020] In the above application, the glioma may be an astrocytoma.

[0021] The present invention also protects a product containing any of the above-mentioned substances that inhibit CDYL activity and / or expression; the functions of the product are at least one of the following C1) to C6): C1) preventing glioma; C2) treating glioma; C3) inhibiting the growth of glioma; C4) inhibiting the proliferation of glioma cells; C5) inhibiting the migration of glioma cells; C6) reducing the invasive ability of glioma cells.

[0022] The product may specifically be composed of any of the above-mentioned substances that inhibit CDYL activity and / or expression.

[0023] In the above product, the glioma may be an astrocytoma.

[0024] Any of the above-mentioned substances that inhibit CDYL activity and / or expression also falls within the scope of protection of the present invention.

[0025] Any of the above-mentioned gliomas may be subcutaneously transplanted or in situ gliomas.

[0026] In any of the above applications, the product may be a medicine.

[0027] Any of the above-mentioned glioma cells can be human glioma cell line U87MG or human glioma cell line U251.

[0028] Experiments have shown that CDYL expression in tumor tissue of glioma patients is higher than in adjacent brain tissue. At the cellular level, knocking down CDYL inhibits the proliferation, migration, and invasion of U87MG cells. At the animal level, knocking down CDYL in U87MG cells significantly inhibits the growth of subcutaneous U87MG xenografts. The effect of knocking down CDYL on glioma growth in animals is more pronounced than that in vitro. Furthermore, intratumoral injection of AAV-shCDYL interferes with CDYL expression in U87MG xenografts, significantly inhibiting their growth. This invention provides a potential new target for the clinical treatment of gliomas and offers new ideas for combined treatments and immunotherapy approaches in the clinical treatment of gliomas. This invention has potential clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1Figure 2 shows the correlation between CDYL expression and survival in patients with malignant glioma. A shows a Kaplan-Meier survival analysis of CDYL mRNA levels and patient survival based on glioma patient survival data from the TCGA database; P < 0.001; the blue curve indicates a CDYL FPKM value > 6; the red curve indicates a CDYL FPKM value ≤ 6. B shows a Kaplan-Meier survival analysis of CDYL mRNA levels and patient survival based on glioma patient survival data from the CGGA database; P < 0.001; the blue curve indicates a CDYL FPKM value > 8; the red curve indicates a CDYL FPKM value ≤ 8.

[0030] Figure 2 This indicates elevated CDYL content in tumor tissue from patients with astrocytoma. The scale bar at low magnification is 400 mm; the scale bar at high magnification is 50 mm. + represents a qualitative or semi-quantitative staining intensity result based on a comprehensive evaluation of cell color intensity and the number of positive cells. The average value of at least 5-10 randomly observed high-power fields (HPFs) was taken.

[0031] Figure 3 Western blot analysis of CDYL in the human astrocyte cell line NHA and human glioma cells U251 and U87MG. Representative Western blot images on the left, corresponding statistical graphs on the right. n = 4 independent experiments. * indicates P < 0.01, ** P < 0.001 compared with NHA, one-way ANOVA.

[0032] Figure 4 Knockdown of CDYL inhibits the proliferation, migration, and invasion of U87MG cells. A shows Western blot analysis of CDYL expression in U87MG cells in the control and CDYL knockdown groups. B shows CCK8 assay proliferation curves of U87MG cells in the control and CDYL knockdown groups after 24, 48, 72, 96, 120, and 144 hours of culture, respectively. 6 hours represents baseline cell count. Six replicate wells were used for the same treatment. This is a representative image from three independent experiments. ***P < 0.001, one-way ANOVA. C shows a schematic diagram of the cell migration assay. D shows cell migration of U87MG cells in the control and CDYL knockdown groups after 18 hours of culture, respectively. n = 3 independent experiments. *P < 0.05, **P < 0.01, one-way ANOVA. E shows a schematic diagram of the cell invasion assay. F shows the cell invasion of U87MG cells in the control and CDYL knockdown groups after 18 hours of culture; n = 3 independent experiments, ns, not significant, ***P < 0.01, one-way ANOVA. In the above statistical figures, * represents comparison with control shRNA.

[0033] Figure 5 Knockdown of CDYL significantly inhibited the growth of subcutaneous tumors in U87MG nude mice. A shows that equal numbers of U87MG cells from the control and CDYL knockdown groups were inoculated subcutaneously on the right posterior dorsal surface of nude mice. On days 2, 9, and 23 after inoculation, the total fluorescence intensity of the tumor site was observed using a small animal in vivo imaging system to represent tumor size. Day 2 represents the background cell count. This is a representative graph from two independent experiments. B shows a statistical graph of the fluorescence intensity of subcutaneous transplanted tumor tissue. Mice in which tumor cells were not successfully inoculated subcutaneously were not included in the final statistics. * indicates *P<0.05, **P<0.01 compared with Control shRNA, two-way analysis of variance. C shows that tumor tissue was sampled and photographed 35 days after subcutaneous inoculation of U87MG cells. Scale bar is 1 cm.

[0034] Figure 6 Intratumoral injection of AAV-shCDYL into U87MG subcutaneous tumors inhibits tumor growth. A shows the experimental timeline. B shows Western blot analysis of U87MG cells infected with AAV-shCDYL. C shows U87MG cells injected subcutaneously 14 days after formation of subcutaneous tumors. Tumor size was monitored weekly and samples were collected 28 days later. Scale bar, 1 cm. D shows tumor volume at 7 and 14 days after intratumoral injection of AAV at multiple sites. n = 7, *P < 0.05, two-way ANOVA, tumor volume = (longest diameter of tumor × shortest diameter of tumor²) / 2. E shows tumor weight. n = 7, * indicates comparison with control AAV, *P < 0.05, unpaired t-test.

[0035] Figure 7 U87MG cells in the control or CDYL knockdown groups were injected into the striatum of nude mice. The size of the orthotopic glioma was monitored at 4, 7, 14, and 21 days. A shows small animal MRI images of the intracranial tumors of one nude mouse from each of the control and experimental groups at different time points. The red arrows indicate the intracranial tumors. B shows the statistical analysis of orthotopic glioma size monitored by MRI. n = 6, ***P < 0.001, two-way ANOVA. C shows a representative image of an orthotopic glioma sample obtained and stained with HE staining at day 25 of growth. Scale bar, 800 μm. D shows immunohistochemical analysis of CDYL in orthotopic glioma tissue. The red arrows indicate CDYL expression in tumor cells. Scale bar, 700 μm at low magnification; 20 μm at high magnification.

[0036] Figure 8Orthotopic gliomas in U87MG nude mice were harvested and paraffin-embedded for immunohistochemical staining on day 25 of growth. A shows Ki67 expression in tumor tissue. The left panel shows a representative histochemical staining image, and the right panel shows a statistical analysis of eight random fields of view. Low-power scale bar: 700 μm; high-power scale bar: 20 μm; ***P < 0.001, unpaired t-test. B shows CD34 expression in tumor tissue. The left panel shows a histochemical staining image, and the right panel shows a statistical analysis of eight random fields of view from the two staining groups. Low-power scale bar: 50 μm; high-power scale bar: 20 μm; ***P < 0.001, unpaired t-test. In the above statistical figures, * indicates comparison with the control shRNA group. Red arrows indicate positively expressing cells. DETAILED DESCRIPTION

[0037] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0038] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0039] Example

[0040] 1. Test materials and methods

[0041] 1. Immunohistochemistry to detect the expression of CDYL molecules on brain astrocytoma and brain tissue combination chips

[0042] 1.1 Main Materials and Reagents

[0043] Brain astrocytoma and brain tissue combination array: Contains 62 tumor tissue samples from human astrocytoma patients of different grades and 10 adjacent brain tissue samples, provided by Xi'an Alina Biotechnology Co., Ltd.

[0044] Rabbit anti-CDYL primary antibody: Sigma, product code HPA035578.

[0045] Ready-to-use UltraSensitive TMS-P Ultrasensitive Kit (Mouse / Rabbit): Maixin Biotechnology Co., Ltd., product number KIT-9720. Kit components: Reagent A: Endogenous peroxidase blocker; Reagent B: Animal non-immune serum (goat); Reagent C: Biotinylated goat anti-mouse / rabbit IgG; Reagent D: Streptavidin-peroxidase.

[0046] DAB color development system: liquid DAB enzyme substrate color development kit, Maixin Biotechnology Co., Ltd., product number DAB-0031 / 1031.

[0047] 1.2 Tissue microarray immunohistochemistry

[0048] 1) Bake the slides at 60°C for 30 min, dewax and hydrate as per normal procedure;

[0049] 2) Antigen retrieval: Antigens were retrievaled with 0.01 mol / L citrate buffer (pH 6.0) at high pressure for 2 min, cooled to room temperature, and washed with phosphate buffer saline (PBS) for 5 min × 3 times;

[0050] 3) Block endogenous peroxidase with 3% H2O2 methanol at room temperature for 10 min, and then wash with PBS for 5 min × 3 times;

[0051] 4) Add normal non-immune animal serum dropwise and incubate at room temperature for 10 minutes;

[0052] 5) Remove serum and add primary antibody (1:300) at 4°C overnight;

[0053] 6) Wash with 0.1% Tween-20 PBS for 5 min × 3 times;

[0054] 7) Add biotin-labeled goat anti-rabbit IgG and incubate at room temperature for 10 minutes;

[0055] 8) Wash with 0.1% Tween-20 PBS for 5 min × 3 times;

[0056] 9) Add streptavidin-peroxidase dropwise and incubate at room temperature for 10 minutes;

[0057] 10) DAB color development for 5 min, followed by washing with distilled water to terminate color development;

[0058] 11) After hematoxylin counterstaining, water washing, and differentiation, wash thoroughly with water to return to blue;

[0059] 12) Conventional dehydration and transparency, neutral gum sealing;

[0060] 13) Full-length high-definition scanning for result analysis.

[0061] 1.3. Film Reading Standards

[0062] After locating the staining results point by point on the chip, cells were classified according to color intensity: no staining (negative (-), light brown staining (weakly positive (+), brown staining (positive (++), and tan staining (strongly positive (+++)). The number of positive cells was categorized as follows: (-) indicates less than 10% positive cells, (+) indicates 10-25% positive cells, (++) indicates 26%-49% positive cells, and (+++) indicates greater than 50% positive cells. Finally, a comprehensive evaluation of the two results was performed to determine the qualitative and semi-quantitative staining intensity results. At least 5-10 randomly observed high-power fields (HPF) were averaged.

[0063] Note: Invalid tissue or defects are indicated by * in the color intensity column.

[0064] 2. Preparation of Lentivirus (Control shRNA, CDYL shRNA 2#, or CDYL shRNA 3#) and Adeno-associated Virus (Control AAV and AAV-shCDYL)

[0065] (1) Preparation of Lentivirus (Control shRNA, CDYL shRNA 2#, or CDYL shRNA 3#)

[0066] Using nucleic acid molecule A: 5'-GGCTGTTCTACCGTTATGT-3' (SEQ ID NO: 1) as the target and LV16 (U6 / Luciferase17&Puro) as the vector, a recombinant lentivirus was constructed by Shanghai Jima Pharmaceutical Technology Co., Ltd. and named CDYLshRNA 2#.

[0067] Using nucleic acid molecule B: 5'-GGGATGGACTCCATGTTAA-3' (SEQ ID NO: 2) as the target and LV16 (U6 / Luciferase17&Puro) as the vector, a recombinant lentivirus was constructed by Shanghai Jima Pharmaceutical Technology Co., Ltd. and named CDYLshRNA 3#.

[0068] A recombinant lentivirus, named Control shRNA, was constructed by Shanghai Gene Pharmaceutical Technology Co., Ltd. using nucleic acid molecule C: 5'-TTCTCCGAACGTGTCACGT-3' (SEQ ID NO: 3) as the target and LV16 (U6 / Luciferase17 & Puro) as the vector. The Control shRNA is a universal control that does not knock down the expression of any known protein.

[0069] (2) Preparation of adeno-associated viruses (Control AAV and AAV-shCDYL)

[0070] Using nucleic acid molecule A: 5'-GGCTGTTCTACCGTTATGT-3' (SEQ ID NO: 1) as the target and pAAV-Luciferase-shRNA as the vector, a recombinant adeno-associated virus was constructed by Beijing Li Keli Biotechnology Co., Ltd. and named AAV-shCDYL.

[0071] A recombinant adeno-associated virus (AAV) was constructed by Beijing Li Keli Biotechnology Co., Ltd. using the nucleic acid molecule D: 5'-GATCCGCTCGCCTGTCTACTAACTAATTCAAGAGATTAGTTAGTAGACAGGCGAGCTTTTTTA-3' (SEQ ID NO: 4) as the target and pAAV-Luciferase-shRNA as the vector. The AAV was named Control AAV. Control AAV is a universal control that does not knock down the expression of any known protein.

[0072] 3. Cell line culture

[0073] 3.1 Main materials and reagents

[0074] Human glioma cell lines U87MG and U251 were provided by the National Laboratory Cell Resource Sharing Platform.

[0075] 0.25% trypsin (containing EDTA): Gibco, product code 25200056.

[0076] MEM culture medium: Hyclone Company, product code SH30265.01.

[0077] Fetal bovine serum (FBS): Hyclone Company, product code SH30084.03.

[0078] Non-essential amino acids (NEAA) solution: Gibco, product code 11140050.

[0079] U87MG complete culture medium: MEM medium + 10% FBS + 1% NEAA.

[0080] U251 complete medium: MEM medium + 10% FBS.

[0081] Quick Freezing: Beijing Biolong Immunotechnology Co., Ltd.

[0082] 3.2 Cell Recovery

[0083] Remove the cryovial containing cells from the liquid nitrogen tank and quickly place it in a 42°C water bath. Gently shake to thaw. Centrifuge at 900 rpm for 3 minutes and carefully discard the supernatant to obtain a cell pellet. Gently pipette and mix thoroughly with complete culture medium. Aspirate the cell suspension, transfer it to a culture dish, and culture in a cell incubator. Replace with fresh culture medium after 24 hours.

[0084] 3.3 Cell line culture and passaging

[0085] Cells were cultured in complete medium in a 37°C, 5% CO2 incubator. When cells reached approximately 80% confluency, they were digested with 0.05% trypsin (containing EDTA) for 2-3 minutes. The trypsin was removed by aspiration, and complete medium was added to terminate the digestion. The suspension was gently pipetted to form a cell suspension. Centrifuged at 900 rpm for 3 minutes, the supernatant discarded, and fresh medium added. The suspension was split into two plates at a ratio of 1:3-1:5. Cells were cultured in a 37°C, 5% CO2 incubator.

[0086] 3.4 Cell Cryopreservation

[0087] The cell suspension after trypsin digestion was centrifuged at 900 rpm for 3 min, the supernatant was discarded, and the cells were resuspended with Quick Freezing to adjust the cell density to approximately 1×10 6 Transfer the cell suspension to a cryovial, place the cryovial directly at -80°C, and transfer to liquid nitrogen for long-term storage after 48 hours.

[0088] 4. Western blot experiment

[0089] 4.1 Main Materials and Reagents

[0090] 30% acrylamide (100 ml): Weigh 29 g acrylamide (Novon) and 1 g N,N'-methylbisacrylamide, dissolve in 80 ml dd H2O, heat to 37°C to dissolve, dilute to 100 ml, filter through three layers of filter paper, and store in a refrigerator at 4°C away from light.

[0091] 10% ammonium persulfate (APS): Weigh 1 g of APS and dissolve it in 10 ml of dd H2O. Filter the solution after dissolution and store in a refrigerator at 4°C away from light.

[0092] 10% Sodium dodecyl sulfate (SDS): Weigh 10 g of SDS (Sigma) and dissolve it in 80 ml of dd H2O. After dissolution, dilute to 100 ml, filter, and store at room temperature.

[0093] Tris-HCl buffer solution:

[0094] 1 mol / L Tris (500 ml, pH 6.8): Dissolve 60.55 g Tris in 400 ml dd H2O, adjust the pH to 6.8 with hydrochloric acid, and make up to 500 ml.

[0095] 1.5 mol / L Tris (1000 ml, pH = 8.8): Dissolve 181.65 g Tris in 900 ml dd H2O, adjust the pH to 8.8 with hydrochloric acid, and make up to 1000 ml.

[0096] TEMED tetramethylethylenediamine: Genview Company, product code DH338-2.

[0097] Protease inhibitor EDTA-free Cocktail: Roche, product code 4693132001. Prepare 20× stock solution according to the instructions and store in a -20℃ refrigerator after aliquoting.

[0098] Tissue or cell lysis buffer: Enhanced RIPA lysis buffer, Beijing Pulilai Company, product code C1053+.

[0099] 5× Tris-glycine running buffer (1 L): Weigh 15.1 g of Tris, 50 ml of 10% SDS, and 94 g of glycine and dissolve them in 1 L of dd H2O.

[0100] 5×SDS-PAGE electrophoresis loading buffer: Beijing Pulilai Company, product code B1012.

[0101] 10× Transfer Buffer (1 L): Dissolve 52.13 g of Tris, 29.295 g of glycine, and 18.75 μl of 10% SDS in 1 L of dd H₂O. Dilute the mixture to 1× Transfer Buffer using a ratio of 10× Transfer Buffer: anhydrous methanol: dd H₂O of 1:2:7 before use.

[0102] 10×TBS (1 L): Weigh 160 g of NaCl, 4 g of KCl, and 60 g of Tris and dissolve them in 800 ml of dd H2O. After complete dissolution, adjust the pH to 7.4 and make up to 1000 ml.

[0103] 1×TBST: prepared in the ratio of Tween-20 (Dingguo): 10×TBS: dd H2O = 1:200:1800.

[0104] Blocking solution: Weigh 2.5 g skim milk powder and dissolve it in 1×TBST to 50 ml.

[0105] Prestained protein marker (Fermentas, USA, product code 26616): 170, 130, 100, 70, 55, 40, 35, 25, 15 and 10 kDa respectively.

[0106] The preparation of SDS-PAGE polyacrylamide separating gel and stacking gel is shown in Table 1.

[0107] Table 1

[0108] Separation gel (10%) 10ml Concentrated gel (5%) 5ml <![CDATA[ddH2O]]> 4.0ml 3.4ml 30% acrylamide 3.3ml 830 μl 1.5mol / LTris(pH=8.8) 2.5ml — 1mol / LTris(pH=6.8) — 630 μl 10% SDS 100 μl 50 μl 10% APS 100 μl 50 μl TMEMD 4 μl 5μl

[0109] 4.2 Extraction of total cell protein

[0110] 1) When cells reach a certain degree of confluence (generally 70-80% unless otherwise specified), discard the culture medium and wash twice with PBS;

[0111] 2) Add about 200 μl of RIPA lysis buffer, lyse on ice for 30 min, and then transfer to a 1.5 ml EP tube;

[0112] 3) Ultrasonic fragmentation of DNA, followed by placing on ice for 30 min to prevent DNA renaturation;

[0113] 4) Centrifuge at 4°C, 12,000 rpm for 20 min and aspirate the supernatant.

[0114] 4.3 Extraction of total tissue protein

[0115] 1) Rapidly isolate the animal's tumor tissue and store it at -80°C;

[0116] 2) Mince the tissue, add an appropriate amount of the above RIPA lysis buffer, grind with a tissue homogenizer (Pellet pestle motor, Rontes), add 350 μl of lysis buffer after thorough grinding, and suspend at 4°C for 1 hour;

[0117] 3) Centrifuge at 12,000 rpm for 20 min at 4°C and aspirate the supernatant.

[0118] 4.4 SDS-PAGE gel electrophoresis

[0119] 1) Gel Preparation: Prepare SDS-PAGE resolving gel and 5% stacking gel of appropriate concentration based on the molecular weight of the protein to be detected. After the stacking gel solidifies, carefully remove the comb, fix the gel glass on the electrophoresis apparatus, add running buffer, and remove any bubbles at the bottom of the gel.

[0120] 2) Loading: Take an equal amount of protein and add SDS-PAGE loading buffer (containing β-mercaptoethanol). Heat at 100°C for 5 minutes to denature the protein. Immediately place on ice to prevent renaturation. Load 50 μg of total protein per well. Add an appropriate volume of prestained protein molecular weight marker to the first well of the same gel and prepare for electrophoresis.

[0121] 3) Electrophoresis: At 70V, when the voltage reaches the junction of the separation gel and the stacking gel, adjust the voltage to 110V and continue electrophoresis until the dye reaches the bottom edge of the separation gel. Be careful not to let the target protein run out of the gel.

[0122] 4) Transfer:

[0123] 4-1) Cut three layers of filter paper and nitrocellulose filter membrane (NC membrane) to the same size as the gel;

[0124] 4-2) Soak the separation gel in transfer buffer for 15 minutes. After soaking the filter paper with transfer buffer, add filter paper, NC membrane, separation gel, and filter paper in the order from anode to cathode. Transfer the membrane at a constant current of 200 mA for 1.5-2 hours. The transfer time will be determined by the molecular weight of the target protein.

[0125] 5) Blocking: After transfer, cut the desired bands under the colorimetric guidance of Ponceau red, rinse in TBST for 1 minute, and then block with 5% skim milk at room temperature for 1.5 hours.

[0126] 6) Apply primary antibody: dilute the primary antibody to a certain ratio with 5% skim milk and incubate at 4°C overnight;

[0127] 7) Wash on a shaker with TBST three times, 10 min each time;

[0128] 8) Apply secondary antibody: dilute the secondary antibody to a certain ratio with 5% skim milk and incubate at room temperature for 70 minutes;

[0129] 9) Wash on a shaker with TBST three times, 10 min each time;

[0130] 10) Mix equal amounts of Solution A and Solution B in the ECL chemiluminescent solution kit, apply evenly to the membrane, place it on the chemiluminescent imaging system, and collect images.

[0131] 5. CCK8 cell proliferation assay

[0132] 1) Cells in the logarithmic growth phase were trypsinized, harvested, and resuspended in culture medium containing 10% FBS. The cell density was adjusted to 3000-5000 cells / 200 μl per well of a 96-well plate. Six replicate wells were treated in each plate (the specific number of plates was determined based on the experimental objectives and monitoring time points).

[0133] 2) After the cells have adhered for 4-6 hours, or after culturing for 24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours, or 144 hours, add 10 μl of CCK8 solution to each well and continue culturing in a 37°C incubator.

[0134] 3) After 1 hour, measure the absorbance at 450 nm using a microplate reader;

[0135] 4) Draw a cell growth curve with time as the horizontal axis and absorbance value as the vertical axis.

[0136] 6. Cell migration assay

[0137] 1) Digest the cells using conventional methods, obtain the cell pellet after centrifugation, and prepare a single-cell suspension in serum-free medium;

[0138] 2) Count the cells using an automatic cell counter and adjust the cell density to 1×10 5 Each group of cells was divided into two parts, one part was used as background for direct adherence culture, and the other part was used for cell migration.

[0139] 3) Using aseptic technique, carefully place a transwell chamber (Millipore, product code PI8P1250) in a 24-well plate using forceps. Add 500-600 μl of culture medium containing 10% FBS to the lower chamber. Avoid creating air bubbles between the chamber and the culture medium, as this will weaken the chemotaxis of the lower chamber to the cells in the upper chamber.

[0140] 4) Add 100 μl of cell suspension (containing 1×10 4 Cells were plated and 200 μl of serum-free medium was added. The cells were cultured at 37°C in 5% CO2 for 18 h. The cells on the surface of the polycarbonate membrane were gently wiped off with a wet cotton swab.

[0141] 5) Dilute D-luciferin sodium salt (Yisheng Bio, product code 40901ES02) in serum-free medium to prepare a luciferin sodium salt working solution with a final concentration of 150 μg / ml.

[0142] 6) Add fluorescein sodium salt working solution to the chamber with the wiped outer surface and incubate for 15-20 minutes.

[0143] 7) The chamber was placed on the small animal in vivo imaging system IVIS Spectrum (PerkinElmer) for imaging and image analysis.

[0144] 7. Cell invasion assay

[0145] 7.1 Preparatory steps

[0146] 1) Matrigel (BD Company, product code 356234) was melted in advance at 4°C;

[0147] 2) Dilute Matrigel with 4°C pre-cooled serum-free medium. The optimal ratio is 1:3. Perform the operation on ice.

[0148] 3) Following aseptic technique, vertically add 100 μl of diluted Matrigel to the center of the bottom of the upper chamber of the Transwell chamber and incubate at 37°C for 1-2 hours to allow the Matrigel to polymerize.

[0149] 4) Place the Matrigel-coated Transwell chamber at 37°C and use within 6 hours. Otherwise, the gel will shrink, resulting in poor results. It is best to prepare and use immediately.

[0150] 7.2 Experimental Procedure

[0151] 1) Digest the cells using conventional methods, obtain the cell pellet after centrifugation, and prepare a single-cell suspension in serum-free medium;

[0152] 2) Count the cells using an automatic cell counter and adjust the cell density to 5×10 5 Each group of cells was divided into two parts, one part was used as background to directly adhere to the wall culture, and the other part was used for cell invasion experiment.

[0153] 3) Remove the 24-well plate containing the Matrigel-coated Transwell chamber and add 500-600 μl of culture medium containing 10% FBS to the lower chamber. Be sure to avoid bubbles between the chamber and the culture medium, as the formation of bubbles will weaken the chemotaxis of the lower culture medium to the upper cells.

[0154] 4) Add 100 μl of cell suspension (containing 5 × 104 cells) to the upper chamber of the culture plate and add 200 μl of serum-free medium. Incubate at 37°C, 5% CO2 for 18 h. Gently wipe away any cells on the Matrigel gel and the polycarbonate membrane using a damp cotton swab.

[0155] 5) Dilute D-luciferin sodium salt with serum-free medium to prepare a luciferin sodium salt working solution with a final concentration of 150 μg / ml.

[0156] 6) Add fluorescein sodium salt working solution to the chamber with the wiped outer surface and incubate for 15-20 minutes.

[0157] 7) The chamber was placed on the IVIS Spectrum small animal in vivo imaging system for imaging and image analysis.

[0158] 8. Screening of U87MG cell lines with stable knockdown of CDYL

[0159] Lentivirus (Control shRNA and CDYL shRNA) carries the luciferase gene and puromycin resistance and is stored in aliquots at -80°C to avoid repeated freezing and thawing.

[0160] 1) One day before lentiviral transfection, adherent cells were seeded in 24-well plates. The cell confluency reached 50-60% at the time of transfection, so that the number of cells at the time of lentiviral transfection was 2×10 5 / hole around.

[0161] 2) Add 10 μg / ml of polybrene (polybrene is a membrane-breaking agent that destroys the cell membrane, allowing the virus to penetrate the damaged membrane and enter the cell to exert its effect). Set an appropriate virus concentration gradient. For a 24-well plate, set up five gradients, adding 0, 1, 2, 4, 6, and 8 μl of virus, respectively. Incubate in serum-free medium at 37°C.

[0162] 3) After 4-6 hours, replace the medium with fresh serum-containing culture medium and continue culturing. After 48 hours, use a small animal in vivo imaging system to observe the fluorescence intensity and select the optimal MOI value for lentiviral transfection.

[0163] 4) Adherent cells were seeded in 12-well plates and puromycin was added to the culture medium at different concentrations. The culture was performed for 7 days. The lowest concentration that killed all cells within 7 days was the optimal puromycin concentration.

[0164] 5) Using the optimal viral load determined from the screening, transfect the cells using the same transfection conditions as previously described. After 24 hours of culture, add the optimal puromycin concentration described above to the culture medium (the control group consisted of cells not transfected with the lentivirus). Culture for 7 days, replacing the culture medium with fresh media every 24 hours. When all cells in the control group undergo apoptosis, the remaining cells are screened as puromycin-resistant U87MG cells harboring either the control lentivirus or the CDYL knockdown lentivirus.

[0165] 6) Use Western blot to verify whether the screened cells can effectively knock down the expression of CDYL.

[0166] 9. Tumor formation model in immunodeficient animals

[0167] 9.1 Experimental Animals

[0168] Nu / nu nude mice, CB17 SCID mice, and NOD SCID mice were all female, 4-5 weeks old, with uniform body weight (no more than 2 g fluctuation), SPF-grade, purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., and housed in the barrier system of the Laboratory Animal Center of Peking University Health Science Center. All experimental animal procedures adhered to the Laboratory Animal Ethics Code and Operational Standards established by the Laboratory Animal Committee of Peking University Health Science Center.

[0169] 9.2. Establishment of a U87MG glioma model in Nu / nu nude mice by subcutaneous ectopic inoculation and tumor monitoring

[0170] 1) Cell preparation

[0171] Three stably transfected cells carrying the luciferase gene in the logarithmic growth phase, including the control group and the CDYL knockdown group, were gently digested with papain (Yisheng Bio, product code 40506ES60). The cell suspension was collected and centrifuged at 800 rpm for 3 min to obtain a cell pellet. The pellet was diluted with serum-free MEM and the cell density was adjusted to 5 × 10 6 / ml, based on the number of cells inoculated subcutaneously per animal, 1×10 6 / 200μl serum-free MEM, estimate the required cell amount, store on ice, and be sure to inject into the animal within 3 hours.

[0172] 2) Animal Preparation

[0173] Nu / nu nude mice, female, 4-5 weeks old, weighing 18-20 g, SPF grade, were randomly divided into three groups, with 3 mice in each group. Nude mice are hairless and do not require shaving before cell inoculation.

[0174] 3) Subcutaneous injection of cells

[0175] The animal was grasped skillfully and disinfected with a 75% alcohol cotton ball on the dorsal side near the right hind limb. The three prepared cell suspensions were extracted with a 1 ml microsyringe and 1×10 cells were injected subcutaneously into each nude mouse. 6 Inject 200 μl of U87MG cells and leave the needle in place for 2 minutes. Return the mice to their cages and observe the growth of the tumor weekly.

[0176] 4) In vivo analysis in mice

[0177] A 15 mg / ml D-luciferin sodium salt / DPBS solution was prepared and sterilized by filtration through a 0.2 μm filter. The solution was aliquoted and stored in the dark until ready for use. Thaw immediately before use. According to the monitoring schedule for the animal experiment, the luciferase substrate D-luciferin sodium salt solution was injected intraperitoneally (the injection volume was calculated at 10 μl / g per mouse, approximately 200 μl per mouse). Ten minutes after injection, imaging was performed using the IVIS Spectrum small animal in vivo imaging system, and images with a photon intensity scale were obtained. The glioma cells inoculated into immunodeficient mice in this experiment were all tumor cells carrying the luciferase gene. Therefore, the bioluminescent photon intensity monitored after intraperitoneal injection of the substrate D-luciferin sodium salt solution represents the number of cells within the subcutaneous tumor tissue and can be used to estimate tumor size.

[0178] 5) Intratumoral injection of AAV-shCDYL

[0179] Each animal was inoculated with 1×10 6 Nude mice were subcutaneously inoculated with U87MG cells (note that the cells inoculated here were not the previously established fluorescently labeled stably transfected cell line, but untreated U87MG cells). 14 days later, visible tumor tissue appeared at the inoculation site, and subcutaneous tumors were established. The mice were randomly divided into two groups, each with 7 mice, and received multi-site injections of control AAV and AAV-shCDYL, respectively, with 10 μl of AAV injected per mouse. Subcutaneous tumor size was monitored weekly with a vernier caliper. After 28 days, the mice were sacrificed, samples were collected, photographed, and weighed. Tumor volume was calculated using the formula: volume = (longest diameter of tumor × shortest diameter of tumor squared) / 2.

[0180] 9.3 Establishment of an Orthotopic U87MG Glioma Model in Nu / nu Nude Mice and Tumor Monitoring

[0181] 1) Cell preparation

[0182] Two stably transfected U87MG cells in the logarithmic growth phase were gently digested, the cell suspension was collected, and the cell pellet was obtained by centrifugation at 800 rpm for 3 min. The cell pellet was resuspended in serum-free MEM and injected intracranially with 5×10 5 Dissolve the required number of cells in 5 μl of serum-free MEM, estimate the required cell amount, store on ice, and inject into the animal within 8 hours.

[0183] 2) Animal Preparation

[0184] Female nu / nu nude mice, 4-5 weeks old and weighing uniformly (18-20 g), were randomly divided into two groups: a control group of 6 mice and a CDYL knockdown group of 6 mice. A 0.5% sodium pentobarbital / normal saline (w / v) solution was prepared for anesthesia during the experiment, with each mouse receiving an intraperitoneal injection of 105 μl per 10 g of body weight.

[0185] 3) In situ cell injection

[0186] The anesthetized nude mouse was placed on the operating table of a stereotaxic instrument. The scalp was disinfected with 75% alcohol. The scalp was cut along the midline to expose the anterior and posterior bregmas. The position of the left striatum was located using a stereotaxic instrument with the anterior bregma as the origin: AP 0.97 mm; ML -1.8 mm; DV -3.5 mm. After the unified parameters were located, 5×10 cells were injected using a microinjection pump micro4. 5 Each mouse was injected with a volume of 5 μl within 3 minutes. The needle was left in place for 3 minutes after the injection. After removing the microinjection pump, the scalp was sutured.

[0187] 4) Small animal MRI monitoring of intracranial tumor growth

[0188] Four days after the in situ inoculation of U87MG glioma cells into the brain of nude mice, the first round of small animal MRI was performed. Subsequently, imaging was performed using a small animal MRI system at 7 days, 14 days, and 21 days to observe the growth of the tumor in the brain of the nude mice. The specific operation is as follows: before MRI, the nude mice were anesthetized with isoflurane gas. After the mice were anesthetized, the nude mice were connected to the gas anesthesia ventilation tube and placed together in a 20mm coil for fixation. The parameters of the 3.0T Siemens small animal MRI were: 20mm coil; T2-weighted (TR 5960MS, TE 89ms); field of view (FOV) 6.4cm×3.9cm; matrix 256×100; number of layers 15; layer spacing 0.9mm; relaxation enhancement rapid acquisition technology was used to scan the sagittal and coronal planes of the nude mice. The method for calculating the volume of the intracranial tumor is: volume = sum of the areas of different layers × layer spacing.

[0189] 9.4. Establishment of U87MG Glioma Models in CB-17SCID and NOD SCID Mice by Subcutaneous Inoculation and Tumor Monitoring

[0190] 1) Cell preparation

[0191] The digestion of cells and the number of cells inoculated per mouse were consistent with the methods and parameters of the previous nude mouse subcutaneous tumor model. The difference was that only one type of U87MG cell (2#) with stable transfection and knockdown of CDYL was used as the experimental group in this experimental model.

[0192] 2) Animal Preparation

[0193] CB-17 SCID mice, female, 4-5 weeks old, weighing 16-18g, SPF-grade, were randomly divided into two groups of 6; NOD SCID mice, female, 4-5 weeks old, weighing 16-18g, SPF-grade, were randomly divided into two groups of 6. Both types of mice have fur and require shaving before cell inoculation.

[0194] 3) Subcutaneous cell inoculation and tumor measurement

[0195] The injection site and method were similar to those used in the previously described nude mouse subcutaneous tumor model. Starting from day 14 of cell inoculation, subcutaneous tumor size and growth were monitored twice weekly. Tumor volume was calculated using a vernier caliper using the same formula as previously described.

[0196] 9.5 Tumor Tissue Collection

[0197] I) Subcutaneous tumor tissue sampling

[0198] In accordance with the ethical and operational requirements of experimental animals, tissue sampling was performed when the maximum diameter of the subcutaneous tumor of the nude mouse was close to 2 cm. The animals were anesthetized with 0.5% sodium pentobarbital / normal saline (w / v), and each nude mouse was injected with 105 μl per 10 g of body weight via intraperitoneal injection. After all animals were under anesthesia, they were photographed. Before sampling, the animals were killed by dislocating the neck, and then the skin was disinfected. After cutting the skin with surgical scissors, the tumor tissue was removed, the tumor was photographed in sequence, weighed, and the tissue was quickly frozen in liquid nitrogen and stored at -80°C.

[0199] II) Brain tissue collection and paraffin section preparation for intracranial in situ glioma

[0200] 1) 25 days after intracranial in situ inoculation of U87MG cells, brain tumor tissue was collected;

[0201] 2) Mice were deeply anesthetized by injection of 1% sodium pentobarbital. Anesthesia was considered satisfactory if the mouse's limbs did not respond when clamped with hemostatic forceps. The mouse's heart was exposed with surgical scissors, the right pericardium was ruptured, and 30 ml of pre-chilled PBS was rapidly injected into the apex of the left ventricle to perform systemic perfusion.

[0202] 3) Continue pre-fixation with 4% paraformaldehyde (PFA) fixative;

[0203] 4) Immediately and carefully remove the brain tissue, transfer it to a 12-well plate containing 5 ml of fixative, and post-fix it in a 4°C refrigerator for 24 hours.

[0204] 5) Discard the fixative solution and replace with 20% and then 30% sucrose solution for dehydration. Incubate at 4°C for 24 hours in each solution, avoiding light.

[0205] 6) Remove the mouse brain, excise excess tissue in front and behind the tumor, trim it, and place the trimmed tissue in a clearly labeled dehydration box;

[0206] 7) Place the dehydration box into the hanging basket and dehydrate in the dehydrator according to the concentration gradient of alcohol;

[0207] 8) embedding the above tissues;

[0208] 9) Place the trimmed and embedded wax block on a paraffin microtome and slice into 3-4 μm slices;

[0209] 10) Remove the tissue using a glass slide and bake in a 60°C oven. Once the wax has melted, remove the tissue and store at room temperature until ready for use.

[0210] 10. Immunohistochemical staining of different immune cell infiltration

[0211] 10.1 Primary Antibody Preparation

[0212] CD11c antibody: Proteintech, product number 17342-1-AP.

[0213] CD20 antibody: OriGene, product number TA800385.

[0214] Ki67 antibody: Proteintech, product code 27309-1-AP.

[0215] F4 / 80 antibody: Proteintech, product code 28463-1-AP.

[0216] CD68 antibody: Proteintech, product code 28058-1-AP.

[0217] CD34 antibody: Abcam, product code ab81289.

[0218] TMEM119 antibody: Cell signaling, product code #90840.

[0219] CD206 antibody: Abcam, product code ab64693.

[0220] 10.2 Immunohistochemical Staining

[0221] 1) Dewaxing and hydration: Bake the tissue sections in a 60°C thermostat for 20 min, then soak in the following sequence: xylene (10 min) → xylene (10 min) → anhydrous ethanol (5 min) → 95% ethanol (5 min) → 75% ethanol (5 min) → deionized water (2 min) → deionized water (2 min);

[0222] 2) Wash with PBS three times, 5 min each time;

[0223] 3) 3% H2O2 (prepared with 80% methanol) was added dropwise to the tissue, allowed to stand at room temperature for 10 minutes, and then washed with PBS three times, each time for 5 minutes.

[0224] 4) Antigen retrieval (EDTA heat retrieval), cooling to room temperature;

[0225] 5) Wash with PBS three times, 5 min each time;

[0226] 6) Add normal goat serum blocking solution dropwise and incubate at room temperature for 20 minutes, then discard the excess liquid;

[0227] 7) Add 50 μl of primary antibody, incubate at room temperature for 1 hour, and then incubate at 4°C overnight;

[0228] 8) Incubate at 37°C for 45 min;

[0229] 9) Wash with PBS three times, 2 min each time;

[0230] 10) Add 50 μl of secondary antibody and incubate at 37°C for 1 h;

[0231] 11) Wash with PBS three times, 5 min each time;

[0232] 12) DAB color development for 1-5 minutes, and the degree of color development was determined under a microscope;

[0233] 13) Rinse with PBS or tap water for 10-15 minutes;

[0234] 14) Nuclear counterstaining was performed with hematoxylin for 2 minutes, and hydrochloric acid alcohol differentiation was used;

[0235] 15) Rinse with tap water for 10-15 minutes;

[0236] 16) Dehydration, transparency, and sealing;

[0237] 17) Observe under a microscope and take photos at 10x and 20x magnification respectively.

[0238] 11. Bioinformatics analysis of TCGA and CGGA

[0239] The cancer genome atlas (TCGA) database contains 153 glioma patient tumor tissue samples. The CDYL transcription levels and patient survival data were downloaded from the following website: https: / / www.proteinatlas.org / ENSG00000153046-CDYL / pathology

[0240] A research team led by Professor Jiang Tao of the Department of Neurosurgery at Tiantan Hospital has established the Chinese Glioma Genome Atlas (CGGA), significantly facilitating basic glioma research. Data from the CGGA database, accessed as mRNAseq_693, contains tumor tissue samples from 409 glioma patients. CDYL transcription levels and patient survival data can be downloaded from the following website: http: / / www.cgga.org.cn /

[0241] For the above data information, the Kaplan–Meier survival analysis method in Prism software was used to analyze the correlation between CDYL mRNA levels and patient survival.

[0242] 12. Data processing and statistical testing

[0243] All experimental results were statistically analyzed using Prism version 7.0 software and expressed as mean ± standard error (SEM). Unpaired t-tests were used to compare the means of two samples, one-way analysis of variance was used to compare the means of multiple groups, and two-way analysis of variance was used to compare the means of multiple groups at multiple time points.

[0244] All statistical analyses were performed with α = 0.05 as the standard for statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001 indicated statistically significant differences; ns, no significance, indicated no significant differences.

[0245] 2. Results and Analysis

[0246] 1. CDYL expression is upregulated in human glioma tumor tissues and cell lines

[0247] (1) The level of CDYL in glioma is correlated with patient survival

[0248] To understand the relationship between CDYL content in glioma tissue and patient prognosis, the inventors consulted the TCGA (Tumor Genetics and General Glioma Database) and the CGGA (Cognitive Glioma Database). Based on transcriptomic analysis and patient survival data from 153 glioma tissue samples from the TCGA database and 409 Chinese glioma patients from the CGGA database, they performed a survival analysis to correlate CDYL expression with patient survival.

[0249] The results of TCGA database showed that the higher the CDYL mRNA level in the tumor tissue of glioma patients, the longer the survival of the patients, P < 0.001, which was statistically significant (see Figure 1 The results of the CGGA database showed that the higher the CDYL mRNA level in the tumor tissue of glioma patients, the shorter the patient's survival time, P < 0.001, which was statistically significant (see Figure 1 These results suggest that CDYL may be involved in the occurrence and development of glioma, but its specific role remains to be studied.

[0250] (2) CDYL content in tumor tissue of glioma patients is elevated

[0251] Gliomas are classified according to their histological cytology into astrocytomas, medulloblastomas, oligodendrogliomas, ependymomas, and other mixed gliomas, with astrocytomas being the most common. Astrocytomas are divided into four grades, with grade I being the least malignant and grade IV being the most malignant. The most common type of grade IV astrocytoma is glioblastoma (GBM).

[0252] To observe the expression of CDYL in glioma tumor tissue, the inventors of the present invention purchased tumor tissue microarrays. Considering the complexity of glioma tissue cell types, only samples of glioma patients with astrocytoma type were selected, and the malignancy grade was based on the diagnostic criteria of the World Health Organization. CDYL immunohistochemical staining of tumor tissue microarrays was performed on a combination microarray containing adjacent brain tissue (n=10) and tumor tissue from patients with astrocytoma of different grades (n=62). The results were comprehensively evaluated based on the cell color intensity and the number of positive cells (see the methods section for details), with the scores from low to high being -, +, ++, and +++. The chi-square test was performed on the histochemical staining results of the above cases to observe whether CDYL expression was correlated with the malignancy of glioma. The statistical method was the Kruskal-Wallis test, and P<0.001 indicated statistical significance.

[0253] The results of immunohistochemical staining were shown in Figure 2 The results showed that the expression of CDYL in tumor tissue of glioma patients was higher than that in adjacent brain tissue.

[0254] The color intensity of the staining results was determined and analyzed using a chi-square test. The results are shown in Table 2. The results showed that CDYL expression was correlated with the malignancy of glioma.

[0255] Table 2. Statistical analysis of CDYL expression in adjacent brain tissues and tumor tissues of patients with different grades of astrocytoma

[0256]

[0257] (3) CDYL expression is elevated in human glioma cell lines

[0258] Western blot was used to detect the expression of CDYL in the human astrocyte cell line NHA and human glioma cell lines U251 and U87MG.

[0259] Test results are shown in Figure 3 The expression of CDYL in glioma cell lines was significantly higher than that in normal astrocytes, with statistical significance.

[0260] 2. Knockdown of CDYL inhibits the proliferation, migration and invasion of glioma U87MG cells

[0261] The development and progression of gliomas is a complex, multi-step process, primarily involving the proliferation, migration, and invasion of in situ tumor cells. The inventors discovered that CDYL is elevated in tumor tissues and cell lines from glioma patients. To further clarify the role of CDYL in glioma development and progression, they investigated the effects of CDYL on tumor cell proliferation, migration, and invasion at the cellular level.

[0262] At the cell line level, the inventors observed significantly elevated CDYL levels in both U251 and U87MG cells. Given that U87MG cells exhibit superior tumorigenicity to U251 in immunodeficient animals, and to facilitate systematic investigation of the role and mechanism of CDYL, subsequent studies used the glioma cell line U87MG. U87MG, a poorly differentiated, highly malignant glioblastoma cell line, is a common target for basic glioma research, as it is a type of astrocytoma.

[0263] The CCK8 cell proliferation assay is a very sensitive and convenient method for detecting tumor cell proliferation. In the experiment, three types of U87MG cells stably transfected with luciferase, Control shRNA (abbreviated as control group), CDYL shRNA 2# (abbreviated as 2#), and CDYL shRNA 3# (abbreviated as 3#), were established through lentiviral transfection and puromycin resistance screening, and Western blot verification was performed (see Figure 4The control group and the U87MG cell line stably transfected with CDYLshRNA were cultured for 6h, 24h, 48h, 72h, 96h, 120h and 144h respectively. The results showed that compared with the control group, the proliferation ability of 2#U87MG cells was weakened, while the proliferation ability of 3#U87MG cells did not change significantly ( Figure 4 (B) Transwell chambers can be used to detect the migration ability of tumor cells. Figure 4 Figure C is a schematic diagram. In the experiment, the control group and U87MG cells stably transfected with CDYL shRNA were cultured for 18 hours, and then the bioluminescence intensity was used to semi-quantitatively represent the number of migrating cells. The results showed that compared with the control group, the migration ability of U87MG cells stably transfected with CDYL shRNA was weakened ( Figure 4 The migration ability of 2#U87MG cells was reduced by 52.5%, P<0.01; the migration ability of 3#U87MG cells was reduced by 36.9%, P<0.05. The invasion ability of tumor cells can be detected using a Transwell chamber covered with a layer of matrigel. Figure 4 Figure E is a schematic diagram. Similarly, the control group and U87MG cells stably transfected with CDYL shRNA were cultured for 18 hours, and the number of invasive cells was semi-quantitatively represented by bioluminescence intensity. The results showed that compared with the control group, the invasive ability of 2#U87MG cells was weakened (reduced by 44.1%; P < 0.001), while the invasive ability of 3#U87MG cells did not change significantly ( Figure 4 Middle F).

[0264] These results show that stable transfection with a lentivirus containing sequence #2 inhibited the proliferation, migration, and invasion of U87MG cells. Stable transfection with a lentivirus containing sequence #3 had no significant inhibitory effect on the proliferation, migration, and invasion of U87MG cells. These results suggest that knockdown of CDYL can inhibit the proliferation, migration, and invasion of glioma cells to a certain extent. The effect of stable transfection with a lentivirus containing sequence #3 on U87MG cells was relatively weak, possibly due to the ineffective knockdown of CDYL by sequence #3. Therefore, further animal studies are needed to investigate the effects of CDYL on gliomas.

[0265] 3. Reducing the expression of CDYL can significantly inhibit the growth of U87MG subcutaneous transplanted tumors

[0266] (1) Reducing CDYL expression in U87MG cells significantly inhibited the growth of subcutaneous transplanted tumors in nude mice

[0267] Compared to the cellular level, whole-animal studies better simulate the environment in which gliomas develop and progress in clinical patients. To further clarify the impact of CDYL on glioma development and progression, the inventors conducted a whole-animal evaluation using a nude mouse subcutaneous xenograft model. Equal numbers of the three stably transfected U87MG cells (control group and CDYL knockdown group, cells 2# and 3#) obtained from the aforementioned screening were injected subcutaneously into nu / nu nude mice to form tumors. The total fluorescence intensity of the tumor sites was observed using a small animal in vivo imaging system. Tumor volume was semi-quantitatively estimated based on the total fluorescence intensity of the tumor tissue.

[0268] See the results Figure 5 The results showed that the subcutaneous xenograft volumes of both U87MG cell lines in the CDYL knockdown group were significantly smaller than those in the control group. Group 2 showed an 85.48% reduction (*P<0.05), while group 3 showed a 91.93% reduction (**P<0.01), both statistically significant. These results demonstrate that CDYL knockdown significantly inhibits the growth of U87MG xenografts in whole animals.

[0269] Combined with cell-based experiments, while CDYL knockdown had little effect on U87MG tumor cells in vitro, multiple animal studies in this study demonstrated that CDYL knockdown significantly inhibited the growth of U87MG xenografts. Considering that CDYL in glioma patients also influences the development and progression of gliomas at the global level, the results observed in whole animals are closer to the truth than those observed in vitro.

[0270] These results demonstrate that knocking down CDYL can inhibit glioma growth, suggesting that elevated CDYL levels in gliomas can promote glioma development and progression. In this experiment, lentiviral vectors were first used to disrupt CDYL expression in U87MG cells, establishing stable transfected U87MG cells. These cells were then inoculated subcutaneously into nude mice for tumorigenesis and tumor assessment. Therefore, this strictly constitutes preemptive intervention.

[0271] (2) Intratumoral injection of AAV-shCDYL inhibits tumor growth of U87MG subcutaneous transplanted tumors

[0272] To further clarify whether CDYL could serve as a target for clinical intervention, AAV (control AAV or AAV-shCDYL) was injected into tumor tissue to observe its effects. In the experiment, U87MG cells were first inoculated subcutaneously into nu / nu nude mice at equal cell numbers to establish tumors. Fourteen days later, AAV was injected at multiple sites, and the size of the subcutaneous xenografts in the nude mice was monitored weekly thereafter.

[0273] Test results are shown in Figure 6The results showed that reducing the CDYL content in U87MG subcutaneous transplanted tumors by infecting them with AAV-shCDYL significantly inhibited tumor growth. Compared with the control group (Control AAV), the tumor volume of the CDYL knockdown group (AAV-shCDYL) decreased by 66.74%, *P<0.05; the tumor weight decreased by 60.83%, *P<0.05; both were statistically significant. This shows that CDYL has certain target therapeutic significance and can be used as a potential intervention target for the clinical treatment of glioma patients.

[0274] 4. Knockdown of CDYL inhibits the growth, cell proliferation and microvascular formation of intracranial in situ gliomas

[0275] (1) Knockdown of CDYL inhibits the growth of intracranial U87MG orthotopic glioma in nude mice

[0276] To further clarify the impact of CDYL on the development and progression of gliomas, the inventors established an orthotopic U87MG glioma model in the brains of nu / nu nude mice and observed tumor growth. In this experiment, U87MG cells from both the control and CDYL knockdown groups were inoculated into the striatum (a region of the brain characterized by loose tissue and abundant blood vessels, making it a favorable location for transplanted cells to survive) within the nude mice's skulls. Small animal magnetic resonance imaging (MRI) was used to monitor intracranial tumor growth.

[0277] See the results Figure 7 The results showed that after knocking down CDYL, the growth of U87MG in situ glioma was significantly inhibited, and the tumor volume was reduced by 67.65%; ***P<0.001, which was statistically significant (see Figure 7 CDYL histochemical staining of tumor tissues was performed as a quality control. The staining results showed that CDYL expression in the orthotopic gliomas established by U87MG cells in the CDYL knockdown group was significantly lower than that in the control group, indicating that CDYL expression was indeed suppressed in the CDYL knockdown U87MG cells injected intracranially, and the observed difference was caused by the reduction of CDYL content (see Figure 7 Middle D).

[0278] To further confirm the results, tissue morphology staining was used to detect tumor-related molecular markers to more intuitively observe the differences within the tumor tissues of the two groups.

[0279] (2) Knockdown of CDYL inhibits tumor cell proliferation and microangiogenesis in glioma in situ

[0280] Tumor cell proliferation and angiogenesis can directly reflect the progression of the tumor. To further clarify the effect of CDYL on U87MG in situ glioma, immunohistochemical staining was performed on in situ glioma tissues, using Ki67 as a proxy for tumor cell proliferation and CD34 as a proxy for tumor tissue angiogenesis.

[0281] The staining results showed that after CDYL knockdown, the number of Ki67+ cells in tumor tissues was significantly reduced (reduced by 28.59%; ***P<0.001; Figure 8 Middle A), microvessel density was significantly decreased (reduced by 59.19%; ***P < 0.001; Figure 8 B) were statistically significant.

[0282] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims. <110> Beijing University <120> A product for preventing and / or treating glioma formation <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 19 <212> DNA <213> Artificial sequence <400> 1 ggctgttcta ccgttatgt 19 <210> 2 <211> 19 <212> DNA <213> Artificial sequence <400> 2 gggatggact ccatgttaa 19 <210> 3 <211> 19 <212> DNA <213> Artificial sequence <400> 3 ttctccgaac gtgtcacgt <210> 4 <211> 63 <212> DNA <213> Artificial sequence <400> 4 gatccgctcg cctgtctact aactaattca aggregate gcgagctttt tta 63

Claims

1. Use of a substance that inhibits CDYL activity and / or expression level in the preparation of a product; the functions of the product are at least one of the following C1) to C3): C1) Prevent glioma; C2) Treat glioma; C3) Inhibit the growth of glioma. The substance that inhibits CDYL activity and / or expression level is z1) or z2): z1) shRNA synthesized by an shRNA expression system targeting nucleic acid molecule A, and the nucleotide sequence of the nucleic acid molecule A is as shown in SEQ ID NO:1; z2) shRNA synthesized by an shRNA expression system targeting nucleic acid molecule B, and the nucleotide sequence of the nucleic acid molecule B is as shown in SEQ ID NO:

2.

2. The application according to claim 1, wherein: The shRNA expression system is a lentiviral expression system or an adeno-associated virus expression system.

3. The application according to claim 1 or 2, characterized in that: The glioma is astrocytoma.

4. A product containing the substance that inhibits CDYL activity and / or expression level described in claim 1 or 2; the functions of the product are at least one of the following C1) to C3): C1) Prevent glioma; C2) Treat glioma; C3) Inhibit the growth of glioma.

5. The product according to claim 4, characterized in that: The glioma is astrocytoma.

6. The substance that inhibits CDYL activity and / or expression level described in any one of claims 1 to 3.

7. The application according to claim 1 or the product according to claim 4, characterized in that: The glioma is in-situ glioma.

Images