Application of astrocyte YAP in preparation of anti-glioblastoma drug or prognosis evaluation kit

Abstract

The application discloses application of YAP of astrocytes in preparation of an anti-glioblastoma drug or a prognosis evaluation kit, and relates to the technical field of biological medicine.The YAP of the astrocytes is used for regulating DHRS3 to inhibit growth of glioblastoma, and the YAP of the astrocytes is targeted to the astrocytes in a tumor microenvironment instead of being targeted to the glioblastoma itself to inhibit the growth of the glioblastoma; meanwhile, the YAP of the astrocytes is applied to a kit for treating or evaluating prognosis of the glioblastoma; and a new idea is provided for preparation of the anti-glioblastoma drug and the kit.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of astrocyte YAP in the preparation of anti-glioblastoma drugs or prognostic assessment kits. Background Technology

[0002] Glioma is a primary brain tumor of the central nervous system, originating from the cells that make up the brain itself. It is the most common primary intracranial tumor, accounting for about half of all primary intracranial tumors. The World Health Organization classifies gliomas into four grades, with glioblastoma (GBM) belonging to grade IV, the highest grade form of glioma and one of the deadliest cancers. Current clinical treatments for GBM mainly include surgery, radiotherapy, and oral temozolomide chemotherapy. However, due to the high invasiveness of GBM cells, complete surgical resection is difficult, and they easily develop resistance to radiotherapy and chemotherapy, resulting in limited efficacy, high recurrence rates, poor prognosis, and a 5-year survival rate of less than 5%. Therefore, there is an unmet clinical need for treatment of GBM, and in-depth exploration of effective therapeutic targets and strategies is of great significance.

[0003] Traditional cancer treatments typically target tumor cells, but non-tumor cells in the tumor microenvironment also play a crucial role in regulating tumor progression. Unlike tumor cells, which undergo continuous mutations during tumor progression, non-tumor cells in the tumor microenvironment possess genetic stability. Therefore, these non-tumor cells have a lower risk of developing drug resistance and causing tumor recurrence, making them highly attractive targets for cancer therapy. Astrocytes are the most abundant cell type in the central nervous system, controlling multiple processes in both health and disease pathology, from maintaining brain metabolic homeostasis to regulating neuroinflammation. In the GBM tumor microenvironment, astrocytes are activated, exhibiting a reactive astrocyte state characterized by upregulated expression of glial fibrillary acidic protein (GFAP), and are closely involved in GBM progression.

[0004] Yes-associated protein (YAP) and its homologous transcriptional co-activator with a PDZ-binding motif (TAZ) are core downstream molecules of the Hippo signaling pathway, and their activity is negatively regulated by the Hippo signaling pathway through phosphorylation. In the central nervous system, YAP is highly expressed in astrocytes, and multiple studies have shown that astrocyte YAP plays various functions under physiological and pathological conditions. However, the role and mechanism of astrocyte YAP in the progression of GBM remain unclear.

[0005] This invention explores the role of YAP in astrocytes in GBM progression at the molecular and cellular levels. It discovers that YAP is highly expressed in GBM-associated astrocytes and inhibits GBM growth by regulating dehydrogenase / reductase (SDR family) member 3 (DHRS3) signaling. This provides new insights for the preparation of anti-GBM drugs, as well as for diagnosis and prognostic assessment. Summary of the Invention

[0006] The first objective of this invention is to propose the application of astrocyte YAP in the preparation of drugs to inhibit the progression of glioblastoma, wherein astrocyte YAP inhibits glioblastoma growth by upregulating DHRS3.

[0007] The second objective of this invention is to provide a method for inhibiting glioblastoma growth based on the above-mentioned application, specifically by administering an activator that activates the expression of tumor-associated astrocytes (YAP) to a subject. The activated YAP inhibits glioblastoma growth by upregulating DHRS3.

[0008] A third objective of this invention is to propose the application of astrocyte YAP in the preparation of kits for the diagnosis or prognostic assessment of glioblastoma.

[0009] A fourth objective of this invention is to provide the application of a reagent for detecting tumor-associated astrocytes (YAPs) in the preparation of a kit for the diagnosis or prognostic assessment of glioblastoma.

[0010] The kit for the diagnosis or prognostic assessment of glioblastoma includes reagents for detecting the expression level of YAP in tumor-associated astrocytes.

[0011] The reagents used in the kit to detect glioblastoma-associated astrocytes (YAPs) are selected from: probes with specificity for detecting YAPs and gene chips.

[0012] A fifth objective of the present invention is to provide a pharmaceutical composition comprising a glioblastoma-associated astrocyte YAP agonist.

[0013] The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0014] This invention inhibits glioblastoma growth by regulating DHRS3 through YAP in astrocytes. It does not target glioblastoma itself, but rather targets glioblastoma-associated astrocytes in the tumor microenvironment to inhibit glioblastoma growth. This provides a new biomarker for the early diagnosis and prognosis of glioblastoma and offers new ideas for the preparation of anti-glioblastoma drugs and reagents. Attached Figure Description

[0015] Figure 1 The following figures illustrate the results of YAP staining experiments on astrocytes in clinical human glioblastoma samples and mouse orthotopic glioblastoma samples, as shown in the examples: Figure A shows the expression level and cell localization of YAP in astrocytes of human glioblastoma samples after staining with GFAP & YAP antibodies to examine tumor tissue, peritumoral tissue, and healthy tissue far from the tumor; Figure B shows the expression level and cell localization of GFAP in A. + The proportion of YAP-nucleated cells in astrocytes; C in the figure represents GFAP in A. + Total YAP expression in astrocytes; Figure D shows the expression level and cellular localization of YAP in astrocytes of mouse glioblastoma samples using immunofluorescence staining with GFAP & YAP antibody co-staining, examining tumor tissue, peritumoral tissue, and healthy tissues far from the tumor; Figure E shows the expression level of GFAP in D. + The proportion of YAP-nucleated cells in astrocytes; F in the figure represents GFAP in D. + Total YAP expression in astrocytes; scale bar: 10 μm. **p<0.01;

[0016] Figure 2 Figure 1 shows the experimental results of detecting the effect of YAP knockout mice on the growth of glioblastoma in situ in the examples: Figure A is the in vivo experimental model: using stereotaxic brain technology, mouse-derived GBM cells GL261-Luciferase (GL261-Luc) labeled with luciferase were orthotopically transplanted into control mouse YAP cells. f / f YAP knockout mice with astrocytes GFAP- Establishment of a mouse syngeneic GBM model and phenotypic detection using the CKO striatum; Figure B shows the in vivo growth of GBM tumors observed by small animal imaging 3 weeks after orthotopic transplantation of GL261-Luc cells, via intraperitoneal injection of the substrate fluorescein into tumor-bearing mice; Figure C shows the statistical analysis of the bioimaging of B; Figure D shows YAP f / f With YAP GFAP - Typical HE staining image of brain sections from CKO tumor-bearing mice, scale bar: 1 mm; E in the image represents statistical analysis of tumor area at point D; F in the image represents YAP. f / f With YAP GFAP -Statistical analysis of survival rate of CKO tumor-bearing mice; *p<0.05, **p<0.01;

[0017] Figure 3 The following figure shows the results of detecting the effect of YAP (yellow astrocytes) on the proliferation and migration of glioblastoma in vitro, as illustrated in the example: Figure A shows the in vitro experimental model: cultured YAP. f / f With YAP GFAP Primary astrocytes from CKO mice were stimulated with glioblastoma-conditional medium (GBM-CM) to obtain tumor-astrocyte conditional medium (T-ACM). The effect of T-ACM on the in vitro proliferation and migration ability of GL261 cells was detected. Figure B shows the YAP (yellow-average astrocyte count) at 24 h as measured by the CCK8 assay. f / f and YAP GFAP - Effect of T-ACM on GL261 activity in the CKO group; C in the figure represents YAP detected by PH3 immunofluorescence assay. f / f and YAP GFAP - Effect of T-ACM on GL261 proliferation in the CKO group, scale bar: 25 μm; D in the figure represents the statistical effect of C; E in the figure represents the YAP detected by the scratch test. f / f and YAP GFAP - Effect of T-ACM on GL261 migration in the CKO group, scale bar: 50 μm; F in the figure represents the statistical effect of E; G in the figure represents the YAP detected by Transwell experiment. f / f and YAP GFAP - Effect of co-culture of CKO primary astrocytes and GL261 cells on the ability of GL261 cells to cross chambers; H in the figure represents the statistical effect of G, *p<0.05, **p<0.01;

[0018] Figure 4The following figures illustrate the verification results of detecting the binding of YAP and Dhrs3 in astrocytes and the regulation of glioblastoma growth by DHRS3 signaling in the examples: Figure A shows the direct binding of YAP and Dhrs3 in cultured primary astrocytes of C57BL / 6 mice detected by the CHIP experiment; Figure B shows the effect of YAP... f / f With YAP GFAP - Schematic diagram of qRT-PCR and Western blot analysis of GBM peritumoral samples from CKO tumor-bearing mice; C in the diagram represents qRT-PCR detection of YAP. f / f With YAP GFAP - Dhrs3 transcription level in peritumoral samples from CKO tumor-bearing mice (GBM); D in the figure represents YAP detected by Western blot. f / f With YAP GFAP - DHRS3 protein expression level in peritumoral samples of CKO tumor-bearing mice (GBM); E and F in the figure are statistical plots against D; G in the figure is the YAP level detected by immunofluorescence staining. f / f With YAP GFAP - DHRS3 expression in peritumoral astrocytes from CKO mouse Sham and GBM groups, scale bar: 25 μm; H in the figure represents statistical analysis against G, and I in the figure represents the detection of YAP in vitro by Western blot. f / f With YAP GFAP - Overexpression of DHRS3 in primary astrocytes of CKO mice, with DHRS3 and mCherry expressed as a fusion; J in the figure shows the immunofluorescence staining detection of YAP cultured in vitro. f / f With YAP GFAP - Overexpression of DHRS3 in primary astrocytes of CKO mice. mCherry-positive cells are transfected cells. Scale bar: 10 μm; K in the figure represents YAP detected by the CCK8 assay. GFAP - The effect of T-ACM overexpression of DHRS3 in primary CKO mouse astrocytes on reversing YAP knockout to promote GL261 cell proliferation; L in the figure represents the cell scratch assay for YAP. GFAP - The effect of T-ACM reversal of YAP knockout after DHRS3 overexpression in primary astrocytes of CKO mice on promoting GL261 cell migration ability. Scale bar: 200 μm. M in the figure represents the statistical analysis of L. *p<0.05, **p<0.01.

[0019] Figure 5Figure 1 shows the results of an experiment detecting the inhibition of glioblastoma growth by astrocyte YAP through DHRS3 signaling, as illustrated in the example. Figure A shows the expression of YAP in primary astrocytes activated by XMU-MP-1 detected by Western blot; Figure B shows the statistical analysis of A; Figure C shows the expression of YAP in primary astrocytes activated by XMU-MP-1 detected by immunofluorescence (scale bar: 25 μm); Figure D shows the experimental model verifying the inhibition of mouse GBM growth by astrocyte YAP activation; Figure E shows the results of an experiment detecting the effect of astrocyte YAP activation on GBM growth in vivo using small animal imaging; Figure F shows the statistical analysis of Figure E; Figure G shows the results of an experiment detecting the effect of astrocyte YAP activation on mouse GBM growth in vivo using HE staining (scale bar: 1). mm; H in the figure represents the statistical analysis of Figure G; Figure I shows the detection of whether YAP in activated astrocytes upregulates DHRS3 by co-staining with GFAP and YAP immunofluorescence, indicating that the inhibition of GBM growth by YAP activation in astrocytes is achieved by upregulating DHRS3, scale bar: 25 μm; J in the figure represents the statistical analysis of Figure I; K in the figure represents the survival curve analysis; *p<0.05, **p<0.01. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific examples. The following description of the embodiments is intended to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be easily made to these embodiments, and the general principles described herein can be applied to other embodiments without creative effort. Therefore, the present invention is not limited to the following embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

[0021] In this embodiment, in vivo in situ experiments and in vitro cell experiments using genetically modified mice clearly demonstrated that the YAP-DHRS3 axis of astrocytes regulates the progression of glioblastoma, and proved that YAP astrocytes can inhibit glioblastoma growth by regulating DHRS3.

[0022] The CDS coding region gene sequence of YAP in this embodiment is shown in SEQ ID NO.1 below:

[0023]

[0024] In this embodiment, the WT mice used were C57BL / 6 mice, and the genetically modified mice were genetically modified mice in the C57BL / 6 mouse background, including the control mouse YAP. f / f YAP knockout mice with astrocytes GFAP -CKO, all animals were housed in a specific pathogen-free (SPF) environment with strictly controlled indoor temperature (20-26℃) and humidity (40%-70%). The selected glioblastoma cell line was mouse-derived glioblastoma cell line GL261. Cells used for establishing the mouse orthotopic glioblastoma model were either GL261 cells or luciferase-labeled GL261 cells. Primary astrocytes were cultured mouse primary astrocytes, sourced from C57BL / 6 or YAP mice according to experimental requirements. f / f , or YAP GFAP -CKO mice.

[0025] The carbon dioxide (CO2) concentration in the cell culture incubator was 5%, the incubator temperature was 37°C, and the cell culture medium was DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin.

[0026] To investigate the correlation between astrocyte YAP and the development and progression of glioblastoma, GBM patient tissues were obtained from clinical sources, and an orthotopic GBM model was established in C57BL / 6 mice. The expression pattern changes of YAP in GBM-related astrocytes were detected and analyzed by immunofluorescence co-staining with astrocyte markers GFAP and YAP.

[0027] First, immunofluorescence staining was used to stain clinical human glioblastoma samples and mouse orthotopic glioblastoma samples to determine the nuclear localization of YAP, which was elevated and activated in glioblastoma-associated astrocytes; the details are as follows:

[0028] 1) Detection of YAP expression patterns in clinical GBM-related astrocytes: Clinically obtained human GBM samples were first fixed in 4% paraformaldehyde solution, then precipitated with 30% sucrose solution. After the tissue dehydrated and settled to the bottom, it was sectioned using a cryostat to a thickness of 14 μm. The sections were then dried and placed in a 90°C water bath with 2% sodium citrate antigen retrieval solution for 30 minutes. After cooling to room temperature, the sections were washed with PBS. The sections were then blocked and permeabilized for 1 hour at room temperature in PBS containing 0.3% Triton X100 and 5% BSA. Primary antibodies GFAP (1:500, MAB360, Sigma-Aldrich) and YAP (1:200, GTX129151, Genetex) were incubated overnight at 4°C. The next day, the sections were washed with PBS. A fluorescent secondary antibody was prepared using 5% BSA solution and incubated at room temperature for 1 hour, followed by washing with PBS. Finally, nuclear staining was performed using DAPI. Finally, the slides were mounted using anti-attenuation fluorescent mounting medium, photographed using a confocal fluorescence microscope, and the experimental results were observed. ImageJ was used to perform quantitative analysis of the fluorescence.

[0029] 2) Detection of YAP expression pattern in mouse GBM-related astrocytes: First, a mouse orthotopic GBM model was established: Mouse-derived GBM cells GL261 were cultured in DMEM medium supplemented with 10% FBS, Gibco, and 1% penicillin / streptomycin at 37˚C in an incubator containing 5% CO2. After reaching a suitable density, they were digested with trypsin for cell counting and diluted with DMEM to a density of 1x10⁻⁶. 5 Cells / μL. Six- to eight-week-old C57BL / 6 mice were anesthetized and placed in a stereotaxic apparatus. The mouse head was carefully fixed, and the skin above the skull was carefully scraped open with a razor. Using Bregma's point as the origin, the coordinates were determined as follows: anterior / posterior: +0.8 mm, medial / lateral: +1.6 mm. A hole was drilled at this location using a cranial drill. Then, 1 μL of the prepared cells was injected into the mouse brain tissue using a microinjection pump to a depth of +2.8 mm (dorsal / ventral). Three weeks later, the mice were perfused, and the brain was harvested, fixed, glycosylated, embedded, and sectioned using a cryostat. The sections were then subjected to GFAP and YAP immunofluorescence co-staining according to the previously described staining procedure and analyzed.

[0030] Experimental results are as follows Figure 1 As shown, the results indicate that in the GBM tumor microenvironment, the total expression level of YAP and the proportion of YAP nuclear localization (activated state) in GBM-related astrocytes are significantly increased, suggesting that astrocyte YAP may be involved in the regulation of GBM progression.

[0031] Then, in YAPf / f and YAP GFAP - An orthotopic GBM model was constructed in the brains of CKO mice. Small animal imaging, HE staining, and mouse survival analysis were used to examine the effect of YAP knockout of astrocytes on the in vivo growth of glioblastoma. Details are as follows:

[0032] 1): Preparation of YAP f / f and YAP GFAP -CKO mouse orthotopic syngeneic GBM model: The GBM mouse model was prepared in the same manner as described above, using YAP mice. f / f and YAP GFAP -CKO, injected with GL261-Luc cells, 1x10⁻¹ per mouse. 5 One cell per 1 μL. Three weeks after cell injection, mice were intraperitoneally injected with a fluorescein substrate, and tumor growth in vivo was monitored and statistically analyzed using a small animal imaging system (PHOTON IMAGERTMOPTIMA, Biospace lab, France). Survival curve analysis and HE staining analysis were subsequently performed on the mice.

[0033] 2) Hematoxylin-eosin (HE) staining: To further confirm the effect of YAP deficiency in astrocytes on the growth of GBM in mice in vivo, YAP was... f / f and YAP GFAP Frozen sections of brain tissue from CKO tumor-bearing mice were prepared. After washing, the sections were carefully immersed in hematoxylin staining solution for approximately 3-5 minutes. The sections were then rinsed continuously under tap water until all residual hematoxylin staining was completely removed from the brain tissue surface. Subsequently, the sections were rapidly immersed in eosin staining solution for 3-5 seconds. After staining, the sections were immediately immersed in tap water and rinsed repeatedly until all residual eosin staining was completely removed, resulting in a clear staining effect. The sections were then sequentially immersed in 75%, 95%, and 100% ethanol solutions, with each concentration used for 5 minutes. After ethanol gradient dehydration, the sections were immersed in xylene solution for clearing for 5 minutes. After removal, the sections were immersed again in fresh xylene solution for 5 minutes to further enhance the clearing effect. The sections were mounted using neutral resin. The sections were then air-dried in a well-ventilated area. Once completely dry, the sections were observed under a microscope, and the tumor size was statistically analyzed.

[0034] Experimental results are as follows Figure 2 As shown, the results indicate that YAP knockout of astrocytes significantly promotes in situ growth of GBM and significantly reduces the survival rate of tumor-bearing mice.

[0035] pass Figure 1 and Figure 2 It can be concluded that in the GBM tumor microenvironment, GBM-associated astrocytes show significantly increased YAP expression and exhibit activated nuclear localization signals, and astrocyte YAP knockout can promote GBM growth in vivo.

[0036] Subsequently, in vitro experiments were conducted for further verification: glioblastoma cell lines and YAP cells were cultured separately in a cell culture incubator. + / + and YAP - / - Primary astrocytes were used to obtain GBM-CM and T-ACM. The effects of T-ACM on the proliferation and migration of glioblastoma cells were detected by cell proliferation and migration assays.

[0037] 1) Obtaining and culturing primary mouse astrocytes: Primary astrocytes were obtained from newborn mice aged 0 to 3 days (P0-P3). The neocortex was carefully isolated and placed in a solution containing 0.25% trypsin. The cells were incubated at 37°C for 15 minutes, and then mechanically suspended into single-cell suspensions. These cells were then seeded into culture dishes coated with poly-L-lysine (0.1 mg / mL, Sigma-Aldrich) and cultured in DMEM medium containing 10% FBS. After 6 to 10 days of culture, microglia and oligodendrocytes were removed by shaking at 250 r / min for 4 to 6 hours.

[0038] 2) Obtaining GBM-CM and T-ACM: 2×10 6 GL261 cells were seeded in 10 cm culture dishes and cultured for 48 hours. After removing the original culture medium, 8 mL of DMEM medium containing 1% FBS was added. After 24 hours of culture, the supernatant was collected, centrifuged at 500 g for 10 minutes, and then filtered through a 0.22 μm microporous membrane to obtain GBM-CM for subsequent experiments. To obtain T-ACM, firstly, a total of 5 × 10⁶ cells were seeded in a 10 cm culture dish and cultured for 48 hours. 6 The primary astrocytes obtained by the above method were seeded into 10 cm culture dishes and cultured in DMEM medium containing 1% FBS for 12 hours. After removing the medium, 8 mL of GBM-CM was added. After culturing for 24 hours, the supernatant was collected, which is T-ACM. It was then centrifuged at 500 g for 10 minutes at 4 °C, aseptically filtered through a 0.22 μm filter, and stored at -80 °C.

[0039] 3) CCK8 cell viability assay: GL261 cells were seeded evenly at a density of 3000 cells per well into 96-well plates and cultured in a cell culture incubator. After 24 hours, the cells adhered fully and grew. The normal culture medium in the plates was then replaced with medium containing 2% FBS, and the cells were cultured for another 24 hours to starve the cells and eliminate the interference of various components in the serum on the subsequent experimental results. Subsequently, YAP... + / + and YAP - / - T-ACM cells were cultured for 12 and 24 hours, and cell viability was assessed using the CCK8 assay. The specific assay method was as follows: the original culture medium in the wells was carefully discarded, and 100 μL of a solution consisting of CCK8 reagent and DMEM culture medium at a 1:9 ratio was added to each well. The mixture was allowed to react for 2 hours. The absorbance of each well at 450 nm was measured using a microplate reader. Finally, based on the measured absorbance data, the relative cell viability of each well compared to the control group was calculated to assess the changes in cell viability under different conditions.

[0040] 4) Scratch healing assay: GL261 cells were evenly seeded in 12-well plates. When the cell healing rate reached approximately 90%, scratches were created using a 200 μL pipette tip. Subsequently, the cells were placed in YAP... + / + and YAP - / - The cells were cultured in T-ACM for 24 hours. The scratch sites were imaged using an optical microscope (CKX53, Olympus) at 0, 12, and 24 hours. Finally, the healing rate of the cell scratches was statistically analyzed using ImageJ software.

[0041] 5) Transwell assay: Add an appropriate amount of complete culture medium to the bottom of each well of a 24-well plate, then carefully place the Transwell chamber on top of the 24-well plate, ensuring that the bottom of the chamber is in full contact with the lower layer of complete culture medium. Select GL261 cells in the logarithmic growth phase for counting, dilute the cells with serum-free medium, and use 1 × 10⁶ cells per well. 4 Cells were seeded at a density of [number] cells per well in the upper layer of the Transwell chamber. Simultaneously, YAP was evenly seeded into the wells of a 24-well plate. + / + and YAP - / -Astrocytes. After cell adhesion, the chambers were inserted into their respective positions, and the entire cell culture plate was placed in a cell culture incubator for incubation. After 24 hours, the upper and lower layers of culture medium in the chambers were discarded, and the cells were washed with PBS. Then, an appropriate amount of 4% PFA was added to the chambers, and the cells were fixed for 20 to 30 minutes. The fixative was discarded, and the cells were washed again with PBS. Finally, 500 μL of 0.1% crystal violet solution was added to the lower layer of the chambers, and staining was performed for 20 minutes. The cell surface was then carefully rinsed with PBS to remove any residual crystal violet. The crystal violet in the upper membrane of the chambers was gently and carefully wiped with a cotton swab, avoiding damage to the cells and membrane structure. After wiping, the chambers were placed in a well-ventilated area to air dry. Once dry, the chambers were photographed, and the number of migrating cells was counted and statistically analyzed.

[0042] Experimental results are as follows Figure 3 As shown, the results indicate that the astrocyte YAP knockout group T-ACM significantly promoted the in vitro proliferation and migration of GBM cells, consistent with the aforementioned in vivo results.

[0043] Subsequently, the binding of YAP to Dhrs3 was verified by chromatin immunoprecipitation (CHIP) assay. Using in vivo mouse samples, qRT-PCR, Western blot, and immunofluorescence staining were used to verify that YAP regulates Dhrs3 expression at both the transcriptional and protein levels, and that overexpression of DHRS3 could reverse the glioblastoma cell growth-promoting phenotype induced by YAP knockout in astrocytes. Details are as follows:

[0044] 1) Chromatin Immunoprecipitation (CHIP) Assay: Primary cultured astrocytes from C57BL / 6 mice were cross-linked with 1% formaldehyde at 37°C for 10 minutes, followed by treatment with 0.125 M glycine at room temperature for 5 minutes to terminate the reaction. Cells were then washed twice with pre-chilled PBS containing benzyl sulfonyl fluoride (PMSF, ST506, Beyotime Biotechnology Co., Ltd., Shanghai, China), and collected using sodium dodecyl sulfate (SDS) lysis buffer from the ChIP assay kit (P2078, Beyotime Biotechnology Co., Ltd., Shanghai, China).

[0045] Samples were sonicated at 4°C using a cell sonicator with a power of 260 watts for 25 cycles, each cycle consisting of 30 seconds of sonication followed by a 60-second pause. Whole-cell lysates were pre-cleaned at 4°C for 30 minutes using Protein A+G agarose / salmon sperm DNA. After extracting 2% of the input sample, the remaining lysate was divided into two aliquots. One aliquot was incubated overnight with anti-YAP antibody (CST, catalog number 14074S, dilution 1:200), and the other aliquot was incubated with control IgG antibody (AC011, Wuhan Aibotek Biotechnology Co., Ltd., China). Protein A+G agarose / salmon sperm DNA was then added, and the mixture was incubated at 4°C for 2 hours.

[0046] The magnetic beads were washed sequentially with low-salt, high-salt, and lithium chloride immune complex wash buffers, followed by two washes with Tris-EDTA (TE) buffer, each with a rotation period of 5 minutes at 4°C. The DNA-protein complex was eluted with 1% SDS and 0.1 M sodium bicarbonate (NaHCO3) elution buffer. After adding 0.2 M sodium chloride (NaCl), the mixture was heated at 65°C for 4 hours to decrosslink. The protein was digested with proteinase K at 45°C for 1 hour, and then the DNA fragments were purified using a DNA purification kit (D0033, Beyotime Biotechnology Co., Ltd., Shanghai, China) for subsequent qRT-PCR.

[0047] 2) qRT-PCR: Take YAP f / f and YAP GFAP- Tissue from CKO-bearing mice containing GBM-related astrocytes around the tumor. 25 mg of brain tissue was added to 0.5 mL of RNA-easy solution, and the tissue was thoroughly disrupted using an ultrasonic homogenizer. 200 μL of enzyme-free water was added, and the mixture was inverted and incubated for 5 min at room temperature. The tissue was then centrifuged at 12000 g for 15 min at room temperature. The sample separated into two layers: an upper aqueous phase (containing RNA, approximately 640 μL) and a dark lower precipitate (containing proteins, DNA, polysaccharides, etc.). Approximately 500 μL of the upper aqueous phase was carefully transferred to a new RNAase-free centrifuge tube, and an equal volume of isopropanol was added. The mixture was gently shaken and incubated for 10 min at room temperature. Centrifugation at 12000 g for 10 min at room temperature was performed; a white precipitate was usually observed. The supernatant was carefully discarded. 0.5 mL of 75% ethanol was added to the precipitate, the bottom of the tube was gently tapped to resuspend the precipitate, and the tube was inverted several times. The precipitate was then centrifuged at 7500 g for 5 min at room temperature. After rinsing for 1 minute, discard the supernatant, being careful not to aspirate the precipitate; allow to air dry at room temperature, add 20 μL of RNase-free water, and dissolve the RNA completely at 55-60 ℃; measure the concentration using Nanodrop, and the RNA 260 / 280 ratio for quality control should be between 1.9 and 2.2; prepare 20 mL of 1% gel, heat at 90V for 15 minutes, load 5 μL of sample, and further test the sample quality.

[0048] The RNA obtained above was reverse transcribed: Using a reverse transcription kit, the following mixture was prepared in an enzyme-free EP tube: 1 μg RNA, 4 μL 4×gDNAwiperMix, and enzyme-free water to a volume of 16 μL. Genomic DNA was removed by incubating at 42°C for 2 min in a PCR instrument. The resulting RNA was then mixed with 4 μL 5×HiScriptIIIqRTSuperMix and incubated at 37°C for 5 min, followed by incubation at 85°C for 5 sec. Then, the cDNA obtained above was subjected to qRT-PCR to detect the Dhrs3 transcriptional expression level. The following mixture was added to the PCR tube: 5 μL qPCR SYBR Green Master Mix, 1 μL Dhrs3-F, 1 μL Dhrs3-R, 2 μL double-distilled water, and 1 μL cDNA template. The mixture was pipetted and mixed; pre-denatured at 95°C for 5 min, followed by cycling at 95°C for 10 sec and 60°C for 30 sec. The results were calculated and statistically analyzed.

[0049] The Dhrs3-F sequence is SEQ ID NO.2: CAATCACTCCCCAGCCGTG;

[0050] The Dhrs3-R sequence is SEQ ID NO.3:GTGATGAGGACTGACTCCCG.

[0051] 3) Western blot: Take YAP f / f and YAP GFAP -Detection of DHRS3 protein expression in brain tissue of CKO mice (Sham group) and tissues enriched with GBM-related astrocytes around tumors in tumor-bearing mice: Tissue samples were placed in 1.5 mL EP tubes and RIPA lysis buffer containing protease inhibitors was added. Cells were then lysed using an ultrasonic disruptor at 30% power, with a 2-second interval between sonication sessions and a 3-second pause, for a total duration of 1 minute. Cells were lysed on ice for 15 min after sonication, then centrifuged at 14000 g at 4 °C for 15 min. The supernatant was transferred to a clean 1.5 mL EP tube, and protein was quantified using a BCA quantitative kit. Finally, 1× loading buffer was added and mixed. The mixture was boiled at 100 °C for 5 min, then loaded onto an SDS-PAGE gel. The protein was then transferred to a PVDF membrane, blocked with 5% skim milk powder for 1 h, and incubated with primary antibody YAP (1:1,000, 14074S, CST) and DHRS3 (1:500, 15393-1-AP, Proteintech) overnight at 4 °C. The next day, the cells were warmed to room temperature for 30 min, washed three times with TBST, incubated with secondary antibody for 1 h, and developed with ECL. β-Tubulin (1:10,000, ET1602-4, HUABIO) was used as an internal control, and statistical analysis was performed.

[0052] 4) Immunofluorescence staining: for YAP f / f and YAP GFAP - CKO group sham and tumor-bearing mouse brain tissue sections were subjected to GFAP & DHRS3 immunofluorescence co-staining. The immunofluorescence staining procedure for frozen sections was as described above, and the results were statistically analyzed.

[0053] 5) Construction of DHRS3 overexpression plasmid: mRNA was extracted from primary astrocytes of C57BL / 6 mice using the methods described above and reversed to cDNA. The Dhrs3-cDNA sequence was amplified by PCR using specific primers. The amplification primers used were: Dhrs3-cDNA-F sequence is SEQ ID NO.4: 5'-G GAATTC The GCCACCATGGTGT-3', Dhrs3-cDNA-R sequence is SEQ ID NO.5: 5'-CG ACCGGTThe obtained sequence, GTTGTCCTCCCCTTAAAGGTGT-3', was purified by PCR and then double-digested with restriction endonucleases EcoRI and AgeI. Simultaneously, the vector pLVX-mcherry-IRES-Neo was digested with the same enzymes. After gel recovery, ligation was performed using T4 ligase. Competent cells were transformed, plated, and single clones were picked. Sequencing confirmed successful plasmid construction, yielding the DHRS3 overexpression plasmid, pLVX-DHRS3-mCherry-IRES-Neo (abbreviated as DHRS3-OE). The control plasmid was pLVX-mcherry-IRES-Neo (abbreviated as Ctrl).

[0054] 6) Lentiviral packaging and primary astrocyte transfection: 293T cells were cultured in DMEM medium supplemented with 10% FBS at 37˚C in an incubator containing 5% CO2. Before virus packaging, the medium was replaced with antibiotic-free DMEM medium. Two EP tubes were prepared: one tube was mixed with Opti-MEM and Lipo3000, and the other tube was mixed with Opti-MEM, p3000, helper plasmids PSPAX2 and PMD2G, and the target plasmid (the control plasmid and DHRS3 overexpression plasmid mentioned above). The two tubes were mixed separately and incubated at room temperature for 5 min. The 293T medium was removed, and the above-mentioned mixing reagents were added. After 8-10 h, antibiotic-free DMEM medium was added. After 36-48 h, the first virus was harvested, and DMEM medium was added. After 24-36 h, the second virus was harvested. The virus was filtered using a 0.22 μm filter and stored at -80℃ for transfection with YAP. + / + and YAP - / - Primary astrocytes were used, and Western blot and immunofluorescence staining confirmed that DHRS3 was overexpressed.

[0055] 7) CCK8 assay to detect the reversal effect of DHRS3 overexpression on the accelerated proliferation of GBM cells induced by YAP knockout in astrocytes: YAP was obtained. + / + -Ctrl T-ACM, YAP - / - -Ctrl T-ACM, YAP + / + -DHRS3-OE T-ACM, YAP - / - The effects of the four T-ACM groups on the proliferation of GL261 cells were detected using the CCK8 assay and statistical analysis was performed.

[0056] 8) Tranwell assay to detect the reversal effect of DHRS3 overexpression on the accelerated migration of GBM cells induced by YAP knockout in astrocytes: YAP was obtained + / + -Ctrl T-ACM, YAP - / - -Ctrl T-ACM, YAP+ / + -DHRS3-OE T-ACM, YAP - / - -DHRS3-OE T-ACM, using Transwell assay to detect the effects of the above four groups of T-ACM on the migration of GL261 cells.

[0057] Experimental results are as follows Figure 4 As shown, considering that YAP is a co-transcription factor, chromatin immunoprecipitation revealed that YAP in astrocytes binds to Dhrs3. GFAP - The expression level of Dhrs3 in GBM-related astrocytes of CKO tumor-bearing mice was significantly downregulated. Figure 4 BH displays YAP GFAP -CKO tumor-bearing mice showed significantly decreased DHRS3 expression, similar to the transcriptional expression level ( Figure 4 Consistent with C), it was also observed that, compared to the Sham group, the expression levels of YAP and DHRS3 were significantly increased in the tumor-bearing group. Figure 1 The upregulation of YAP expression in GBM-associated astrocytes was observed to be consistent with that in other studies. Figure 4 The study also observed that YAP positively regulates Dhrs3 expression.

[0058] Figure 4 IM shows YAP cultured in vitro - / - Overexpression of DHRS3 in primary astrocytes demonstrated that overexpression of DHRS3 could reverse the phenotype of promoting glioblastoma cell growth induced by YAP knockout.

[0059] The above experimental results demonstrate that the YAP-DHRS3 axis of astrocytes inhibits the growth of GBM. It clarifies that YAP in astrocytes inhibits GBM growth by binding to dehydrogenase / reductase (SDR family) member 3 (Dhrs3) and positively regulating DHRS3 signaling.

[0060] Finally, the inhibition of glioblastoma growth by YAP cells in astrocytes via DHRS3 was verified. The specific procedures are as follows:

[0061] 1) Activation of YAP in primary cultured astrocytes with XMU-MP-1: Primary mouse astrocytes were cultured and treated with 2.5 μM XMU-MP-1 (an inhibitor of Hippo kinase MST1 / 2). The protein expression of YAP was detected by Western blot and GFAP & YAP immunofluorescence co-staining.

[0062] 2) In situ modeling of GBM in C57BL / 6 mice to detect the inhibitory effect of activated astrocyte YAP on in vivo growth: The in situ modeling of GBM was performed as described above. The experiment was divided into 3 groups. The first group: GL261-Luc cell injection group, injection volume was 1x10 5 Group 1: 100 cells; Group 2: GL261-Luc+ primary cultured astrocytes injected, with an injection volume of 1 x 1010 GL261 cells. 5 1, 2 x 10 astrocytes 5 Group 3: Primary astrocytes injected with GL261-Luc + XMU-MP-1-activated YAP cells, with an injection volume of 1 x 10^9 GL261-Luc cells. 5 XMU-MP-1 stimulates 2 x 10 YAP astrocytes 5 Three weeks later, mice were injected intraperitoneally with the substrate luciferase to detect tumor growth in vivo. After the mice were collected, brain tissue sections were taken and stained with hematoxylin and eosin (HE) to further determine the size of the tumor in vivo and to generate survival curves.

[0063] 3) The above frozen sections were subjected to GFAP & DHRS3 immunofluorescence co-staining to verify whether DHRS3 in astrocytes of the XMU-MP-1-activated YAP group was significantly upregulated in in vivo tumor tissues.

[0064] To demonstrate whether activating YAP in astrocytes inhibits GBM progression by upregulating DHRS3 signaling, a mouse orthotopic GBM model was constructed by co-injecting primary astrocytes with GL261-Luc and XMU-MP-1-activated YAP. The results are as follows: Figure 5 As shown in Figure AC, XMU-MP-1 stimulates YAP expression in primary astrocytes. DK shows that astrocytes can promote GBM growth in vivo, while stimulating YAP in primary astrocytes can upregulate DHRS3 and inhibit GBM growth, indicating that targeting the YAP-DHRS3 axis of astrocytes may be beneficial for GBM treatment.

[0065] In summary, this invention targets tumor-associated astrocytes involved in glioblastoma progression within the tumor microenvironment. The high expression of YAP by astrocytes can inhibit GBM growth by regulating DHRS3, providing a new approach for finding novel targeted therapies for glioblastoma.

[0066] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0067] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. Application of YAPs in the preparation of anti-glioma drugs.

2. The application as described in claim 1, characterized in that, Targeting astrocytes in the tumor microenvironment of glioblastoma, the growth of glioblastoma is inhibited by upregulating DHRS3 through tumor-associated astrocyte YAP.

3. A method for inhibiting the growth of glioblastoma, characterized in that, By administering an activator that activates the expression of tumor-associated astrocytes (YAPs) to subjects, the activated YAPs inhibited glioblastoma growth by upregulating DHRS3.

4. Application of YAPs of astrocytes in the preparation of kits for the diagnosis or prognostic assessment of glioblastoma.

5. The use of a reagent for detecting glioblastoma-associated astrocytes (YAPs) in the preparation of kits for the diagnosis or prognostic assessment of glioblastoma.

6. A kit for the diagnosis or prognostic assessment of glioblastoma, characterized in that, This includes reagents for detecting YAP expression levels in tumor-associated astrocytes.

7. The kit according to claim 6, characterized in that, The reagents used to detect glioblastoma-associated astrocytes (YAPs) are selected from: probes with specificity for detecting YAPs and gene chips.

8. A pharmaceutical composition, characterized in that, Including YAP agonists for glioblastoma-associated astrocytes.

9. The pharmaceutical composition according to claim 8, characterized in that, It also includes pharmaceutically acceptable carriers.

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