Diagnostic marker combination for glioblastoma prognosis and application thereof

Abstract

The invention provides a diagnostic marker combination for glioblastoma prognosis. The diagnostic marker combination comprises a three-structural-domain protein 21 (TRIM21) and a platelet-derived growth factor receptor alpha (PDGFRA) and application of the diagnostic marker combination. According to the invention, the fact that the PDGFRA and the TRIM21 are jointly used as a group of biomarkers is proposed for the first time, and a new thought is provided for performing typing diagnosis, treatment scheme selection or prognosis evaluation on the glioblastoma. And a more accurate choice is provided for selection and application of the PDGFRA inhibitor in personalized treatment of patients with glioblastoma.

Description

Technical Field

[0001] The present invention relates to the technical field of biological diagnosis, and in particular to a prognostic diagnostic marker for glioblastoma. Background Art

[0002] Glioblastoma (GBM) is the most common primary malignant tumor of the central nervous system in adults, originating from glial cells. Epidemiologically, glioblastoma can occur in all age groups and is more common in men. Treatment options include surgical resection, radiotherapy, chemotherapy, and electric field therapy. However, due to the highly invasive nature of glioblastoma, it is difficult to completely remove the tumor tissue that is unclearly separated from normal brain tissue. Therefore, the postoperative survival time of glioblastoma patients is still very short, with a median overall survival of approximately 15 months, and less than 5% of patients survive for more than 5%, seriously threatening the life and health of patients.

[0003] The development and progression of glioblastoma is a multi-step process involving multiple genes. In-depth analysis of whole-genome sequencing results from a large sample of glioblastoma tissues has identified a series of genetic alterations closely associated with glioblastoma, including gene point mutations, gene methylation modifications, gene amplification, and gene deletions. These discoveries have deepened our understanding of the mechanisms of glioblastoma development and provided a solid theoretical foundation for the development of more effective treatments. Among these genetic alterations, platelet-derived growth factor receptor alpha (PDGFRA) gene copy number amplification is one of the most frequent. Further studies have revealed that the majority of low-grade gliomas and the majority of glioblastomas that have evolved from low-grade gliomas exhibit PDGFRA gene copy number amplification. Furthermore, in glioma patients with a history of intracranial radiotherapy, PDGFRA gene copy number amplification is the most prominent genetic alteration, suggesting that abnormal PDGFRA expression may be a key factor in glioma development. Mouse experiments have also confirmed that overactivation of the PDGF / PDGFRA signaling pathway in glial cells can lead to the occurrence of glioblastoma. PDGFRA plays an important role in the molecular classification of glioblastoma, especially in transcriptomic classification. PDGFRA gene copy number amplification is one of the key molecular markers of the proneuronal type, combined with the IDH mutation type, which helps to distinguish other subtypes (mesenchymal type, classical type). However, the application of PDGFRA in the personalized treatment of glioblastoma under the guidance of molecular pathology is still unclear. But in fact, a single PDGFRA gene copy number amplification cannot be used as a glioma classification and prognostic factor. Therefore, methods targeting PDGFRA in clinical applications need further analysis. Summary of the Invention

[0004] The present invention provides a new approach for using TRIM21 and PDGFRA together as a group of biomarkers for typing diagnosis, treatment plan selection or prognosis evaluation of glioblastoma.

[0005] The research of the present invention found that TRIM21 (three-domain protein 21) is the E3 ubiquitin ligase of platelet-derived growth factor receptor α (PDGFRA), which can transport PDGFRA to the proteasome for degradation through ubiquitination modification. This modification cannot be reflected at the RNA level, but directly affects the metabolism and function of PDGFRA as a post-translational modification. Patients with amplified PDGFRA gene copies can be further more clearly divided into those with proneuronal glioblastoma with a better prognosis. TRIM21 recognizes and binds to PDGFRA through the PRYSPRY domain and realizes the ubiquitination modification function through the RING-finger domain. The present invention proposes for the first time to use PDGFRA and TRIM21 together as a type of diagnosis and treatment option for glioblastoma, providing a more accurate choice for the selection and application of PDGFRA inhibitors in the personalized treatment of glioblastoma patients.

[0006] The present invention first provides a diagnostic marker combination for the prognosis of glioblastoma, which comprises tri-domain protein 21 (TRIM21) and platelet-derived growth factor receptor alpha (PDGFRA).

[0007] The present invention also provides a diagnostic preparation for glioblastoma prognosis, which comprises a first antibody targeting tri-domain protein 21 (TRIM21) and a second antibody targeting platelet-derived growth factor receptor alpha (PDGFRA).

[0008] In one embodiment according to the present invention, the first antibody is a humanized antibody, a mouse antibody, a rabbit antibody or a nanobody; and / or the second antibody is a humanized antibody, a mouse antibody, a rabbit antibody or a nanobody.

[0009] The present invention further provides use of the above-mentioned diagnostic marker combination or the above-mentioned diagnostic preparation in preparing a diagnostic kit for glioblastoma prognosis.

[0010] In one embodiment of the present invention, the above-mentioned use is achieved by detecting the expression level of three-domain protein 21 (TRIM21) and the expression level of platelet-derived growth factor receptor alpha (PDGFRA) in the sample.

[0011] In one embodiment according to the present invention, the expression level of the three-domain protein 21 (TRIM21) and the expression level of the platelet-derived growth factor receptor alpha (PDGFRA) are achieved by one or more methods selected from immunoblotting (Western Blot), enzyme-linked immunosorbent assay (ELISA), immunofluorescence chromatography and microfluidic chip.

[0012] The beneficial effects of the above technical solution of the present invention are as follows:

[0013] This study discovered that TRIM21, as the E3 ubiquitin ligase for PDGFRA, can induce ubiquitination and degradation of PDGFRA, thereby interfering with PDGFRA expression at the protein translation level. This study, for the first time, proposes the combined use of PDGFRA and TRIM21 as a panel of biomarkers, providing a new approach for glioblastoma classification, diagnosis, treatment selection, and prognostic assessment. This approach provides a more accurate approach for selecting and applying PDGFRA inhibitors in personalized treatment for glioblastoma patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Figure 2 shows the interaction between TRIM21 and PDGFRA, which induces ubiquitination of PDGFRA. HA-Ub, PDGFRA-Flag, TRIM21-GFP, and an empty GFP plasmid were co-transfected into HEK293FT cells. At 48 hours post-transfection, MG132 was added and treated for 4 hours. The cells were then harvested for IP.

[0015] Figure 2 The following is a descending sort of the TRIM21 expression ratio in various tumors and normal tissues. The expression differences of TRIM21 in various tumors and normal tissues were analyzed through the database.

[0016] Figure 3 Figure 2 shows the survival analysis and prognostic graphs for PDGFRA High / TRIM21 High and PDGFRA High / TRIM21 Low in the TCGA-GBM database. A shows a comparative survival graph for patients with PDGFRA High / TRIM21 High and PDGFRA High / TRIM21 Low in the TCGA / GBM database. B / C show comparative prognostic graphs for patients with PDGFRA High / TRIM21 High and PDGFRA High / TRIM21 Low in the TCGA / GBM database, respectively.

[0017] Figure 4Figure 1 shows the GSEA gene enrichment analysis of PDGFRA High / TRIM21 High and PDGFRA High / TRIM21 Low in the TCGA-GBM database. Figure A shows the analysis of genes associated with aggressiveness in patients with PDGFRA High / Trim21 High and PDGFRA High / Trim21 Low in the TCGA / GBM database; Figure B shows the analysis of genes associated with the signaling axis in patients with PDGFRA High / Trim21 High and PDGFRA High / Trim21 Low in the TCGA / GBM database.

[0018] Figure 5 Figure 1 shows the results of a TRIM21-induced ubiquitination and degradation assay for PDGFRA via the K48 pathway. A shows the results of co-transfection of PDGFRA-Flag, TRIM21-GFP, and empty GFP plasmids with HA-Ub, HA-Ub-K48R, and HA-Ub-K63R into HEK293FT cells. MG132 was added at 48 hours and treated for 4 hours before the cells were harvested for IP. B shows the results of IP experiments in glioblastoma cells LN18 and PDGFRA-overexpressing LN229 treated with DMSO, CQ, and MG132 for 3 hours, respectively. C shows the results of IP experiments in glioblastoma cells U251 stably overexpressing TRIM21 and a control group treated with DMSO, CQ, and MG132 for 3 hours, respectively.

[0019] Figure 6 Figure 1 shows the effects of stable overexpression or knockdown of TRIM21 on PDGFRA. A. U251 glioblastoma cells stably overexpressing TRIM21 were serum-starved overnight and then treated with PDGF-AA. Cells were harvested at time points 0, 15, 30, and 60 minutes for Western blot analysis. B. U251 glioblastoma cells stably knocked out TRIM21 were serum-starved overnight and then treated with PDGF-AA. Cells were harvested at time points 0, 15, 30, and 60 minutes for Western blot analysis.

[0020] Figure 7Overexpression of TRIM21 shortens the half-life of glioblastoma U251 cells. Figures A and B show glioblastoma U251 cells treated with CHX (control and TRIM21-overexpressing cells), harvested at 0, 1, 2, 3, 4, 5, and 6 hours, and analyzed by Western blot analysis (Figure A). Figures C and D show glioblastoma LN18 cells treated with CHX (control and TRIM21-overexpressing cells), harvested at 0, 1, 2, 3, 4, and 5 hours, and analyzed by Western blot analysis (Figure C).

[0021] Figure 8 Figure 1 shows the results of a TRIM21 ubiquitination assay using its PRYSPRY domain to bind to PDGFRA and its RING domain to ubiquitinate. A is a schematic diagram of the construction of four TRIM21 truncations based on the TRIM21 domain structure. B shows the results of an IP assay in which PDGFRA-Flag and TRIM21-Myc-WT, △1, △2, △3, and △4 were transfected into HEK293FT cells, treated with MG132 for 4 hours at 48 hours, and then harvested for IP. C shows the results of an IP assay in which PDGFRA-Flag, HA-Ub, and a Myc empty vector were co-transfected with TRIM21-WT, △3, and △4 at 48 hours, and then harvested for IP.

[0022] Figure 9 Figure 1 shows the significant effect of TRIM21 knockdown on the migration of glioblastoma cells. Figure A shows U251 cells stably overexpressing TRIM21 and controls, plated into transwell chambers, and after 24 hours, the chambers were closed for crystal violet staining. Figure B shows U251 cells stably underexpressing TRIM21 and controls, plated into transwell chambers, and after 24 hours, the chambers were closed for crystal violet staining. Figure C shows LN18 cells stably overexpressing TRIM21 and controls, plated into transwell chambers, and after 24 hours, the chambers were closed for crystal violet staining. Figure D shows LN18 cells stably underexpressing TRIM21 and controls, plated into transwell chambers, and after 24 hours, the chambers were closed for crystal violet staining. DETAILED DESCRIPTION

[0023] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0024] Example 1

[0025] IP detection of whether TRIM21 affects the ubiquitination of PDGFRA

[0026] (1) Cell plasmid transfection experiment

[0027] HEK293FT cells (purchased from Prosper) in good condition were cultured. The complete medium in a 10 cm dish was replaced with 5 ml of Opti-MEM medium. The target plasmids PDGFRA-Flag, TRIM21-GFP, empty GFP vector, and Ub-HA (10 μg total) and 10 μl of Lipo 2000 were co-transfected into the cells. After 6 hours, the cells were replaced with complete medium and cultured. At 48 hours, MG132 was added for 4 hours, and the cells were harvested for subsequent IP experiments.

[0028] (2) CO-IP experiment

[0029] ① Total cell protein extraction: Aspirate the culture medium, add 5ml of PBS, gently shake to wash the culture medium, and discard the PBS. After adding 1ml of PBS, scrape the transfected HEK293FT cells with a cell scraper and transfer them to a 1.5ml centrifuge tube using a pipette. Centrifuge at 800g for 3 minutes at 4°C, discard the supernatant, and retain the cell pellet. Add approximately 10 volumes of RIPA (weak) lysis buffer containing PMSF to the cell pellet and lyse on ice for 30 minutes. After lysis, place the cell lysate in a 4°C centrifuge and centrifuge at 25,000g for 15 minutes. Transfer the supernatant after centrifugation to a fresh 1.5ml centrifuge tube to obtain the total cell protein.

[0030] ② Lysate pre-clearing: Discard the remaining liquid by aspirating 20µl of Protein A+G magnetic beads per dish of cells, then resuspend in RIPA (weak) lysis buffer. Add the magnetic bead suspension to the lysate sample and place in a rotating incubator at 4°C for 1 hour. From the pre-cleared lysate sample, take 75µl as the input group and add 25µl of 4× loading buffer. Mix thoroughly, then heat in a 95°C metal bath for 5 minutes. After heating, place in a -20°C refrigerator until ready to use.

[0031] ③ Antibody magnetic bead incubation: Dispense 20µl of antibody magnetic beads and IgG magnetic beads per dish of cells, discard the original liquid, and resuspend in RIPA (weak) lysis buffer. Place the pre-cleared lysate sample on a magnetic rack to aspirate the liquid and transfer it to two centrifuge tubes, one for the labeled antibody group and one for the IgG negative control group. Add the corresponding magnetic beads to each tube, place in a 4°C refrigerator in a rotating incubator, and rotate overnight.

[0032] ④ Magnetic bead wash: After overnight incubation with antibody-magnetic beads, remove the sample, place it on a magnetic rack, and discard the liquid. Re-add RIPA (weak) lysis buffer, return to the 4°C refrigerator, and place in a rotating incubator. Rotate for 30 minutes, repeat three times. Then, wash with ice-cold PBS three more times by inversion. Finally, discard the liquid and retain the magnetic beads.

[0033] ⑤ Sample preparation: Resuspend the magnetic beads in 100 μl of 1× loading medium, mix thoroughly, and heat in a 95°C metal bath for 5 minutes. Cool on ice after heating. Verify the sample using Western blot.

[0034] (3) Western blot experiment

[0035] ①SDS-PAGE electrophoresis: Place the protein gel in an electrophoresis tank and add sufficient electrophoresis buffer. Add 30 μg of protein sample to each well. Run at 120V for 20 minutes, then increase to 160V and run for approximately 40 minutes until bromophenol blue runs out of the gel. Terminate the electrophoresis and proceed to transfer to the membrane.

[0036] ② Transfer: Use the sandwich method to place the filter paper, PVDF membrane and electrophoresis gel into the clamp in sequence, place the clamp into the electrotransfer tank, and transfer the membrane at a constant current of 0.36A for 2 hours.

[0037] ③Immunolysis: Block the membrane in 5% skim milk at room temperature on a shaker for 1 hour. Wash the membrane with PBST and incubate in the primary antibody at 4°C overnight. After incubation with the primary antibody, remove the membrane from the antibody and incubate in PBST three times, 10 minutes each, at room temperature on a shaker. Then, incubate the membrane in the secondary antibody at room temperature on a shaker for 1 hour. Repeat the same procedure to wash the secondary antibody three times, 10 minutes each.

[0038] ④ Chemiluminescence: Prepare developer, drain excess liquid on the membrane, add appropriate amount of developer, place in a developing instrument for development and take pictures.

[0039] (4) The main protein that binds to PDGFRA in glioblastoma cells

[0040] After transfection of the PDGFRA-Flag plasmid into the glioblastoma cell line LN18 (purchased from Pronocell), the cells were harvested for IP-Flag experiments, and magnetic beads linked to PDGFRA and its associated proteins were obtained. The beads were then sent to Jingjie Biotechnology Company for mass spectrometry detection, and the ranking of the main proteins associated with PDGFRA was obtained (Table 1).

[0041] Table 1

[0042] Proteins that bind to PDGFRA Number of specific peptides Protein peak area# TMCC1 1 <![CDATA[5.02×10 8 ]]> UBB 9 <![CDATA[2.44×10 8 ]]> hspa1b 4 <![CDATA[1.21×10 8 ]]> HSP90AB1 7 <![CDATA[3.37×10 7 ]]> ZYX 4 <![CDATA[3.04×10 7 ]]> HBB 2 <![CDATA[2.73×10 7 ]]> PIK3R1 6 <![CDATA[2.4×10 7 <!-- 4 -->]]> YWHAE 2 <![CDATA[1.69×10 7 ]]> H1FX 2 <![CDATA[1.19×10 7 ]]> BAG2 4 <![CDATA[1.18×10 7 ]]> CST6 1 <![CDATA[1.12×10 7 ]]> CANX 5 <![CDATA[9.31×10 6 ]]> hla-a 3 <![CDATA[9.24×10 6 ]]> GNAT1 1 <![CDATA[8.66×10 6 ]]> RNASE7 1 <![CDATA[8.22×10 6 ]]> BUB3 3 <![CDATA[7.96×10 6 ]]> RBMX 2 <![CDATA[7.63×10 6 ]]> DNAJA1 1 <![CDATA[7.56×10 6 ]]> SCGB1D2 1 <![CDATA[7.37×10 6 ]]> HLA-DRB5 1 <![CDATA[6.69×10 6 ]]> TRIM21 2 <![CDATA[6.52×10 6 ]]>

[0043] #Protein peak area represents the relative protein content. The larger the peak area value, the higher the protein content

[0044] Figure 1 As shown in A, it is demonstrated that TRIM21 binds to PDGFRA and TRIM21 can induce ubiquitination of PDGFRA. Figure 1Figure B further demonstrates that increasing TRIM21 expression leads to increased ubiquitination of PDGFRA. Table 1 shows that among the proteins that bind to PDGFRA, those with the ubiquitin UBB and TRIM21 are more strongly bound. These findings suggest that TRIM21 may be an E3 ubiquitin ligase for PDGFRA, inducing its ubiquitination.

[0045] Example 2

[0046] Analysis of the expression ratio of TRIM21 between various tumors and normal tissues

[0047] Enter TRIM21 on the GEPIA website to obtain the differences in TRIM21 gene expression in various tumors and their corresponding normal tissues. The height of the bar represents the median expression of a specific tumor type or normal tissue ( Figure 2 ). Figure 2 The expression ratios of TRIM21 in various tumors and normal tissues are sorted in descending order. The expression differences of TRIM21 in various tumors and normal tissues were analyzed using the database.

[0048] Figure 2 As shown in the database analysis, the expression level of TRIM21 in glioblastoma is most different from that in normal tissues, suggesting that TRIM21 may play an important role in glioblastoma and may become an effective therapeutic target.

[0049] Example 3

[0050] When PDGFRA is highly expressed, the effect of changes in TRIM21 expression on the survival and prognosis of glioblastoma patients.

[0051] STAR-counts data and corresponding clinical information for glioblastoma were downloaded from the public database TCGA-GBM (https: / / portal.gdc.cancer.gov / ). Data in TPM format were extracted and normalized using log2(TPM+1). Finally, samples with high PDGFRA expression and both RNAseq data and clinical information were retained, resulting in a total of 267 samples for further analysis to distinguish high and low TRIM21 expression. The GTEx data used (https: / / gtexportal.org / home / datasets) were from version V8. Statistical analysis was performed using R software version 4.0.3. Results were considered statistically significant when the P value was less than 0.05.

[0052] The results are as follows Figure 3As shown in A, under the premise of high expression of PDGFRA, high expression of TRIM21 is associated with shorter survival, while low expression of TRIM21 is associated with longer survival. Figure 3 As shown in Figures BC, high expression of both PDGFRA and TRIM21 is significantly detrimental to patient prognosis. This suggests that while PDGFRA plays a crucial role in glioblastoma and has been investigated as a key therapeutic target in previous studies, targeting PDGFRA alone may not necessarily lead to effective treatment. These results suggest that we should increase research on co-therapy with TRIM21.

[0053] Example 4

[0054] Gene enrichment analysis

[0055] The data of Example 3 can be uploaded to the website (https: / / db.cngb.org / genomics / tools / gsea) for gene enrichment analysis to obtain relevant pathways with enrichment significance or signal axis information with high correlation.

[0056] The results are as follows Figure 4 As shown in Figure A, overexpression of both PDGFRA and TRIM21 is enriched for gene pathways associated with cell invasion, and is indeed highly correlated with the PDGF signaling axis. Previous studies have shown that overexpression of PDGFRA can induce the early onset of gliomas, but no further progression has been observed. However, overexpression of TRIM21 increases the invasiveness of PDGFRA-overexpressing glioblastoma cells, correlating with the diffuse nature of glioblastoma. This suggests that simultaneously targeting PDGFRA and TRIM21 is of great significance as therapeutic targets.

[0057] Example 5

[0058] TRIM21 induces ubiquitination and degradation of PDGFRA via the K48 pathway

[0059] (1) Cell plasmid transfection experiment

[0060] Plasmids PDGFRA-Flag, TRIM21-GFP, GFP empty vector, Ub-HA, Ub-K48R and Ub-K63R were co-transfected into HEK193FT cells, respectively. After 48 h, the cells were treated with MG132 for 4 h and then harvested to prepare protein samples for Western Blot analysis (see Example 1 for specific methods).

[0061] (2) Construction of stable cell lines

[0062] ① Overexpression of TRIM21 and control plasmids were purchased from Sino Biological Co., Ltd. Human, PDGF alpha receptor / PDGFRAcDNA ORF Clone, C-DYKDDDDK tag Overexpression Plasmid, Human, TRIM21 Lentiviral cDNA ORF Clone, C- tag overexpression plasmid and pLV-C-GFPSpark Lentivirus ControlPlasmid control plasmid.

[0063] ② Virus packaging: Using healthy HEK293FT cells, replace the complete medium in a 10 cm dish with 5 ml of Opti-MEM medium. Transfect the packaging plasmid and target plasmid into the cells. After 6 hours, replace the complete medium with the culture medium and continue culturing. Collect the supernatant at 48 and 72 hours, filter through a 0.45 μm filter, and store in aliquots at -80°C until use.

[0064] ③ Target cell infection and screening: Use glioblastoma cells in good growth condition, add virus solution and complete culture medium, and culture for 48 hours. Then, digest the cells to make a cell suspension, and screen the labeled cells by loss for culture.

[0065] (3) MG132, CQ, and DMSO control treatment experiments

[0066] Three dishes each of LN18 glioblastoma cells cultured in a 10 cm dish, LN229 cells overexpressing PDGFRA constructed using the above method, and U251 cells overexpressing TRIM21 and control cells were used. The old culture medium was discarded and replaced with complete culture medium containing MG132 (proteasome inhibitor), CQ (autophagy inhibitor), and DMSO (control solvent). After 4 hours of treatment, the cells were collected and protein samples were prepared for Western Blot experiments.

[0067] The results are as follows Figure 5 As shown, transfection of ubiquitin plasmids with point mutation K48 directly affects the ubiquitination of PDGFRA induced by TRIM21, while transfection of ubiquitin plasmids with point mutation K63 has no effect ( Figure 5A) Treatment of glioblastoma cells LN18, LN229-RA, and U251 with a proteasome inhibitor, an autophagy inhibitor, and a control solvent revealed that proteasome inhibition increased PDGFRA expression, while autophagy inhibition did not significantly alter PDGFRA expression. This suggests that PDGFRA is primarily degraded via the proteasome. While ubiquitination at the K48 site targets proteasomal degradation, ubiquitination at the K63 site typically stabilizes the protein. These results suggest that TRIM21 primarily induces ubiquitination and degradation of PDGFRA at the K48 site.

[0068] Example 6

[0069] Effect of TRIM21 expression on glioblastoma cells stimulated by PDGFRA ligands

[0070] Prepare four dishes of healthy U251 glioblastoma cells stably overexpressing or knocking out TRIM21, as well as controls. Serum-starve the cells at approximately 90% confluence by replacing them with 10 ml of serum-free medium overnight for 16 hours. The next day, replace the medium with serum-free medium supplemented with 100 ng / ml PDGF-AA. Harvest cells at time points 0, 15, 30, and 60 minutes for protein preparation and Western blotting.

[0071] The results are as follows Figure 6 As shown in Figure 3, the expression of PDGFRA protein in glioblastoma cells U251, which stably overexpress TRIM21, was significantly reduced, and the stimulation response to its ligand PDGF-AA was low ( Figure 6 A). In glioblastoma cells U251 with stable knockout of TRIM21, the protein expression of PDGFRA was significantly increased, and the stimulation response to its ligand PDGF-AA was higher ( Figure 6 B) Further evidence indicates that TRIM21 induces ubiquitination and degradation of PDGFRA at the K48 site, significantly reducing its ligand response to PDGFRA, suggesting that PDGFRA and TRIM21 could be combined as therapeutic targets for glioblastoma.

[0072] Example 7

[0073] Cycloheximide (CHX) tracking assay

[0074] Prepare well-conditioned glioblastoma cells U251 and LN18 that stably overexpress or knock out TRIM21 at a culture density of approximately 80%. Discard the old culture medium and replace it with 5 ml of new complete culture medium containing 50 ng / ml CHX. Harvest cells at time points 0, 1, 2, 3, 4, 5, and 6 h to prepare protein samples and perform Western Blot experiments (see Example 1 for specific methods). The images obtained after development were analyzed for band grayscale values using Image J software, and the grayscale values obtained were statistically plotted using Graphpad Prism software ( Figure 7 ABCD).

[0075] pass Figure 7 ABCD observed that overexpression of TRIM21 in glioblastoma cells and subsequent treatment with the protein synthesis inhibitor CHX resulted in a sharp decrease in PDGFRA expression in the cells, suggesting that TRIM21 significantly shortens the half-life of PDGFRA protein.

[0076] Example 8

[0077] Identification of the domains of TRIM21 that bind to PDGFRA and induce its ubiquitination

[0078] (1) Construction of protein truncation based on the domain structure of TRIM21

[0079] Searching for TRIM21 on https: / / www.uniprot.org / provides the loci for its protein domains and allows the design of plasmids for corresponding domain truncations, including those lacking the CC and PRYSPRY domains, those lacking the PRYSPRY domain, those lacking the RING domain, and those lacking the RING, B-BOX, and CC domains.

[0080] (2) Determine the binding domains

[0081] PDGFRA-Flag, Ub-HA and TRIM21 wild type or truncated 1 / 2 / 3 / 4 plasmids were co-transfected into healthy HEK293FT cells. After 48 h, the cells were treated with MG132 for 4 h and then harvested for CO-IP and Western blot experiments (see Example 1 for specific methods).

[0082] (3) Identify the domain that induces ubiquitination

[0083] PDGFRA-Flag, Ub-HA and TRIM21 wild type or truncated forms 3 and 4 plasmids were co-transfected into healthy HEK293FT cells. After 48 h, the cells were treated with MG132 for 4 h and then harvested for CO-IP and Western blot experiments (see Example 1 for specific methods).

[0084] pass Figure 8 As can be observed in A and B, the magnetic beads enriched with PDGFRA-Flag interacted with the wild type and truncations 3 and 4 of TRIM21, suggesting that TRIM21 interacted with PDGFRA through the common region PRYSPRY between the wild type and truncations 3 and 4. Figure 8 As can be seen in Figures A and C, although TRIM21 binds to PDGFRA via the PRYSPRY domain, it clearly lacks the ability to induce its ubiquitination, suggesting that TRIM21 induces PDGFRA ubiquitination through the RING domain, a region present in the wild type but absent in truncations 3 and 4. These results indicate that TRIM21 binds to PDGFRA via the PRYSPRY domain and induces PDGFRA ubiquitination via the RING domain.

[0085] Example 9

[0086] Cell Transwell migration assay

[0087] ① Cell suspension preparation: Stably overexpressed or knocked-down glioblastoma cells (U251 and LN18) and their corresponding control cells (see Example 5 for specific methods) cultured for 3-10 generations were trypsinized and digested with complete medium. The cell suspension was collected and placed in a 15 ml centrifuge tube. Centrifuged at 800 g for 3 minutes at room temperature, the supernatant discarded, and the cell pellet retained. Resuspend the cells in 2 ml of complete medium and count using a cell counting plate.

[0088] ② Cell plating: Place approximately 100,000 cells into each transwell chamber. Perform at least three replicates for each experiment. Calculate the required volume of cell suspension based on the cell density indicated on the cell counting plate. Remove the appropriate cell suspension and place it into a 15ml centrifuge tube. Centrifuge for 3 minutes, discard the supernatant, and retain the cell pellet. Resuspend the suspension in serum-free medium and plate it onto the upper chamber of each transwell chamber. Then, aspirate 1ml of complete medium into the lower chamber of each chamber. Incubate in an incubator for 16 hours.

[0089] ③ Fixation and staining of chambers: Remove the chambers, discard the culture medium, and rinse three times with 500 μl of PBS per well. Add 500 μl of tissue fixative to the outside of the chambers and fix at room temperature for 20 minutes. Aspirate the fixative and rinse three times with 500 μl of PBS. Add 500 μl of crystal violet solution to the outside of the chambers and stain at room temperature for 30 minutes. Aspirate the crystal violet solution and rinse three times with 500 μl of PBS. Finally, remove any remaining cells from the inside of the chambers with a cotton swab.

[0090] ④ Chamber imaging and statistical analysis: Place the chamber under an upright microscope, take a picture of each chamber in 5 fields of view, and then use Image J to count the number of cells and analyze the differences between groups ( Figure 9 ABCD).

[0091] The results are as follows Figure 9 As shown in Figure 2, the migration ability of glioblastoma cells LN18 and U251 overexpressing TRIM21 was significantly enhanced compared with the control group, with statistical significance ( Figure 9 A, C). Compared with the control group, the migration ability of glioblastoma cells LN18 and U251 in which TRIM21 was knocked down was significantly weakened, with statistical significance ( Figure 9 B, D). This suggests that TRIM21 can affect the migration ability of tumor cells, which may be related to the diffuse nature of glioblastoma. This suggests that targeting TRIM21 has positive implications as a therapeutic target.

[0092] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

A diagnostic marker combination for the prognosis of glioblastoma, comprising tri-domain protein 21 (TRIM21) and platelet-derived growth factor receptor alpha (PDGFRA).

2. A diagnostic preparation for the prognosis of glioblastoma, comprising a first antibody targeting tri-domain protein 21 (TRIM21) and a second antibody targeting platelet-derived growth factor receptor α (PDGFRA).

3. The diagnostic preparation according to claim 2, wherein The first antibody is a humanized antibody, a mouse antibody, a rabbit antibody or a nanobody; and / or the second antibody is a humanized antibody, a mouse antibody, a rabbit antibody or a nanobody.

4. Use of the diagnostic marker combination according to claim 1 or the diagnostic preparation according to claim 2 or 3 in the preparation of a diagnostic kit for the prognosis of glioblastoma.

5. The use according to claim 2, which is achieved by detecting the expression levels of three-domain protein 21 (TRIM21) and platelet-derived growth factor receptor alpha (PDGFRA) in the sample.

6. The use according to claim 1 or 2, wherein The expression levels of the three-domain protein 21 (TRIM21) and the platelet-derived growth factor receptor alpha (PDGFRA) are achieved by one or more methods selected from Western Blot, enzyme-linked immunosorbent assay (ELISA), immunofluorescence chromatography, and microfluidic chip.

Images