Glioma marker and application thereof

Abstract

The invention discloses a marker of glioma, and the marker of glioma is PQLC2 protein. The invention reveals the association between PQLC2 expression and glioma for the first time. The PQLC2 expression quantity is accurately investigated and analyzed, so that whether an individual suffers from glioma or not can be effectively judged, meanwhile, the severity of glioma can be clearly determined, and the prognosis condition of a patient can be evaluated.

Description

Technical Field

[0001] The present invention belongs to the technical field of precision cancer medicine, and in particular relates to a glioma marker and its application. Background Art

[0002] Gliomas are typical malignant brain tumors characterized by diffuse infiltration, lack of distinct borders, unlimited proliferation, and high invasiveness. Based on their histomorphological characteristics, the World Health Organization (WHO) classifies gliomas into grades I to IV for diagnosis, treatment planning, and prognostic assessment. Glioblastoma (GBM) is an aggressive glioma that exhibits resistance to treatment and a high recurrence rate; GBM patients typically have a short survival time. GBM accounts for 50% to 60% of all brain tumors and 12% to 15% of all intracranial tumors. The survival rate of GBM patients is low. Due to the various difficulties in treating gliomas, clinicians are pessimistic about the treatment prospects of this disease. Therefore, doctors and researchers are actively searching for cancer-related molecular markers so that they can combine the patient's pathological classification to ultimately determine the most effective treatment.

[0003] PQ-loop repeat-containing protein (PQLC2) is a heptahelical protein with a repeating PQ-loop structure that functions as an amino acid transporter, mediating the pH-dependent export of cationic amino acids (arginine, histidine, and lysine) from lysosomes. PQLC2 is thought to play a role in the lysosomal export of mixed cystamine-cysteine disulfides formed after cystamine treatment in patients with cystinosis, a disease characterized by the accumulation of cysteine within lysosomes.

[0004] At present, the relationship between PQLC2 gene and glioma has not been reported domestically or internationally. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art and provide a glioma marker and application thereof.

[0006] In order to solve the above technical problems, the present invention provides a glioma marker, wherein the glioma marker is PQLC2 protein. Furthermore, the glioma is glioblastoma.

[0007] Based on a general technical concept, the present invention provides an application of the glioma marker in the preparation of a drug for treating glioma.

[0008] Based on a general technical concept, the present invention provides an application of the glioma marker in preparing a glioma detection kit.

[0009] Based on a general technical concept, the present invention provides an application of the glioma marker in preparing a detection kit for glioma prognosis judgment.

[0010] The above application further predicts the prognosis of glioma patients by detecting the expression level of the PQLC2 gene.

[0011] Compared with the prior art, the advantages of the present invention are:

[0012] This study provides a glioma marker: PQLC2. This study reveals for the first time the association between PQLC2 expression and glioma. Accurately examining and analyzing PQLC2 expression can effectively determine whether an individual has glioma, help determine the severity of glioma, and assess the patient's prognosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0014] Figure 1 This is a waterfall plot of gene expression obtained from 168 tumor samples in the present invention.

[0015] Figure 2 Statistical results of PQLC2 expression in 163 GBM tumor tissues and 207 normal tissues.

[0016] Figure 3 These are the statistical results of survival of patients with high and low PQLC2 expression.

[0017] Figure 4 The statistical results of PQLC2 expression in different grades of gliomas (WHOⅡ, Ⅲ, Ⅳ).

[0018] Figure 5 Statistical results of PQLC2 expression in wild-type and mutant IDH.

[0019] Figure 6 Statistical results of PQLC2 expression in males and females.

[0020] Figure 7 The statistical results of PQLC2 expression at different age stages.

[0021] Figure 8 The statistical results of PQLC2 expression in first-episode gliomas and recurrent gliomas are shown.

[0022] Figure 9 These are the statistical analysis results of R-4.2.1.

[0023] Figure 10 Statistical results of cell survival rates in the PQLC2 high expression group and low expression group.

[0024] Figure 11 These are the immunohistochemical staining results of adjacent tumors, low-grade and high-grade gliomas.

[0025] Figure 12 Statistical results of PQLC2 expression in adjacent cancer tissues, low-grade and high-grade gliomas. DETAILED DESCRIPTION

[0026] The present invention will be further described below in conjunction with specific preferred embodiments, but the scope of protection of the present invention is not limited thereby.

[0027] The materials, reagents, and instruments used in the following examples can all be purchased from commercial sources. The experimental methods in the following examples, unless otherwise specified, are all conventional methods in the art.

[0028] The main reagents involved in the experiment are listed in Table 1.

[0029] Table 1: Main equipment involved in the experiment.

[0030]

[0031]

[0032] Example 1

[0033] A glioma marker of the present invention: PQLC2 protein.

[0034] Experiment 1: Investigate the gene expression in GBM (glioblastoma) patients.

[0035] We obtained gene expression profiles and clinical data from 163 tumor tissues and 207 normal tissues from GBM (glioblastoma) patients from the Cancer Genome Atlas (TCGA) database. Records lacking sufficient age and overall survival information were excluded from subsequent processing. To make the RNA sequencing data comparable with the microarray data, the raw count data were first converted. Survival analysis and survival curve analysis (Kaplan-Meier curve analysis) were performed using a survival analysis software package, using the median as the cutoff point.

[0036] Figure 1Figure 1 shows waterfall plots of gene expression obtained from 168 tumor samples. Figure A shows a waterfall plot for patients with high PQLC2 gene expression, while Figure B shows a waterfall plot for patients with low PQLC2 gene expression. As can be seen from the figures, mutation frequencies of genes such as TP53, NFI, and MUC16 were significantly higher in patients with high PQLC2 gene expression than in patients with low PQLC2 gene expression. This significant difference provides valuable clues, suggesting that the pathogenesis of glioblastoma may involve a complex regulatory network of multiple genes.

[0037] Figure 2 The statistical results of PQLC2 expression in 163 GBM tumor tissues and 207 normal tissues are shown in the figure. As can be seen from the figure, PQLC2 expression in GBM patients is significantly higher than that in normal tissues compared with the control group, demonstrating that PQLC2 expression can be used as an indicator to distinguish gliomas from normal tissues.

[0038] Experiment 2: Survival and expression analysis in GEPIA analysis.

[0039] 2.1. We validated the relationship between PQLC2 expression and GBM clinicopathological data using GEPIA (http: / / gepia.cancer-pku.cn / index.html). GEPIA analyzes RNA sequencing data from 9736 cancer samples and 8587 normal samples collected from the GTEx and TCGA projects. Using GEPIA, we evaluated survival curves for different PQLC2 expression patterns to determine the association between gene expression and GBM patient prognosis.

[0040] Figure 3 The survival statistics of patients with high and low PQLC2 expression are shown in the figure.

[0041] Among patients with established GBM, those with low PQLC2 expression had significantly longer survival than those with high PQLC2 expression, demonstrating that PQLC2 expression can be used to predict patient survival. GEPIA analysis showed that PQLC2 expression was significantly elevated in GBM patients compared to the control group, and that patients with high PQLC2 expression had significantly shorter survival.

[0042] 2.2. The differences in PQLC2 expression in different pathological stages were compared using staging diagrams, and the differential expression of PQLC2 was analyzed by drawing box plots (with disease status as the independent variable).

[0043] Figure 4The following table shows the statistical results of PQLC2 expression in gliomas of different grades (WHO II, III, and IV). The results in the figure show that the expression level of PQLC2 gene is relatively low in low-grade gliomas, and the higher the glioma grade, the higher the expression level of PQLC2 gene.

[0044] Figure 5 The statistical results of PQLC2 expression in wild-type and mutant IDH are shown in the figure. The results in the figure show that PQLC2 expression in wild-type IDH is significantly lower than that in mutant IDH, and in different grades of glioma (WHO II, III, and IV), PQLC2 expression in wild-type IDH is significantly lower than that in mutant IDH.

[0045] 2.3 R-4.2.1 Statistical Analysis: TCGA data were analyzed using R-4.2.1. Logistic regression was used to investigate the relationship between PQLC2 expression and clinical data. Multivariate Cox analysis was used to assess the impact of PQLC2 expression and other variables (sex and age) on survival. The significance threshold was set at 0.05.

[0046] Figure 6 The statistical results of PQLC2 expression in males and females show that there is no statistical difference in PQLC2 expression between males and females.

[0047] Figure 7 The following table shows the statistical results of PQLC2 expression at different age groups. The results in the figure show that PQLC2 expression is higher in patients aged 42 and above, but there is no statistical difference in PQLC2 expression between high-grade and low-grade tumors.

[0048] Figure 8 The statistical results of PQLC2 expression in first-episode gliomas and recurrent gliomas are shown in the figure. As can be seen from the results in the figure, there is no statistical difference in the expression of PQLC2 in first-episode gliomas and recurrent gliomas.

[0049] Figure 9 This is the statistical analysis result of R-4.2.1. It can be seen from the figure that age and PQLC2 are both risk factors for GBM.

[0050] Experiment 3: Cell experiment.

[0051] The MTT method was used to detect cell proliferation. B cells, CD8+T cells, CD4+T cells, macrophages, neutrophils, and dendritic cells in the logarithmic growth phase were digested with trypsin, resuspended in complete culture medium to form a cell suspension, and counted. For the PQLC2 high- and low-expression groups, 2000 cells / well were seeded into six wells of a 96-well plate, using a culture medium of 100 μL / well. During the plating process, the cells were plated to ensure a consistent number of cells per well. Six blank wells containing only 100 μL of complete culture medium were also set up as baseline values. At the start of the experiment, five plates were plated and incubated in a 37°C, 5% CO2 cell culture incubator. Starting on the second day of incubation, one plate was assayed daily using the MTT assay. The MTT assay method involved adding 20 μL of 5 mg / mL MTT to each well 4 hours before terminating the incubation period. No medium change was required. After 4 hours, the culture medium was completely aspirated, taking care not to remove any formazan particles at the bottom of the plate. 100 μL of DMSO was added to dissolve the formazan particles. The plate was shaken for 2–5 minutes, and the OD values were measured at 490 / 570 nm using a microplate reader. Cox regression analysis was performed.

[0052] Figure 10 The survival rate of cells in the PQLC2 high-expression group and the PQLC2 low-expression group is statistically analyzed. As can be seen from the figure, the survival period of cells in the PQLC2 low-expression group was significantly longer than that in the PQLC2 high-expression group.

[0053] Experiment 4: Immunohistochemical analysis.

[0054] Tumors were graded according to the 2016 WHO classification, and 15 samples were analyzed using immunohistochemistry (IHC). Immunohistochemistry was performed using the streptavidin-peroxidase method, and signal distribution and expression levels were observed microscopically. The specific steps were:

[0055] (1) Sample selection: 15 frozen clinical tissue samples, including 5 adjacent cancer tissues, 5 low-grade gliomas, and 5 high-grade gliomas.

[0056] (2) OTC embedding of 15 tissue samples and frozen sections for later use.

[0057] (3) Randomly select two samples for IHC testing of PQLC2 immunohistochemistry. After the preliminary experiment is confirmed, complete IHC staining of the remaining tissue samples. Photographs are taken under white light, and each slide provides three high-magnification and three low-magnification field images.

[0058] (4) Rewarming of frozen sections: After taking the frozen sections out of the refrigerator, place them at room temperature to rewarm, then soak and wash them in pure water for 3 to 5 minutes.

[0059] (5) Antigen retrieval: Place the tissue sections in a retrieval box filled with citric acid antigen retrieval buffer (pH 6.0) in a microwave oven for antigen retrieval. Heat on medium heat for 8 minutes until boiling, then turn off the heat for 8 minutes to keep warm and then switch to medium-low heat for 7 minutes. During this process, prevent excessive evaporation of the buffer and do not allow the slides to dry out. After cooling naturally, place the slides in PBS (pH 7.4) and wash them three times on a decolorizing shaker, shaking for 5 minutes each time.

[0060] (6) Blocking endogenous peroxidase: Place the sections in 3% hydrogen peroxide solution and incubate at room temperature in the dark for 25 minutes. Place the slides in PBS (pH 7.4) and wash them three times on a decolorizing shaker, each time for 5 minutes.

[0061] (7) Serum blocking: After the sections are slightly dried, use a histochemical pen to draw a circle around the tissue (to prevent the antibody from escaping). Add 3% BSA to the circle to evenly cover the tissue and block at room temperature for 30 minutes. (If the primary antibody is goat-derived, use rabbit serum for blocking; if the primary antibody is from other sources, use BSA for blocking).

[0062] (8) Add primary antibody: Gently shake off the blocking solution, add PQLC2 antibody diluted 1:100 in PBS to the slice, and incubate the slice flat in a humidified box at 4°C overnight (add a small amount of water to the humidified box to prevent the antibody from evaporating).

[0063] (9) Adding secondary antibody: Place the sections in PBS (pH 7.4) and wash them three times on a decolorizing shaker, each time for 5 minutes. After the sections are slightly dried, add a secondary antibody (HRP-labeled) of the same species as the primary antibody to the circle to cover the tissue and incubate at room temperature for 50 minutes.

[0064] (10) DAB staining: The sections were placed in PBS (pH 7.4) and washed three times on a decolorizing shaker, each time for 5 minutes. After the sections were slightly dried, freshly prepared DAB staining solution was added dropwise to the circle. The staining time was monitored under a microscope. A positive color was brownish yellow. The sections were rinsed with tap water to terminate the staining.

[0065] (11) Re-staining of cell nuclei: Re-stain with hematoxylin for about 3 minutes, rinse with tap water, differentiate with hematoxylin differentiation solution for a few seconds, rinse with tap water, return to blue with hematoxylin bluing solution, and rinse with running water.

[0066] (12) Dehydration and sealing: Place the slices in 75% alcohol for 5 minutes, 85% alcohol for 5 minutes, anhydrous ethanol for 5 minutes, anhydrous ethanol for 5 minutes, and xylene for 5 minutes to dehydrate and make them transparent. Take the slices out of xylene and let them dry slightly, then seal them with neutral gum.

[0067] (13) The dried sections can be observed under a microscope or images can be collected.

[0068] Figure 11Images of ICH in adjacent, low-grade, and high-grade gliomas. Immunohistochemistry results on frozen sections show that hematoxylin stains nuclei in blue, while DAB stains positive nuclei in brown.

[0069] Figure 12 Statistical results of PQLC2 expression in adjacent cancer tissues, low-grade and high-grade gliomas.

[0070] from Figure 11 and Figure 12 It can be seen that there is a significant difference in the expression level of PQLC2 in adjacent cancer tissues and in high-grade GBM tissues, and the expression of PQLC2 in high-grade GBM tissues is significantly increased.

[0071] The above description is only a preferred embodiment of the present invention and does not constitute any formal limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can, without departing from the spirit and technical solution of the present invention, use the methods and technical contents disclosed above to make many possible changes and modifications to the technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention, still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A glioma marker, characterized in that: The glioma marker is PQLC2 protein.

2. Use of the glioma marker according to claim 1 in the preparation of a drug for treating glioma.

3. Use of the glioma marker according to claim 1 in preparing a glioma detection kit.

4. Use of the glioma marker according to claim 1 in preparing a detection kit for glioma prognosis judgment.

5. The use according to claim 4, characterized in that The prognosis of glioma patients can be predicted by detecting the expression level of the PQLC2 gene.

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