Use of m6a methylation level in lncrna-wee2-as1 as a glioma marker

Abstract

The present application relates to the m 6 Application of methylation level of A as glioma marker, the present application confirms the m 6 The methylation modification level is related to the prognosis condition of brain glioma, and the increase of methylation abundance and expression level means worse prognosis. In addition, the present application researches and confirms that the expression of lncRNA-WEE2-AS1 is related to the formation of drug resistance of anti-tumor drugs, and inhibiting the expression of the lncRNA helps to improve the resistance of dasatinib, and provides a feasible treatment scheme for the clinical targeted treatment of drug-resistant patients.

Description

Technical Field

[0001] This invention belongs to the technical field of glioma markers and therapeutic drugs, specifically relating to m in lncRNA-WEE2-AS1. 6 The application of A methylation level as a marker for glioma and the application of lncRNA-WEE2-AS1 inhibitors as sensitizers for dasatinib. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Glioblastoma (GBM) is the most aggressive and deadliest brain tumor with the worst prognosis. The current standard treatment for glioblastoma is surgery, followed by oral chemotherapy with temozolomide and concurrent radiotherapy; however, the median survival is approximately 14 months. Therefore, identifying new targets and strategies for glioblastoma treatment is crucial.

[0004] Recent research evidence suggests that long non-coding RNAs (lncRNAs), as key regulators of gene expression, have complex regulatory relationships with tumor-associated gene expression at the epigenetic, transcriptional, and post-transcriptional levels, which are closely related to tumorigenesis and malignant progression. The tissue-specific and condition-specific expression patterns of lncRNAs indicate that they are potential biomarkers and provide a theoretical basis for their clinical targeting. N6-methyladenosine (m... 6 A) represents the methylation modifications of the most common mRNA and lncRNA in eukaryotes, regulating almost every aspect of mRNA metabolism, including RNA splicing, translation, stability, and degradation, and modulating gene expression at the posttranscriptional level. Given m 6 The functional importance of A-methylation modification in normal biological processes is increasingly supported by validation studies, indicating that RNAm 6 Dysregulation of A-modification also contributes to the initiation, progression, and resistance of GBM. However, to date, m 6 The distribution and function of A-modified lncRNAs, particularly the role of elevated lncRNAs in GBM tissues, have not been fully elucidated. Understanding m 6 The role of A-regulated lncRNAs in the pathogenesis of GBM will provide important theoretical basis for the clinical diagnosis and targeted therapy of GBM patients. Meanwhile, selecting targeted drugs and interfering RNA therapies against this lncRNA will bring new approaches to the treatment of glioblastoma. Summary of the Invention

[0005] This invention screened glioma-related lncRNAs using high-throughput sequencing and obtained the lncRNA-WEE2-AS1, which is associated with a poor prognosis in gliomas. This invention confirmed the correlation between methylation modification of this lncRNA and glioma development, clarified the corresponding regulatory mechanism, and provided the application of lncRNA methylation as a glioma biomarker. Furthermore, to clarify the clinical application of the above-mentioned lncRNA, this invention also explored the relationship between lncRNA expression and clinical drugs. Inhibiting the expression of this lncRNA helps improve the sensitivity of corresponding drugs and helps improve clinical drug resistance.

[0006] Based on the above research findings, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides m from lncRNA-WEE2-AS1. 6 Application of A methylation level as a marker for glioma.

[0008] This invention is the first to use m 6 A high-throughput sequencing (m 6 A-seq reported on the m in the GBM organization 6 A-modified lncRNAs exhibit high specificity compared to normal brain tissue. Among them, lncRNA-WEE2-AS1 shows high specificity in m 6 Both the modification and transcriptional levels of A were significantly upregulated, and its high expression was associated with a poor prognosis of GBM. Functionally, this invention demonstrates that WEE2-AS1 promotes the pathogenesis and malignant development of GBM both in vitro and in vivo. Mechanistically, METTL3 mediates the m-modification of WEE2-AS1. 6 A modification enhances its expression in an IGF2BP3-dependent manner. Furthermore, WEE2-AS1 promotes RPN2 protein stability by blocking CUL2-mediated RPN2 K322 ubiquitination, thereby activating the AKT signaling pathway to promote GBM malignant progression.

[0009] Preferably, the application as a glioma marker includes, but is not limited to, its use as a marker for glioma diagnosis or prognosis, and the specific application methods include, but are not limited to, any of the following:

[0010] (1) The application of the lncRNA-WEE2-AS1 methylation level detection reagent as a reagent for the preparation of glioma diagnostic or prognostic reagents;

[0011] (2) The prognostic level of glioma patients was assessed by detecting the methylation level of lncRNA-WEE2-AS1 in the clinical samples of the subjects;

[0012] (3) By detecting the methylation level of lncRNA-WEE2-AS1 in the clinical samples of the subjects, it is possible to determine whether the subjects have glioma.

[0013] In the above application methods (1)-(3), the methylation level in the lncRNA-WEE2-AS1 includes m 6 A methylation abundance and expression level; preferred lncRNA-WEE2-AS1 m 6 The site of A methylation is exon 7 (chr7:141,704,725 to Ch7:141,705,148).

[0014] In a second aspect, the present invention provides a glioma prognosis or diagnostic kit, the kit comprising reagents for detecting methylation levels in lncRNA-WEE2-AS1.

[0015] Preferably, the reagents for detecting the methylation level of lncRNA-WEE2-AS1 include, but are not limited to, reagents for detecting the methylation level of the above sites based on methods such as MeRIP-seq, miCLIP-seq, SCARLET, LC-MS / MS, etc. The kit may include reagents for extracting nucleic acid components from clinical samples, immunomagnetic beads, etc.

[0016] In translational medicine, this invention found that blocking WEE2-AS1 expression improves the sensitivity to dasatinib treatment. This work highlights WEE2-AS1 as a potential prognostic biomarker and therapeutic target in GBM, and its knockout significantly improves the efficacy of dasatinib in GBM, providing a promising strategy for improving targeted combination therapy in GBM patients.

[0017] In a third aspect, the present invention provides the use of lncRNA-WEE2-AS1 inhibitors as dasatinib sensitizers.

[0018] In the third aspect mentioned above, the lncRNA-WEE2-AS1 inhibitor includes, but is not limited to, small molecule compounds, nucleic acids, active peptides or hormones that can inhibit the expression of lncRNA-WEE2-AS1. It can also be reagents involved in knocking out or knocking down lncRNA-WEE2-AS1 in the body through genetic engineering. The genetic engineering means include, but are not limited to, methods of modifying the target site through homologous recombination, plasmid transfection, CRISPER restriction enzyme system, etc.

[0019] In a fourth aspect, the present invention provides a pharmaceutical composition comprising an active dose of an lncRNA-WEE2-AS1 inhibitor and dasatinib.

[0020] In the above composition, dasatinib should be at a therapeutically effective dose, which can be routinely determined based on the physician's clinical experience; the ratio of the lncRNA-WEE2-AS1 inhibitor to dasatinib can be routinely adjusted based on factors such as the subject's tolerance to the above drugs; in a more preferred embodiment, the drug composition is administered to patients with glioma or to tumor patients who have developed resistance to dasatinib.

[0021] In a fifth aspect, the present invention provides a method for reversing dasatinib resistance, the method comprising administering an active dose of a lncRNA-WEE2-AS1 inhibitor to a glioma patient in need of treatment.

[0022] The beneficial effects of one or more of the above technical solutions are as follows:

[0023] 1. This invention first clarifies the correlation mechanism between lncRNA-WEE2-AS1 expression and poor prognosis of glioma. Compared with the prior art, it further clarifies the application of the methylation modification level of exon 7 in lncRNA-WEE2-AS1 as a disease biomarker. The above results further expand the feasible means for the prognosis or diagnosis of glioma.

[0024] 2. Dasatinib is a multi-target oral inhibitor that can cross the blood-brain barrier, but its monotherapy efficacy is poor, and combination therapy is often used. This invention provides a way to enhance the body's sensitivity to dasatinib. Under the same dosage, it is expected to improve the therapeutic effect of dasatinib or improve dasatinib resistance that occurs during treatment, providing a more ideal clinical treatment option for glioma patients receiving dasatinib. Attached Figure Description

[0025] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0026] Figure 1 To identify and characterize m 6 Results related to the use of A-modified lncRNA WEE2-AS1 as a potential biomarker for GBM;

[0027] in, Figure 1 A is through m 6 A-seq was used to screen for different types of methylated lncRNAs in GBM tumor tissues compared with normal brain tissues (NBTs);

[0028] Figure 1 B represents Log2FC (lncRNA expression) and Log2FC (m6 A lattice plot of A-methylation difference values;

[0029] Figure 1 C is a Venn diagram showing the overlap between the upregulated lncRNAs and the average expression levels of the top 300 lncRNAs in the TCGA and Qilu datasets, as well as lncRNAs in the overregulated quadrant;

[0030] Figure 1 D represents the results of the GEPIA database analysis: WEE2-AS1 was significantly overexpressed in glioma tissues compared to GTEx NBTs;

[0031] Figure 1 E represents the Kaplan-Meier survival curve: WEE2-AS1 is a prognostic risk factor for glioma;

[0032] Figure 1 F represents overexpression of WEE2-AS1 in GBM tissues;

[0033] Figure 1 G represents the Kaplan-Meier survival curve analysis results: showing that WEE2-AS1 is a prognostic risk factor for GBM;

[0034] Figure 1 H indicates that WEE2-AS1 was significantly overexpressed in the GBM group compared to NBTs in the Qilu cohort.

[0035] Figure 1 I indicates that NBTs in GBM tissues were significantly upregulated compared to the Qilu cohort using qRT-PCR.

[0036] Figure 1 J was significantly upregulated in both GBM and GSCs; data are expressed as mean ± SD.

[0037] Figure 1 K represents the subcellular localization of WEE2-AS1(Cy3) in LN229 and U251 GBM cells as shown by RNA-FISH assay; cell nuclei were stained with DAPI (bule); scale bar, 25 μm; statistical significance is shown as *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001;

[0038] Figure 1 L represents the relative expression of WEE2-AS1 in the cytoplasm (GAPDH was used as a cytoplasmic marker) and nucleus (U6 was used as a nuclear marker) by qRT-PCR assay; data are expressed as mean ± SD.

[0039] Figure 2WEE2-AS1 promotes the proliferation, migration, and invasion of GBM cells in vitro and in vivo;

[0040] in, Figure 2 A represents the inhibitory effect of WEE2-AS1 knockout on the proliferation of U118MG and LN229 GBM cells;

[0041] Figure 2 B represents the promoting effect of WEE2-AS1 overexpression on A172 and U251 GBM cells;

[0042] Figure 2 C represents images of tumor sphere formation in the sh-NC or sh-WEE2-AS1 group;

[0043] Figure 2 D represents a representative tumor sphere formation image of GSCs with ov-NC or ov-WEE2-AS1; scale bar, 200 micrometers; histogram representing the mean sphere diameter; data represent the mean ± SD of at least three independent experiments.

[0044] Figure 2 E represents the limiting dilution test of GSCs for sh-NC or sh-WEE2-As1;

[0045] Figure 2 F represents the limiting dilution test of GSCs for ov-NC or ov-WEE2-AS1;

[0046] Figure 2 G represents the migration and invasion capabilities of GBM cells with sh-NC or sh-WEE2-AS1.

[0047] Figure 2 H represents the migration and invasion ability of GBM cells with ov-NC or ov-WEE2-AS1; scale bar, 200 micrometers; quantification histograms represent relative cell numbers; data represent the mean ± SD of at least three independent experiments.

[0048] Figure 2 I represents the invasive ability of GBM cells in the sh-NC or sh-WEE2-AS1 group;

[0049] Figure 2 J represents the invasive ability of GBM cells with ov-NC or ov-WEE2-AS1.

[0050] Figure 2 K represents the tumor size in mice implanted with LN229 cells of sh-WEE2-AS1 or sh-NC.

[0051] Figure 2 L represents the tumor size in mice implanted with U251MG cells of ov-WEE2-AS1 or ov-NC.

[0052] Figure 2 M represents the Kaplan-Meier survival curve of LN229 cells implanted in mice with sh-WEE2-AS1 or sh-NC cells;

[0053] Figure 2 N represents the Kaplan-Meier survival curves of mice expressing U251MG cells of ov-WEE2-AS1 or ov-NC; logarithmic ranking analysis was performed, with n≥4 for each group; statistical significance was indicated by *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

[0054] Figure 3 m mediated by METTL3 6 The A modification enhances the stability of WEE2-AS1 in an IGF2BP3-dependent manner;

[0055] in, Figure 3 A is the m on WEE2-AS1 in the GBM organization and NBTs displayed by IGV software. 6 A. Peak distribution;

[0056] Figure 3 B represents m in glioma tissue from the GEPIA database. 6 Correlation between A methyltransferase METTL3 and WEE2-AS1;

[0057] Figure 3 C indicates that MeRIP detection shows WEE2-AS1 was... 6 Antibody A was highly enriched, and the enrichment was downregulated in METTL3-blocked (left) U251 and (right) GSC267 cells; data represent mean ± SD of at least three independent experiments.

[0058] Figure 3 D represents the relative expression of METTL3 and WEE2-AS1 in (left) U251MG GBM cells and (right) GSC267 GSCs transfected with sh-NC or sh-METTL3, as shown by qRT-PCR assay; data represent the mean ± SD of at least three independent experiments.

[0059] Figure 3 E represents the detection of WEE2-AS1 RNA stability in METTL3 gene knockout (left) U251MG and (right) GSC267 cells by qRT-PCR, and the cells were treated with Act-D for a specified time period.

[0060] Figure 3F represents proteins that interact with WEE2-AS1 as shown by silver staining assays; these proteins were identified by RNA pulldown / mass spectrometry.

[0061] Figure 3 G represents the interaction between WEE2-AS1 and IGF2BP3 in U251 and GSC267 cells as shown by Western blot analysis; WEE2-AS1 antisense beads and beads served as negative controls.

[0062] Figure 3 H represents the relative enrichment of WEE2-AS1 detected by IGF2BP3 antibody as shown by RIP-qPCR assay. Data represent the mean ± SD of at least three independent experiments;

[0063] Figure 3 I represents m in glioma tissue from the GEPIA database. 6 A. Correlation between readings IGF2BP3 and WEE2-AS1;

[0064] Figure 3 J represents the relative expression of IGF2BP3 and WEE2-AS1 in (left) U251MG GBM cells and (right) GSC267 GSCs transfected with sh-NC or sh-METTL3, as shown by qRT-PCR assay; data represent mean ± SD of at least three independent experiments.

[0065] Figure 3 K represents the measurement of WEE2-AS1 RNA stability in IGF2BP3 gene knockout (left) U251MG and (right) GSC267 cells by qRT-PCR, and treatment with Act-D for a specified time period.

[0066] Figure 3 L is a schematic diagram of the Flag marker carrier showing the RRM and KH structures of IGF2BP3;

[0067] Figure 3 M is the structure of FLAG-labeled IGF2BP3 in HEK293T cells as shown by Western blot.

[0068] Figure 3 N represents the enrichment of WEE2-AS1 in HEK293T cells transfected with full-length and truncated FLAG-labeled plasmids by RIP-qPCR analysis; data represent the mean ± SD of at least three independent experiments.

[0069] Figure 3 O is the 7th exon of WEE2-AS1, m 6A schematic diagram of the location of pattern A; a schematic diagram of the P-mutated (GGAC ​​to GGCC) firefly luciferase reporter vector;

[0070] Figure 3 Q represents the luciferase assay results in m in U251MG and GSC267 cells. 6 The major location of A-mediated WEE2-AS1; statistical significance was shown as ns > 0.05, **P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001;

[0071] Figure 4 The results indicate that the interaction between WEE2-AS1 and RPN2 proteins promotes the malignant progression of GBM by activating the AKT signaling pathway.

[0072] in, Figure 4 A indicates that the GSVA results show that pro-cancer signaling pathways are significantly upregulated in GBM samples with high RPN2;

[0073] Figure 4 B represents the correlation between RPN2 and the enrichment scores of cancer marker pathways in the TCGAGBM cohort;

[0074] Figure 4 C represents the TCGA GBM dataset, which shows that classic oncogenic pathways involved in tumor pathogenesis are significantly enriched in the RPN2 high-expression group;

[0075] Figure 4 D shows the interaction between WEE2-AS1 and RPN2 in (top) LN229 GBM cells and (bottom) GSC20 GSCs, as revealed by Western spectroscopy.

[0076] Figure 4 E represents the relative enrichment of WEE2-AS1 detected by RIP-qPCR assay using RPN2 antibody; data represent the mean ± SD of at least three independent experiments.

[0077] Figure 4 F represents RNA. FISH-IF assay showed the co-localization of WEE2-AS1 and RPN2 in GBM cells.

[0078] Figure 4 G represents the secondary structure of WEE2-AS1 predicted by RNAfold WebServer;

[0079] Figure 4 H represents the RNA pull-down experiment performed on different fragments of WEE2-AS1 corresponding to biotinylated RNA transcribed in vitro from LN229 and GSC20 cells.

[0080] Figure 4 I represents the phosphorylation level of AKT in LN229 GBM cells of sh-NC or sh-WEE2-AS as detected by Western spectroscopy.

[0081] Figure 4 J represents the phosphorylation level of AKT in LN229 GBM cells with si-NC or si-RPN2 as detected by Western spectroscopy.

[0082] Figure 4 K represents the CCK-8 assay used to detect the proliferation capacity of GBM cells transfected with ov-NC or ov-WEE2-AS1 and siRPN2;

[0083] Figure 4 L represents a representative tumor sphere formation image of GSCs co-transfected with ov-NC or ov-WEE2-AS1 and si-RPN2; scale bar, 200 μm; quantitative bar chart represents the mean sphere diameter; data represent the mean ± SD of at least three independent experiments.

[0084] Figure 4 M represents the migration and invasion ability of GBM cells co-transfected with ov-NC or ov-WEE2-AS1 and siRPN2 as detected by Transwell migration and invasion assays; scale bar, 200 μm; quantification histograms represent relative cell numbers; data represent the mean ± SD of at least three independent experiments.

[0085] Figure 4 N represents Western blot analysis showing that E-cadherin, N-cadherin, CD44, RPN2, AKT, and p-AKT proteins were expressed in GBM cells co-transfected with ov-NC or ov-WEE2-AS1 and siRPN2; statistical significance was indicated by *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

[0086] Figure 5 The results related to WEE2-AS1 stabilizing RPN2 protein by preventing CUL2-mediated ubiquitin-protein body degradation;

[0087] in, Figure 5 A represents the relative expression in GBM cells transfected with sh-NC or sh-WEE2-AS1 by qRT-PCR; data represent the mean ± SD of at least three independent experiments.

[0088] Figure 5 B shows the Western blot results of RPN2 protein expression in GBM cells transfected with sh-NC or sh-WEE2-AS1.

[0089] Figure 5 C represents the time of GBM cell transfection with sh-NC or sh-WEE2-AS1 and the expression results of RPN2 protein;

[0090] Figure 5 D represents the expression result of RPN2 protein transfected into sh-NC or sh-WEE2-AS1;

[0091] Figure 5 E represents the level of RPN2 ubiquitination in GBM cells transfected with sh-NC or sh-WEE2-AS1, as detected by Co-IP.

[0092] Figure 5 In Figure F, the top figure shows the potential ubiquitination sites of RPN2 predicted by BDM-PUM (http: / / bdmpub.biocuckoo.org / ) and the UbiBrowser database (http: / / ubibrowser.ncpsb.org / ); the bottom figure shows the RPN2 ubiquitination level detected by Co-IP experiment after transfection with Flag-labeled wild-type or mutant RPN2 KR vectors.

[0093] Figure 5 G is used for Co-IP detection of RPN2 ubiquitination levels in GBM cells transfected with wild-type or K322 mutant RPN2 KR vectors transfected with sh-NC or sh-WEE2-AS1 and Flag-labeled;

[0094] Figure 5 In Figure H, the top image shows the crystal structure of the RPN2 protein with K322; the bottom image shows the retention capacity of the K322 ub site on the RPN2 protein.

[0095] Figure 5 I is a silver staining assay showing protein bands interacting with RPN2; the arrows indicate the CUL2 protein bands.

[0096] Figure 5 J represents the co-localization of RPN2 and CUL2 in GBM cells as shown by immunofluorescence staining assay; scale bar, 25 μm;

[0097] Figure 5 K represents the expression of CUL2 and RPN2 proteins in GBM cells transfected with si-NC or si-CUL2, as detected by Western spectroscopy.

[0098] Figure 5 L represents the level of RPN2 ubiquitination in GBM cells transfected with si-NC or si-CUL2 as shown by Co-IP assay;

[0099] Figure 5M represents the interaction strength between RPN2 and CUL2 in GBM cells transfected with ov-NC or ov-WEE2-AS1 by Co-IP assay, and the cells were treated with MG132 at specified time points; statistical significance was indicated by ns>0.05.

[0100] Figure 5 N represents the expression of CUL2 and RPN2 proteins in GBM cells transfected with sh-NC or sh-WEE2-AS1 as shown by Western spectroscopy.

[0101] Figure 6 To characterize the effect of knocking out WEE2-AS1 on improving the efficacy of dasatinib against GBM;

[0102] in, Figure 6 A represents the Spearman correlation analysis between WEE2-AS1 and drug bioavailability (AUC) in the CellMiner database;

[0103] Figure 6 In section B, the top figure shows the Spearman's correlation between WEE2-AS1 and dasatinib AUC; the bottom figure compares the estimated dasatinib AUC between the high and low WEE2-AS1 groups.

[0104] Figure 6 C represents the Pearson correlation between the expression of WEE2-AS1 and PDGFRA;

[0105] Figure 6 D represents the proliferative capacity of GBM cells transfected with sh-NC or sh-WEE2-AS1 in the CCK-8 assay, and the cells were treated with dasatinib (20 nM) at a specified time.

[0106] Figure 6 E represents a representative tumor sphere formation image of GSCs transfected with sh-NC or sh-WEE2-AS1, processed with dasatinib (20 nM) for a specified time; scale bar, 200 μm; quantified histogram representing the mean sphere diameter; data represent the mean ± SD of at least three independent experiments.

[0107] Figure 6F is the left-hand bioluminescence image showing tumor size in mice implanted with fluorescein-labeled LN229 cells expressing sh-WEE2-AS1 or sh-NC and treated with dasatinib (10 mg / kg) for a specified time; the right-hand panel is a quantitative histogram representing bioluminescence flux; data represent mean ± SD, n = 5 per group; G is the Kaplan-Meier survival curve of mice implanted with fluorescein-labeled LN229 cells expressing sh-WEE2-AS1 or sh-NC and treated with dasatinib (10 mg / kg); logarithmic analysis was performed, n = 5 per group.

[0108] Figure 6 H represents the proposed functional working model of WEE2-AS1 in the malignant progression of GBM; statistical significance is shown as ***P<0.001 and ****P<0.0001. Detailed Implementation

[0109] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0110] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0111] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0112] Example 1

[0113] I. Experimental Materials and Methods

[0114] 1. Patients and specimens

[0115] Human GBM tissue and normal brain tissue (cortex from patients undergoing decompression surgery for traumatic brain injury or hypertensive intracerebral hemorrhage) were obtained from patients hospitalized at Qilu Hospital from November 2017 to December 2019. All participants provided written informed consent, and the study was approved by the Scientific Research Ethics Committee of Qilu Hospital, Shandong University (Approval No.: KYLL-2018-324).

[0116] 2. Data Collection

[0117] The Cancer Genome Atlas (TCGA) GBM RNA sequencing (RNA-seq) transcriptome data and corresponding clinicopathological parameters of GBM patients were obtained from the TCGA database (http: / / cancergenome.nih.gov / ). The Wang RNA-seq dataset (FPKM format) and clinical information were extracted from the supplemental data of the article; missing data were obtained using the K-nearest neighbor (KNN) method. RNA-seq data from local samples in this embodiment have been deposited in the Genomic Sequence Archive (GSA), accession number CRA002339. Processed data and basic association analyses will be provided in the supplemental data or, upon reasonable request, to the relevant authors.

[0118] 3. Cell Culture

[0119] Human GBM cell lines (U251MG, U118MG, LN229, and A172) and human embryonic kidney cell line 293T (HEK293T) were purchased from ATCC. Cells were maintained in DMEM supplemented with 10% fetal bovine serum. All patient-derived GSC cell lines, including mesenchymal (MES) subtype GSC cell lines (GSC20 and GSC267), bone marrow (PN) subtype GSC cell lines (GSC11 and GSC8-11), and neural progenitor cells (NPCs), were donated by Dr. Frederick F. Lang and Dr. Krishna PLBhat (University of Texas MD Anderson Cancer Center, Houston, USA). Cells were cultured in DMEM / F12 supplemented with B27 (Invitrogen, California), 20 ng / ml EGF (R&D Systems, USA), and 20 ng / ml bFGF (R&D Systems, California, USA). Cells were cultured in a standard humid atmosphere at 37°C and 5% carbon dioxide.

[0120] 4. Western blot

[0121] Proteins were extracted from GSC or GBM cells. The following primary antibodies were used. GAPDH (CST, 5174), β-actin (CST, 14074), CD44 (Proteintech, 15675-1-AP), AKT (CST, 9272), p-Akt (Ser473, CST, 4060), CDK1 / cdc2 (CST, 9116), p-CDK1 / cdc2 (Tyr15) (CST, 4 539), P21 (CST, 2947), IGF2BP3 (Abcam, ab177477), Ub (CST, 3936), DYKDDDDK-Flag (CS T, 14793), His-Flag (CST, 12698), CUL2 (Santa, sc-166506), and RPN2 (Abcam, ab244399).

[0122] 5. RNA interference and lentiviral transfection

[0123] Following the Lipo3000 (L300015, Invitrogen, USA) protocol, temporary knockout of WEE2-AS1, IGF2BP3, RPN2, and CUL2 was achieved using small interfering RNA (siRNA) from GenePharma (Shanghai, China) or RiboBio (Guangzhou, China). The full-length human WEE2-AS1 sequence and control sequences were cloned into the GV502 lentiviral vector to construct lentiviruses for stable overexpression (Genechem, China). Simultaneously, the WEE2-AS1 gene knockout sequence (shRNA) and the corresponding confounding control (shNC) were cloned into the GV112 lentiviral vector to construct lentiviruses (Genechem, China). The detailed oligonucleotides used in this study include si-NC, si-WEE2-AS1, si-RPN2, si-IGF2BP3, sh-METTL3, si-CUL2, sh-WEE2-AS1, and sh-NC. The siRNA was designed and synthesized by Guangzhou Ruibo Biotechnology Co., Ltd., and the shRNA was designed and synthesized by Shanghai Jikai Gene Medical Technology Co., Ltd.

[0124] 6. RNA extraction and RT-PCR

[0125] According to the manufacturer's agreement, TRIzol (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from cells. Primers used included WEE2-AS1, GAPDH, β-actin, METTL3, RPN2, and IGF2BP3 amplification primers, which were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0126] Quantitative PCR was performed using TB green Premix Ex Taq (Takara; Tokyo, Japan) on a real-time PCR detection system (480II, Roche; Basel, Switzerland).

[0127] 7. Animal Research

[0128] Using a stereotactic apparatus, fluorescein-expressing human GBM cell lines were randomly injected into the frontal lobes of 4-week-old BALB / c nude mice (5 × 10⁻⁶ cells / mL). 5 A xenograft model was established using mice (10 μL PBS). Tumor growth was examined on days 7, 14, and 21 using bioluminescence imaging (IVIS spectrum in vivo imaging system, PerkinElmer; Hopkinton, MA, USA). Mouse brains were harvested by H&E and IHC staining. For animal experiments involving combination therapy, dasatinib (HY-10181, MCE) was dissolved in 90% SBE-β-CD (C871854, MACKLIN) saline. Mice were administered the solution daily by gavage at a concentration of 10 mg / kg. All procedures involving mice were approved by the Board of Institutional Animal Care and Use Committee of Qilu Hospital, Shandong University (Institutional Animal Care and Use Committee Issue No.: DWLL-2021-039).

[0129] II. Research Results

[0130] 1. m 6 A-modified lncRNA WEE2-AS1 is highly expressed in GBM.

[0131] To understand the role of lncRNAs in GBM 6 Due to differences in modification patterns, this embodiment performed m on three human GBM tumor tissues and three normal brain tissues (NBTs). 6 A-seq detection. Results showed a significant number of lncRNAs with m 6 The abundance of A modifications changed significantly, with pseudogenes (24.3%), lincRNAs (36.7%), and antisense (37.8%) lncRNAs accounting for the majority. Figure 1 A). By analyzing m 6 The combined analysis of A-seq and RNA-seq data in this example revealed different methylated m groups in GBM tissues. 6 There is a positive correlation between the abundance of A modification and the corresponding lncRNA expression level. Figure 1B, Pearson correlation: 0.592, P < 0.001). These genes were mainly divided into four groups, including 27 hypermethylated and upregulated genes ("hyper-up"), 58 hypomethylated and downregulated genes ("hypo-down"), 3 hypermethylated but downregulated genes ("hyper-down"), and 6 hypomethylated but upregulated genes ("hypo-up"). Subsequently, this embodiment intersected the lncRNAs with the average expression levels of the top 300 in the TCGA and Qilu datasets with the lncRNAs in the upper quadrant, and found that WEE2-AS1 was the only m 6 lncRNAs with simultaneously upregulated methylation abundance and expression levels (A) Figure 1 C). TCGA data confirmed that WEE2-AS1 expression in gliomas (LGG and GBM) was significantly higher than in normal tissues, with GBM showing the highest expression. Figure 1 D). Patients with high WEE2-AS1 expression have a significantly poorer prognosis. Figure 1 E), and the same result was obtained in another GBM dataset ( Figure 1 F, G). Compared to NBTs in the Qilu queue, WEE2-AS1 was also significantly increased in the GBM organization ( Figure 1 H). In vitro qRT-PCR results also validated these results ( Figure 1 I). Furthermore, compared to the corresponding normal cells, WEE2-AS1 expression was significantly upregulated in both GBM and GSCs (I). Figure 1 J). The function of lncRNAs is mostly related to their intracellular localization. Nuclear-cytoplasmic fractionation experiments and FISH assays showed that WEE2-AS1 is mainly localized in the cytoplasm (J). Figure 1 In summary, these results indicate that WEE2-AS1 is significantly upregulated in GBM tissues and is a predictable risk factor, suggesting its potential involvement in the malignant progression of GBM.

[0132] 2. WEE2-AS1 promotes the proliferation, migration, and invasion of GBM cells in vitro and in vivo.

[0133] To explore the function of WEE2-AS1 in GBM proliferation, metastasis, and invasion, this embodiment aims to describe the changes in cell biological behavior of GBM cells with WEE2-AS1 silencing and overexpression in vitro. Knockout of WEE2-AS1 significantly inhibited the proliferation of U118MG and LN229 GBM cells. Figure 2 A), while overexpression of WEE2-AS1 significantly promoted these cellular behaviors in A172 and U251GBM cells. Figure 2B), such as CCK-8, colony formation, and EdU detection. Furthermore, this embodiment performed neurosphere formation and limiting dilution assays on GSC20 and GSC267 GSCs, and the results showed that WEE2-AS1 significantly promoted tumor sphere expansion in GSCs ( Figure 2 C, D) and sphere-forming ability ( Figure 2 E, F). Furthermore, transpore, wound healing, and three-dimensional collagen spheroid invasion assays showed that WEE2-AS1 knockout impaired the migration and invasion abilities of U118MG and LN229GBM cells, while WEE2-AS1 overexpression significantly promoted these cellular behaviors. Figure 2 GJ).

[0134] Furthermore, in vivo experiments showed that downregulation of WEE2-AS1 significantly inhibited tumor growth and prolonged the survival time of tumor-bearing mice, while overexpression of WEE2-AS1 had the opposite effect. Figure 2 In summary, these results indicate that WEE2-AS1 plays a carcinogenic role in GBM by regulating cell proliferation, migration, and invasion.

[0135] 3. METTL3-mediated m 6 The A modification enhances the stability of WEE2-AS1 in an IGF2BP3-dependent manner.

[0136] m 6 A modifications regulate all stages of the RNA life cycle, such as RNA splicing, stabilization, degradation, and nuclear export, thereby modulating RNA expression and function. Because this embodiment focuses on m... 6 Joint analysis of A-seq and RNA-seq data revealed that, compared with normal tissue, GBM tissue contained m 6 The abundance of A-modification and the gene expression of WEE2-AS1 were both significantly upregulated. This example then explores m 6 The intrinsic mechanism of A-mediated WEE2-AS1 expression. Using IGV software to analyze m... 6 Visualization using A-seq revealed that, compared to NBTs, m on exon 7 of WEE2-AS1 in the GBM organization... 6 A-modification abundance was upregulated ( Figure 3 A). GEPIA database analysis showed that m in glioma tissue 6 There is a significant positive correlation between the expression of A methyltransferases METTL3 and WEE2-AS1. Figure 3 B). Furthermore, MeRIP-qPCR assays showed that WEE2-AS1 can be converted to m 6 Antibody A was significantly enriched, and its enrichment was significantly downregulated in METTL3 knockout GBM cells compared to the NC group. Figure 3C). Furthermore, knockout of METTL3 significantly reduced the expression level of WEE2-AS1 in GBM cells ( Figure 3 D). Therefore, this embodiment proposes that m 6 A may affect the stability of WEE-AS1. To verify this hypothesis, this embodiment evaluated the effect of METTL3 on the stability of WEE2-AS1 using an actinomycin D RNA stability assay. The study found that the half-life of WEE2-AS1 in the METTL3 knockout group was significantly shorter than that in the NC group. Figure 3 E). Subsequent RNA pulldown / mass spectrometry analysis results from this embodiment showed that WEE2-AS1 and m 6 Reader A binds to IGF2BP3, a well-known RNA-binding protein belonging to the insulin-like growth factor 2 mRNA-binding protein (IGF2BP) family, to stabilize its target RNA. Figure 3 F), it has been reported that IGF2BP plays a carcinogenic role in GBM progression. RNA pulldown and RIP-qPCR assays both confirmed that WEE2-AS1 can bind to the IGF2BP3 protein. Figure 3 G, H). GEPIA database analysis also showed a significant positive correlation between the expression of IGF2BP3 and WEE2-AS1 in glioma tissue. Figure 3 I). Consistent with the results for METTL3, knockout of IGF2BP3 significantly reduced the expression level and stability of WEE2-AS1. Figure 3 J, K). IGF2BP3 consists of two RNA recognition motifs (RRMs) and four K homology domains (KHs). Therefore, in this embodiment, a vector with six FLAG tags was subsequently constructed to detect which domains interact with WEE2-AS1. Figure 3 L, M). RIP-PCR experiments confirmed that the KH1-2 domain is crucial for the recognition of WEE2-AS1. Figure 3 N).

[0137] To further clarify m 6 The regulatory mechanism of A on WEE2-AS1 expression was explored in this embodiment. 6 A modification site. m in this embodiment... 6 A-seq data showed that m in the 7th exon region 6 The peak value of A varies from chr7:141,704,725 to Ch7:141,705,148, with two m values. 6 Pattern A (GGACs) was determined ( Figure 3O). This embodiment then uses a luciferase reporter containing firefly luciferase, followed by wild-type WEE2-AS1, mutant 1 (Mut-1), or mutant 2 (Mut-2), where the putative m of exon 7 is... 6 A mutation occurred at site A (from GGAC to GGCC) Figure 3 P). For example Figure 3 As shown in Q, in the case of IGF2BP3 knockout, the luciferase activity reported by wild-type and Mut-2 WEE2-AS fusion was significantly reduced, but there was no significant difference in the luciferase activity reported by Mut-1 WEE2-AS1 fusion. Figure 3 Q) indicates that site 1 in exon 7 of WEE2-AS1 is m 6 The main location of A regulation. In summary, the results of this embodiment show that METTL3-mediated m6A modification of WEE2-AS1 enhances its stability and promotes its expression in an IGF2BP3-dependent manner.

[0138] 4. WEE2-AS1 interacts with RPN2, promoting the malignant progression of GBM by activating the AKT signaling pathway.

[0139] Numerous studies have shown that cytoplasmic lncRNAs can interact with proteins and participate in cellular regulation. Pull-down experiments indicate that WEE2-AS1 can interact with RPN2 protein. GSVA results suggest that pro-tumor signaling pathways are significantly upregulated in GBM samples with high RPN2 compared to GBM samples with low RPN2. Figure 4 A). This embodiment also analyzes the correlation between RPN2 and cancer marker pathways. For example... Figure 4 As shown in Figure B, there is a significant positive correlation between RPN2 and these oncogenic pathways. Furthermore, similar to the GSVA enrichment analysis, the GSEA results also showed that in the TCGA GBM dataset, classic pathways involved in tumor pathogenesis were significantly enriched in the RPN2 high-expression group. Figure 4 C). Therefore, this embodiment hypothesizes that WEE2-AS1 may promote the malignant progression of GBM by affecting the function of RPN2. Then, this embodiment performed RNA pull-down and RIP-qPCR experiments to verify the interaction between WEE2-AS1 and RPN2. Figure 4 D, E). RNA FISH-immunofluorescence (FISH-IF) analysis was also performed, showing that WEE2-AS1 and RPN2 co-occur in the cytoplasm ( Figure 4F). To further investigate the secondary structure of the interaction between WEE2-AS1 and RPN2, the structure of the WEE2-AS1 molecule was predicted using RNAfold WebServer and divided into three main substructures, each containing a base pairing structure and a hairpin structure. Figure 4 G). RNA pull-down results showed that WEE2-AS1#3 bound to RPN2 as effectively as the full-length WEE2-AS1, while other substructures lost their binding ability. Figure 4 H) indicates that nucleotides 1590-2262 are necessary for binding to RPN2. Next, this example explores potential biological pathways downstream of the WEE2-AS / RPN2 axis. Functional enrichment analysis showed that GBM samples, which highly expressed both WEE2-AS1 and RPN2, were significantly enriched in the AKT signaling pathway (H). Figure 4 AC). This embodiment then investigated the phosphorylation levels of AKT in WEE2-AS1 knockout and overexpressing GBM. For example... Figure 4 I and Figure 4 As shown in G, overexpression of WEE2-AS1 enhanced AKT phosphorylation levels, but knockout of WEE2-AS1 inhibited AKT phosphorylation levels. Consistent with the results for WEE2-AS1, knockout of RPN2 also inhibited activation of the AKT signaling pathway. Figure 4 J). Rescue experiments showed that RPN2 knockout could compensate for the increased proliferation, invasion, and migration of GBM cells and the self-renewal capacity of GSCs induced by exogenous overexpression of WEE2-AS1, as well as the activation of the AKT signaling pathway (J). Figure 4 In summary, these results indicate that WEE2-AS1 interacts with RPN2, thereby activating the downstream AKT signaling pathway and promoting the malignant progression of GBM.

[0140] 5. WEE2-AS1 stabilizes RPN2 protein by preventing CUL2-mediated ubiquitin-protein body degradation.

[0141] Next, this embodiment explores the potential mechanism of interaction between WEE2-AS1 and RPN2 proteins. The investigation in this embodiment shows that WEE2-AS1 does not significantly alter the mRNA expression of RPN2. Figure 5 A), but promotes its protein level in GBM cells ( Figure 5 B). This embodiment further observed that, compared to the NC group, overexpression of WEE2-AS1 prolonged the half-life of the RPN2 protein by blocking its synthesis. Figure 5C) indicates that WEE2-AS1 enhances the stability of RPN2 to promote its protein expression. The ubiquitin-protein body system (UPS) is a major pathway for intracellular protein degradation. Further investigation in this example showed that WEE2-AS1 knockout reduced RPN2 protein expression levels, which could be restored by the proteasome inhibitor MG132. Figure 5 D). Meanwhile, compared to the corresponding NC group, RPN2 ubiquitination was increased in WEE2-AS1 knockout GBM cells and increased in WEE2-AS1 major expression GBM cells (D). Figure 5 E), indicating that WEE2-AS1 regulates the stability of the RPN2 protein through ubiquitin-proteasome activity. Next, this example uses BDM-PUM (http: / / bdmpub.biocuckoo.org / ) and the UbiBrowser database (http: / / ubibrowser.ncpsb.org / ) to predict RPN2 ubiquitination sites, identifying seven potential ubiquitination sites ( Figure 5 F). Then, in this embodiment, the potential ubiquitination site was mutated from lysine (K) to arginine (R) to inhibit ubiquitination. IP results showed that, compared with other groups, the mutation at the K322 site significantly reduced RPN2 ubiquitination (F). Figure 5 F), after the K322 site mutation, the decrease in RPN2 protein ubiquitination level induced by WEE2-AS1 knockout disappeared. Figure 5 G) highlights that K322 is the major ubiquitination site of RPN2. Furthermore, this embodiment predicts the structure of RPN2 using the Swiss Model Online website (https: / / swissmodel.expasy.org / ) and visualizes the K322 ubiquitination site, which is highly conserved in mammals. Figure 5 H). Ubiquitin ligase (E3) is one of the most critical and heterogeneous enzymes in the ubiquitination pathway, and there are currently no reports on E3 ubiquitin ligase-mediated RPN2 ubiquitination degradation. To identify the E3 ligase involved in proteasome-mediated RPN2 degradation, this example performed common IP experiments and mass spectrometry analysis, revealing that CUL2 is a protective factor for GBM prognosis. It forms a cullin-RING complex to promote substrate ubiquitination and degradation and can bind to RPN2 (H). Figure 5 I). IF staining assays confirmed the co-localization of RPN2 and CUL2 in GBM cells. Figure 5 J). Furthermore, this embodiment found a sharp increase in RPN2 protein expression compared to the NC group (J). Figure 5 K), and its ubiquitination level was significantly decreased in CUL2 knockout GBM cells ( Figure 5L). Co-IP assay showed that, compared with the NC group, the binding strength of CUL2 to RPN2 was significantly reduced in GBM cells that majorly expressed WEE2-AS1 (L). Figure 5 M), while WEE2-AS1 gene knockout does not affect CUL2 protein expression ( Figure 5 The results indicate that WEE2-AS1 enhances its stability by inhibiting the binding of RPN2 to CUL2. Collectively, these results suggest that WEE2-AS1 stabilizes RPN2 by preventing CUL2-mediated RPN2 K322 ubiquitination, thereby promoting the malignant progression of GBM.

[0142] 6. Knockout of WEE2-AS1 can improve the efficacy of dasatinib against GBM.

[0143] To further understand the impact of WEE2-AS1 on drug response, this example evaluated the association between WEE2-AS1 and drug responses from clinically tested or FDA-approved drugs collected from the CellMiner database (https: / / discover.nci.nih.gov / cellminer / ). Using Spearman correlation analysis, this example identified 21 drugs that were significantly associated with WEE2-AS1. Figure 6 A) Two of these drugs—irofulven and dasatinib—were significantly negatively correlated with WEE2-AS1, suggesting that WEE2-AS1 may inhibit the therapeutic sensitivity of these two drugs. Dasatinib is a potent second-generation competitive inhibitor of adenosine triphosphate (ATP) effective against multiple protein tyrosine kinases, including platelet-derived growth factor receptor (PDGFR) and Src family kinases, and has been approved by the FDA for childhood chronic myeloid leukemia. Interestingly, given its inherent qualities (i.e., lipophilicity, size, and protein binding), dasatinib can cross the blood-brain barrier, highlighting its potential as a central nervous system permeator. Studies have shown that dasatinib improves the efficacy of GBM treatment, particularly for GBM carrying PDGFRA mutations. Recently, scRNA-seq data from dasatinib-resistant GBM showed increased AKT activation. Similarly, GBM cells with high WEE2-AS1 expression also exhibited activation of the AKT signaling pathway. In this embodiment, it was found that the bioavailability of dasatinib was significantly lower in the WEE2-AS1 high expression group compared with the WEE2-AS1 low expression group. Figure 6 B). Further analysis revealed a significant negative correlation between WEE2-AS1 and PDGFRA expression. Figure 6C) indicates that WEE2-AS1 knockout may enhance the sensitivity of GBM cells to dasatinib. Further functional experiments showed that, compared with the control group, the combination of WEE2-AS1 knockout and dasatinib significantly inhibited the proliferation and self-renewal capacity of GBM cells in vitro. Figure 6 D, 6E). Furthermore, in vivo experiments showed that downregulating WEE2-AS1 in combination with dasatinib significantly inhibited tumor growth and prolonged the survival time of tumor-bearing mice. Figure 6 (F, 6G). In summary, the data from this embodiment demonstrate that blocking WEE2-AS1 expression can enhance the therapeutic effect of dasatinib on GBM.

[0144] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. m in lncRNA-WEE2-AS1 6 The application of A methylation level detection reagent in the preparation of prognostic reagents for glioblastoma, characterized in that... The prognostic level of glioma patients was assessed by detecting the methylation level of lncRNA-WEE2-AS1 in clinical samples of subjects.

2. The m in lncRNA-WEE2-AS1 as described in claim 1 6 The application of A methylation level detection reagent in the preparation of prognostic reagents for glioblastoma, characterized in that... The methylation level in lncRNA-WEE2-AS1 includes m 6 A. Methylation abundance and expression level.

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