Application of nup35 gene as a diagnostic, prognostic and therapeutic target for glioblastoma

Abstract

The application discloses application of NUP35 gene as a glioblastoma diagnosis, prognosis and treatment target, and finds that NUP35 gene plays an important role in the proliferation and malignant progression of glioma by regulating fatty acid metabolism of glioma cells. It is found that NUP35 is specifically highly expressed in glioma, and increases with the increase of the pathological grade of patients. Specific knockdown of NUP35 can effectively inhibit the growth of glioma cells and prolong the survival period of tumor-bearing mice. NUP35 accelerates fatty acid metabolism in glioma cells by promoting the expression of fatty acid metabolism related genes, and promotes the growth of glioma. It is found that the expression level of NUP35 can be used as a glioma prognosis marker, and NUP35 can be used as an effective target for glioma treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically involving the application of the NUP35 gene as a target for the diagnosis, prognosis and treatment of glioblastoma. Background Technology

[0002] Glioblastoma multiforme (GBM) is the most common and most malignant primary tumor of the central nervous system. It is a type of astrocytoma, classified as WHO grade IV. Its characteristics include highly aggressive, poorly defined borders, and multifocal growth. It commonly occurs in the deep white matter of the frontal and temporal lobes, but can also involve the basal ganglia and posterior fossa (such as the cerebellar hemispheres and brainstem). Clinical manifestations include symptoms of increased intracranial pressure (headache, projectile vomiting, decreased vision), neurological dysfunction (hemiplegia, aphasia, sensory disturbances), and seizures. The median survival is only 12-16 months, with a five-year survival rate of less than 7%, indicating an extremely poor prognosis.

[0003] Therefore, in-depth research into the molecular mechanisms of GBM development and progression, the discovery of new therapeutic targets, and the development of molecular targeted therapy are major issues in this field and have significant clinical implications. Summary of the Invention

[0004] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments.

[0005] As one aspect of the present invention, the present invention provides the application of the NUP35 gene as a target for the diagnosis, prognosis and treatment of glioblastoma, which includes the NUP35 gene as a specific molecular marker for glioblastoma.

[0006] Specifically, gliomas are diagnosed by detecting the mRNA expression level of the NUP35 gene.

[0007] Specifically, the primers for detecting the NUP35 gene are: upstream primer: 5′-GGCCCGCGTTAGGATCTG-3′; downstream primer: 5′-ATCGAGGTTGTGGAGTCACC-3′.

[0008] Specifically, the NUP35 gene serves as a therapeutic target for glioblastoma, including inhibiting glioma cell growth by suppressing the mRNA and protein expression levels of the NUP35 gene.

[0009] Specifically, the inhibition of NUP35 gene mRNA and protein expression levels includes using nucleic acid molecules, small molecule chemical drugs, peptides, proteins, or interfering lentiviruses to inhibit NUP35 gene mRNA and protein expression levels.

[0010] Specifically, the nucleic acid molecules include antisense oligonucleotides, dsRNA, siRNA, or shRNA.

[0011] Specifically, the shRNA sequence is one or more of SEQ ID NO: 1 to 3.

[0012] As a preferred application of the NUP35 gene described in this invention: the NUP35 gene regulates the expression of key genes in the process of fatty acid synthesis and decomposition, thereby regulating fatty acid metabolism in glioma cells.

[0013] The beneficial effects of this invention: The NUP gene family contains more than 100 genes. This invention discovers that the NUP35 gene plays an important role in the proliferation and malignant progression of gliomas by regulating fatty acid metabolism in glioma cells. This invention finds that NUP35 is specifically highly expressed in gliomas, and this expression increases with the progression of the patient's pathological grade. Functional experiments further confirm that overexpression of NUP35 promotes glioma cell proliferation. Specific knockdown of NUP35 effectively inhibits glioma cell growth and prolongs the survival of tumor-bearing mice. Further transcriptome sequencing analysis and functional experiments reveal that NUP35 accelerates fatty acid metabolism in glioma cells by promoting the expression of fatty acid metabolism-related genes, thereby promoting glioma growth. This invention finds that NUP35 expression levels can serve as a prognostic biomarker for gliomas, and that NUP35 can be an effective target for glioma treatment. Attached Figure Description

[0014] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below, wherein:

[0015] Figure 1 NUP35 is highly expressed in gliomas and is associated with poor patient prognosis.

[0016] Figure 2 NUP35 promotes the growth of glioma cells.

[0017] Figure 3 NUP35 does not regulate glioma cell proliferation by affecting NPC formation and nuclear membrane integrity.

[0018] Figure 4 NUP35 promotes fatty acid metabolism in glioma cells. Detailed Implementation

[0019] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples.

[0020] 1. Obtaining glioblastoma tissue and culturing primary GBM cells: After the glioblastoma tissue was minced and digested with trypsin, it was cultured in DMEM high glucose medium (Bio-channel, catalog number BC-M-005) with 10% FBS (Gibco) to obtain primary GBM cells.

[0021] 2. Plasmid construction and lentivirus transduction:

[0022] shRNA cloning: Three non-overlapping shRNA sequences were designed and synthesized based on the human NUP35 gene (Gene ID: 129401). After annealing to double-stranded oligo sequences, they were ligated into double-digested linearized pSLenti-U6-shRNA-CMV-mCherry-F2A-Puro-WPRE (Shanghai Heyuan Biotechnology) (hereinafter referred to as pSLenti) vectors. These vectors were then transformed into Stbl3 competent cells. After single-colony sequencing verification, high-purity recombinant plasmids were extracted using the QIAGEN PlasmidMidiprep Kit, with a concentration ≥500 ng / μL, to obtain the recombinant pSLenti-shNUP35 plasmid.

[0023] shNUP35#1: 5′-TAGTGATTATCAGGTTATTTC-3′ (SEQ ID NO: 1)

[0024] shNUP35#2: 5′-GCACAGTATGGGAATATCT-3′ (SEQ ID NO: 2)

[0025] shNUP35#3: 5′-GCAGTGACAGATGTGCTTT-3′ (SEQ ID NO: 3)

[0026] OE-NUP35 Cloning: The full-length CDS sequence of NUP35 was synthesized based on human NUP35 (GenBank ID: NM_138285.5), and an EcoR1 restriction site was added. After annealing to double-stranded DNA, it was ligated into the EcoR1-digested linearized GL130pSLenti-CMV-EGFP-3xFLAG-PGK-Puro-WPRE (hereinafter referred to as GL130) vector. This vector was transformed into Stbl3 competent cells, and after single-colony sequencing verification, high-purity recombinant plasmids were extracted using the QIAGEN PlasmidMidiprep Kit at a concentration ≥500 ng / μL. The recombinant GL130-NUP35-WT (hereinafter referred to as NUP35) plasmid was obtained.

[0027] Lentiviral packaging and concentration: Recombinant pSLenti-shNUP35 / NUP35, psPAX2 (Addgene#12260), and pMD2.G (Addgene#12259) were mixed in a ratio of 3:2:1 and transfected into HEK293T cells (70% density) using Polyjet. Viral supernatant was collected twice, at 48h and 72h.

[0028] Virus concentration: The supernatant was filtered through a 0.45 μm filter membrane, centrifuged at 70,000 × g for 2 h at 4 °C, and stored at -80 °C.

[0029] Titer determination: The number of viral particles (TU / mL) was determined by qPCR, with a target titer ≥1×10^8TU / mL.

[0030] Glioma cell infection: U251 / GBM cells were infected with lentivirus at MOi=10, and 4 μg / mL Polybrene was added. After 48 h, selection was performed with 5 μg / mL Puromycin for 3 days. Infection efficiency was detected by fluorescence microscopy.

[0031] Silencing / overexpression efficiency assay: Western blot: RIPA lyses cells, SDS-PAGE isolates proteins, incubates with anti-NUP35 primary antibody at 4°C overnight, HRP secondary antibody is used for imaging, and NUP35 protein levels are detected.

[0032] 3. In vitro functional verification

[0033] EdU, CCK-8, Transwell, and colony formation experiments (Xiuping Zhou, et al. Neuro-Onc0l. 2017).

[0034] 4. In vivo experiments

[0035] Nude mouse xenograft model: GBM cells infected with shNUP35 (3×10^ 6 Tumors were implanted in situ into the right striatum of the brain of nude mice (one per mouse), while the control group received control group cells. Tumor growth was observed by in vivo imaging of small animals on days 7, 14, and 21 post-implantation, and survival curves were plotted based on mouse survival time.

[0036] 5. Transcriptome sequencing and analysis:

[0037] Total RNA was collected from U251 cells with downregulated NUP35 and control group for RNA-seq detection. Differentially expressed genes were screened using a cut-off value of |log2FC|≥1.2 and QValue<0.05. KEGG and GSEA enrichment analyses were combined to screen major pathways and key genes regulated by NUP35, and further detection was performed by qPCR.

[0038] 6. Transmission electron microscopy:

[0039] Cells of downregulated NUP35 and control groups were collected by centrifugation, fixed overnight at 4°C with 2.5% glutaraldehyde solution, rinsed, fixed with 1% osmium tetroxide solution, rinsed, and dehydrated with ethanol of varying concentrations. After treatment with pure acetone, the cells were embedded, prepared into slides, and observed under a transmission electron microscope.

[0040] 7. Tumor Tissue Microarray (TMA) Fabrication and Patient Follow-up:

[0041] Surgical specimens and clinical information, including imaging data and pathological features, were collected from glioma patients. TMA (tumor tissue analysis) was prepared according to our published paper (Yan Wang, et al. Neuro-Oncology. 2022). Two cores with typical pathological changes and a diameter of 1 mm were selected from each sample based on HE staining and arranged in a predetermined order on pre-prepared blank recipient paraffin blocks. The blocks were heated in a 52°C oven to ensure tight adhesion between the tissue cores and the recipient blocks. The paraffin blocks were trimmed using a fully automated tissue sectioner to ensure complete exposure of each glioma core, and sections were prepared at a thickness of 4 μm. The sections were then mounted on adhesive slides. Patients were followed up by telephone every three months postoperatively to understand symptoms, follow-up examinations, and to monitor the patient's survival status and postoperative treatment.

[0042] 8. Immunofluorescence staining and imaging:

[0043] Cell slides were prepared and fixed with 4% paraformaldehyde for 30 min at room temperature after 24 h. The slides were then washed twice with PBS, followed by permeation with 0.5% Triton-X-100 for 10 min, and blocked with a blocking solution of 3% BSA + 0.05% Triton-X-100 at room temperature for 1 h. Primary antibody was incubated overnight at 4°C, followed by 5 washes with PBS, and then secondary antibody binding at room temperature for 1 h. After 5 washes with PBS, the slides were mounted with an anti-fluorescence quencher containing DAPI. Finally, the cells were observed and photographed using a laser confocal microscope (Leica Stellaris 5).

[0044] 9. NUP35 gene expression level detection: The primers for NUP35 gene detection are: upstream primer: 5′-GGCCCGCGTTAGGATCTG-3′; downstream primer: 5′-ATCGAGGTTGTGGAGTCACC-3′.

[0045] Figure 1Analysis revealed high expression of NUP35 in gliomas, which is associated with poor patient prognosis. A. NUP35 mRNA expression levels in non-tumor brain tissue and glioma tissue samples from the TCGA database. B. NUP35 mRNA expression levels in glioma tissue samples of different grades from the CGGA database. C. Correlation analysis of NUP35 mRNA levels and patient survival from the TCGA database. D. Correlation analysis of NUP35 mRNA levels and patient survival from the CGGA database. E. Western blot detection of NUP35 protein expression levels in non-tumor brain tissue and glioma patient samples. F. TMA-immunohistochemical staining detection of NUP35 protein expression levels in non-tumor brain tissue and glioma tissue samples of different grades. G. Statistical results of NUP35 protein expression levels in non-tumor brain tissue and glioma tissue samples (E). Data are expressed as mean ± SD. ＊＊ p < 0.01, t-test. H represents the quantitative statistical results of NUP35-positive cells in the F-plot, comparing the percentage of NUP35-positive cells in non-tumor brain tissue and glioma tissues of different grades. Data are expressed as mean ± SD. ＊＊＊ p < 0.001. I. The relationship between NUP35 expression levels and overall patient survival is shown. J. ROC curve analysis of NUP35 expression levels as a glioma marker. NUP35 as a glioma marker has a diagnostic accuracy of 95%, a diagnostic specificity of 94%, and a sensitivity of 91%.

[0046] Figure 2 To investigate the effect of NUP35 overexpression on the malignant progression of glioma, the following methods were used: A. Western blotting (WB) was used to detect the protein expression level of NUP35 in different glioma cell lines. B. WB was used to verify the NUP35 overexpression results in U87 cells. C. Fluorescence microscopy was used to observe the overexpression of NUP35 and the subcellular localization of NUP35-GFP in U87 glioma cells. D. CCK-8 assay was used to detect the effect of NUP35 overexpression on U87 cell proliferation. Data are expressed as mean ± SD from three independent experiments. ＊＊＊ p < 0.001, t-test. E. EdU assay to detect the effect of NUP35 overexpression on U87 cell proliferation. F. Quantitative statistical results of the number of EdU-positive cells shown in Figure E. Data are expressed as mean ± SD of three independent experiments. ＊＊＊ p < 0.001, t-test. G. Transwell assay to detect the effect of NUP35 overexpression on U87 cell invasion and migration. H. Scratch assay to detect the effect of NUP35 overexpression on U87 cell migration. I. Statistical results of the effect of NUP35 on cell invasion and migration ability are shown in Figure G. Data are expressed as mean ± SD of three independent experiments. ＊ p < 0.05 ＊＊p < 0.01, t-test. J. Presents the quantitative statistical results of NUP35 on cell migration in H. Data are expressed as mean ± SD of three independent experiments. ＊＊ p < 0.01, t-test.

[0047] Figure 3 NUP35 does not regulate glioma cell proliferation by affecting NPC formation or nuclear membrane integrity. A. Western blotting validates NUP35 downregulation in U251 and GBM cells. B. Colony formation assay examines the effect of NUP35 downregulation on the proliferation of U251 and GBM cells. C. Statistical results of cell colony size are shown in Figure B. Data are expressed as mean ± SD from three independent experiments. ＊＊ p < 0.01, ＊＊＊ p<0.001, t-test. D. EdU assay to detect the effect of downregulation of NUP35 on the proliferation of U251 and GBM cells, respectively. E. Statistical results of the number of EdU-positive cells shown in Figure D. Data are expressed as mean ± SD of three independent experiments. ＊＊ p < 0.01, t-test. F. Small animal live imaging showing the size of orthotopic xenografts in mice at different time points after NUP35 downregulation. G. Quantitative analysis of tumor growth using bioluminescence values, data are expressed as mean ± SD, *p < 0.05. ＊＊＊ p<0.001, t-test (n=9 per group). H. Animal survival curves show the overall survival of animals with downregulated NUP35 and the control group. I. mAb414 / NUP35 immunofluorescence results show the nuclear membrane morphology after downregulation of NUP35. J. Transmission electron microscopy results show the NPC status after downregulation of NUP35. K. Statistical results of the number of NPCs in Figure J are shown, with data expressed as mean ± SD of three independent replicates, ns indicating no statistical difference, t-test. L. Statistical results of the diameter of NPCs in Figure J are shown, with data expressed as mean ± SD of three independent replicates, ns indicating no statistical difference, t-test.

[0048] Figure 4NUP35 promotes fatty acid metabolism in glioma cells. A. Heatmap shows differentially expressed genes regulated by NUP35, with 709 genes downregulated and 924 genes upregulated (DEGsCut-off: |log2FC|>1.2, FDR<0.05). B. KEGG pathway enrichment analysis shows the top 10 signaling pathways enriched by DEGs regulated by NUP35. C. Heatmap shows fatty acid metabolism-related genes regulated by NUP35 in DEGs. D. GSEA analysis shows that oxidative phosphorylation pathway was significantly inhibited after NUP35 knockdown. E. RT-qPCR detection of the effect of downregulated NUP35 on the mRNA levels of key fatty acid metabolism enzymes FASN and CPT1A. Data are expressed as mean ± SD of three independent replicates. ＊＊ p < 0.01, t-test. Effect of NUP35 overexpression on the mRNA levels of key fatty acid metabolism enzymes FASN and CPT1A was detected by RT-qPCR. Data are presented as mean ± SD of three independent replicates. ＊＊ p < 0.01, t-test. G. Western blot analysis to detect the effect of NUP35 downregulation or overexpression on the protein expression of key fatty acid metabolism enzymes FASN and CPT1A, with β-actin as an internal control. H. Seahorse energy metabolism analysis to detect the effect of NUP35 downregulation on cellular oxygen consumption rate (OCR). I. Seahorse energy metabolism analysis to detect the effect of NUP35 overexpression on cellular oxygen consumption rate (OCR). J. BODIPY 493 / 503 fluorescence staining to show the changes in lipid droplet content in cells after NUP35 downregulation or overexpression (scale bar: 10 μm). K. CCK-8 assay to detect the effect of Orlistat (20 μM) treatment on cell proliferation in NUP35-overexpressing cells. Data are expressed as mean ± SD of three independent replicates. ＊＊＊ p<0.001, t-test.

[0049] This invention designs shRNA based on the CDS region of the NUP35 gene and packages it into a lentiviral vector, allowing it to enter eukaryotic cells and silence NUP35 expression. The shRNA of this invention can silence NUP35 protein expression, significantly inhibiting immortalization and the proliferation of primary glioma cells. Therefore, a drug with this shRNA as its active ingredient can be used as an anti-tumor agent for the treatment of glioma. This is of great significance in providing a new drug target for the treatment of glioma and can solve the problem of adverse reactions of existing anti-tumor drugs.

[0050] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The application of a reagent for detecting the NUP35 gene in the preparation of diagnostic reagents for glioblastoma, characterized in that: The NUP35 gene serves as a specific molecular marker for glioblastoma.

2. The application according to claim 1, characterized in that: Glioma can be diagnosed by detecting the mRNA expression level of the NUP35 gene.

3. The application according to claim 1, characterized in that: The primers for detecting the NUP35 gene are: upstream primer: 5'-GGCCCGCGTTAGGATCTG-3'; downstream primer: 5'-ATCGAGGTTGTGGAGTCACC-3'.

4. The application of a reagent that inhibits the mRNA and protein expression levels of the NUP35 gene in the preparation of drugs that inhibit the growth of glioma cells, characterized in that: This includes using shRNA to suppress the mRNA and protein expression levels of the NUP35 gene; the sequence of the shRNA is one or more of SEQ ID NO:1~3.

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