CN114381522B

CN114381522B - Application of NUP98 gene as glioma stem cell specific molecular marker and glioblastoma treatment and prognosis target

Info

Publication number: CN114381522B
Application number: CN202111683737.4A
Authority: CN
Inventors: 汪秀星; 慈书圣; 秦浩凯
Original assignee: Nanjing Medical University
Current assignee: Nanjing Medical University
Priority date: 2021-12-30
Filing date: 2021-12-30
Publication date: 2023-09-05
Anticipated expiration: 2041-12-30
Also published as: CN114381522A

Abstract

The application discloses an application of NUP98 gene as a glioma stem cell specific molecular marker and glioblastoma treatment and prognosis target, wherein NUP98 regulation and DNA damage repair is realized by combining with a transcription factor P65 and regulating the transcription activity of P65, and NUP98 overexpression is related to low survival rate of brain tumor patients. The expression level of NUP98 is used as a glioblastoma marker, the diagnosis accuracy of NUP98 serving as the glioblastoma marker is 93 percent, the diagnosis specificity is 100 percent, and the sensitivity is 85.4 percent.

Description

Application of NUP98 gene as glioma stem cell specific molecular marker and glioblastoma treatment and prognosis target

Technical Field

The application belongs to the technical field, and particularly relates to application of NUP98 gene as a molecular marker for diagnosing glioma stem cells and a therapeutic and prognostic target.

Background

Glioblastoma (GBM) is the most malignant glioma among astrocytomas. Tumors are located subcortical, most growing throughout the supratentorial hemispheres. It grows in invasive nature, often invading several brain leaves and deep structures, and can also spread across the contralateral hemispheres of the brain via the corpus callosum. The place of occurrence is most seen in frontal lobe. The glioblastoma has high growth speed, 70-80% of patients have disease course of 3-6 months, and the disease course is only 10% of those of more than 1 year. Longer disease progression may develop from less malignant astrocytomas. Because of rapid tumor growth, extensive cerebral edema and obvious intracranial pressure increase symptoms, all patients have headache and vomiting symptoms. The oedema of the optic disc is manifested by headache, mental changes, weakness of the limbs, vomiting, disturbance of consciousness and speech. Tumor infiltration damages brain tissue, causes a series of focal symptoms, and patients have hemiplegia, hemiparalysis, aphasia, and hemiparalysis to different extents. Examination of the nervous system can reveal hemiplegia, brain nerve damage, off-body sensory disturbance and off-blindness. The incidence of epilepsy is less common than astrocytomas and oligodendrocytomas, and some patients have seizures. Some patients show mental symptoms such as apathy, dementia, mental retardation, etc.

Glioma stem cells (Glioma Stem Cells, GSC) are considered critical for GBM treatment tolerance and tumor recurrence, but clinical treatment regimens targeting GSC alone or in combination have not progressed effectively. Therefore, finding therapeutic targets of Gliobastoma (GBM) and molecular markers for diagnosis and prognosis is a technical problem to be solved.

Disclosure of Invention

This section is intended to outline some aspects of embodiments of the application and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section as well as in the description of the application and in the title of the application, which may not be used to limit the scope of the application.

The application provides an application of NUP98 genes, wherein the NUP98 genes are used as glioma stem cell specific molecular markers.

As a preferable scheme for the application of the NUP98 gene of the application: the NUP98 gene is used as a glioblastoma treatment and prognosis target.

As a preferable scheme for the application of the NUP98 gene of the application: glioblastoma is diagnosed by detecting the mRNA expression level of NUP98 gene.

As a preferable scheme for the application of the NUP98 gene of the application: the NUP98 gene is combined with the transcription factor P65, and the transcription activity of the P65 is regulated to regulate and control DNA damage repair of glioma stem cells.

As a preferable scheme for the application of the NUP98 gene of the application: the expression level of NUP98 gene correlates with glioblastoma patient survival.

As a preferable scheme for the application of the NUP98 gene of the application: NUP98 gene detection primers were, upstream primer: 5'-CTCCACCACTAATTCAGGCTTT-3'; a downstream primer: 5'-GAGGCTGGTAGTCTGCTGATT-3'.

The application has the beneficial effects that: NUP98 gene plays an important role in GSC self-renewal, proliferation and malignant progression of glioma by regulating DNA damage repair of GSC. Through analysis of patient databases and cellular levels we found that NUP98 was specifically highly expressed in GSCs. After NUP98 knockdown we found that GSC self-renewal, proliferation, and tumor progression in vivo could be inhibited. Further by transcriptome sequencing and bioinformatic analysis, we found that NUP98 plays a role in GSC self-renewal, proliferation and inhibition of tumor progression in vivo by modulating DNA damage repair. Finally, through protein mass spectrum detection, we find that NUP98 regulates DNA damage repair to be combined with a transcription factor P65 and regulates the transcription activity of the P65, and NUP98 overexpression is related to low survival rate of brain tumor patients. The expression level of NUP98 is used as a glioblastoma marker, the diagnosis accuracy of NUP98 serving as the glioblastoma marker is 93 percent, the diagnosis specificity is 100 percent, and the sensitivity is 85.4 percent.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art. Wherein:

FIG. 1 is a specific up-regulation of NUP98 in GSCs and GBM.

Figure 2 is NUP98 regulating GSCs growth and self-renewal.

Figure 3 is NUP98 maintains GSCs stem cell markers, viability and glioblastoma growth.

FIG. 4 is a DNA repair pathway regulated by NUP98 in GSCs.

FIG. 5 is a DNA repair pathway of NUP98 interacting with P65 to regulate GSCs.

FIG. 6 is a graph showing that NUP98 and P65 together regulate DNA repair pathways in GSCs.

FIG. 7 is that NUP98 overexpression correlates with low survival in brain tumor patients.

FIG. 8 is a correlation analysis of nuclear pore complex family member genes.

Fig. 9 shows P65 interacting with NUP 98.

FIG. 10 is a DNA repair pathway of P65 regulated GSCs.

Detailed Description

In order that the above-recited objects, features and advantages of the present application will become more apparent, a more particular description of the application will be rendered by reference to specific embodiments thereof.

Example 1:

obtaining and culturing glioblastoma stem cells: glioblastoma tissue used in the experiments of the present application was obtained from redundant surgical resection specimens of Duke university patients, subjected to neuropathological examination and informed consent according to the protocol approved by the institutional review board (090401), all patient studies were conducted as per the declaration of Helsinki. GSC387 and GSC4121 are assigned by the university of duchenne material assignment protocol. All GSCs cells were cultured in Neurobasal medium (Invitrogen) supplemented with B27 (Invitrogen) without vitamin A, and EGF and bFGF (20 ng/mL each; R & D Systems), sodium pyruvate and glutamax were added.

In vivo tumorigenesis model construction: all in vivo tumor development experiments in mice were performed according to animal protocols approved by the institutional committee of use and care animal at san diego university, california. Live GSCs cells were quantified and injected intracranially into NSG mice. NUP98 shRNA experiments were performed by implanting 10,000 human GSCs cells into the right cortex of NSG mice to form intracranial xenografts until significant neurological signs of the mice were observed, and then the mice were sacrificed for further analysis. Mouse brain tissue was obtained, fixed in 4% formaldehyde, cryopreserved in 30% sucrose, frozen and sectioned, and HE stained for histological analysis.

Plasmid and lentiviral transduction: lentiviruses expressing two non-overlapping shRNA targeting human nucleoporin NUP98 (nucleoporin 98 kDa) genes (TRCN 0000046913 and TRCN 0000046914), P65 (TRCN 0000014683 and TRCN 0000014686), and a non-targeting control shRNA (TRCN 00409) were obtained from Sigma-Aldrich, and the knockdown efficiency of shRNA was analyzed with qRCR for subsequent experiments. Human NUP98 full-length gene, various deletion mutants of human NUP98, and P65 full-length gene were prepared by cloning the Open Reading Frame (ORF) of the gene into pCDHMCS-T2A-Puro-MSCV vector (System Biosciences) using In-Fusion HD Cloning Kit (Clontech, 638920) kit, lentivirus was prepared with 293FT cells, and lentivirus transfection was achieved by co-transfection of packaging vector pCMV-dr8.2 dvpr and pCI-VSVG using standard calcium phosphate transfection methods.

Proliferation and neurosphere formation assays: cell proliferation experiments were performed in 96-well plates plated at a density of 1000 cells/well, and repeated 6 times. Cell proliferation was measured using the CellTiter-Glo (Promega, madison, WI, USA) kit, neurosphere formation was measured by in vitro limiting dilution method, reference (Flavahan et al, 2013).

RT-PCR quantification: total cellular RNA was extracted using Trizol reagent (Sigma Aldrich), qScript cDNA Synthesis Kit (Takara) for reverse transcription of RNA into cDNA. Real-time quantitative PCR was performed using a Applied Biosystems 7900HT cycler, SYBR-Green PCR Master Mix (Vazyme). The primers used for qPCR analysis of the application are respectively as follows:

the human NUP98 gene upstream primer: 5'-CTCCACCACTAATTCAGGCTTT-3'; a downstream primer: 5'-GAGGCTGGTAGTCTGCTGATT-3';

human P65 gene upstream primer: 5'-ATGTGGAGATCATTGAGCAGC-3'; a downstream primer: 5'-CCTGGTCCTGTGAGCCATT-3';

primer upstream of SOX2 gene: 5'-AGGGCTGGGAGAAAGAAGAG-3'; a downstream primer: 5'-GGAGAATAGTTGGGGGGAAG-3';

myc gene upstream primer: 5'-TGAGGAGACACCGCCCAC-3'; a downstream primer: 5'-CAACATCGATTTCTTCCTCATCTTC-3';

olig2 gene upstream primer: 5'-CAAGAAGCAAATGACAGAGCCGGA-3'; a downstream primer: 5'-TGGTGAGCATGAGGATGT AGTTGC-3';

the BRCA1 gene upstream primer: forward 5'-GAAACCGTGCCAAAAGACTTC-3'; a downstream primer: 5'-CCAAGGTTAGAGAGTTGGACAC-3';

BRCA2 gene upstream primer: 5'-ACAAGCAACCCAAGTGTCAAT-3'; a downstream primer: 5'-TGAAGCTACCTCCAAAACTGTG-3';

EME1 gene upstream primer: 5'-TCTGAGGAGTTGCCAACATTTG-3'; a downstream primer: 5'-GGCTTCACAATCTGAGATGTCAA-3';

RAD54B gene upstream primer: 5'-GCCAAACACTGATGATTTGTGG-3'; a downstream primer: 5'-CCTGAGAAGAATGCGAGATAGC-3';

the BLM gene upstream primer: 5'-CAGACTCCGAAGGAAGTTGTATG-3'; a downstream primer: 5'-TTTGGGGTGGTGTAACAAATGAT-3';

XRCC2 gene upstream primer: 5'-TGCTTTATCACCTAACAGCACG-3'; a downstream primer: 5'-TGCTCAAGAATTGTAACTAGCCG-3';

BRIP1 gene upstream primer: 5'-CTTACCCGTCACAGCTTGCTA-3'; a downstream primer: 5'-CACTAAGAGATTGTTGCCATGCT-3';

RAD54L gene upstream primer: 5'-AGGCAGGTCCTGTGATGATGA-3'; a downstream primer: 5'-TCAAAGGTTTCCGAAAAGGAGAC-3';

the BRCC3 gene upstream primer: 5'-GAGTCTGACGCTTTCCTCGTT-3'; a downstream primer: 5'-TGTATCATCGTTCAACTCCCCT-3';

the MRE11 gene upstream primer: 5'-ATGCAGTCAGAGGAAATGATACG-3'; a downstream primer: 5'-CAGGCCGATCACCCATACAAT-3';

RAD51 gene upstream primer: 5'-CAACCCATTTCACGGTTAGAGC-3'; a downstream primer: 5'-TTCTTTGGCGCATAGGCAACA-3'.

Western blotting: samples were collected and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150mM nacl,0.5% sodium deoxycholate, 0.1%SDS,100mM NaF,1mM Na3VO4,1mM EDTA,10% (v/v) glycerol, containing PMSF and cocktail inhibitor), incubated on ice for 30 min, bio-Rad kit was used to determine protein concentration, run using NuPAGE Bis-Tris gel (Life Technologies), and then transferred onto PVDF membranes (Millipore). TBST 5% nonfat dry milk was added for blocking. The antibody was incubated overnight at 4 ℃. The antibodies used were as follows: anti-NUP98 (C39A 3), anti-P65 (D14E 12), anti-HA (6E 2), anti-HA (C29F 4), anti-Caspase-3 (9662), anti-clean Caspase-3 (5A 1E), antibodies were purchased from Cell Signaling Technology. Anti-gamma-H2 AX (ab 2893) was purchased from Abcam. The Anti-beta-tubulin (AM 031A) antibody was purchased from Abgent. Anti-PARP1 (13371-1-AP) is purchased from Proteintech. Anti-BRCA1 (A0212), anti-BRCA2 (A2435), anti-Rad51 (A2829), anti-Rad54L (A20181) antibodies were purchased from ABclonal.

Co-immunoprecipitation: cells were lysed using IP buffer (20 mM Tris-HCl, 150mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 1mM DTT) containing PMSF and cocktail inhibitor for 2 hours at 4 ℃. After centrifugation at 4℃for 10 min 14000rpm, the supernatant was further treated with 20. Mu.g/mL DNase I for 30 min, and indicator antibodies and IgG were added to the supernatant as negative controls and incubated overnight at 4 ℃. The supernatant was then immunoprecipitated by incubating Protein-A+G agar beads at 4℃for 2 hours, and for co-immunoprecipitation of Flag tag Protein, the supernatant was incubated with M2 beads (Millipore Sigma) at 4℃overnight,

immunofluorescent staining and imaging: for immunofluorescence microscopy imaging, cells were plated on coverslips coated with matrix gel for 24 hours, fixed with 4% paraformaldehyde twice for 30 minutes at room temperature, washed with PBS, then permeabilized with 0.45% Triton X-100 for 10 minutes, and blocked with 3% BSA for 2 hours at room temperature. Cells were incubated overnight at 4℃with primary antibody, washed three times with PBS, stained with either Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen) conjugated secondary antibody at 37℃for 2 hours, washed with PBS, stained nuclei with DAPI, added with anti-fluorescence quencher, and cells were observed by confocal imaging (Nikon Ti-E-AIR) or Nikon 80I 10-1500X microscopy and treated with imageJ software.

Patient database bioinformatics: to determine the clinical relevance of NUP98 in glioblastoma patients, we queried the cancer genomic profile (TCGA) glioblastoma dataset, with expression levels divided into two groups, with median as demarcation points. The time to live and status of both groups of patients were analyzed using Kaplan-Meier statistical test and log rank test.

Chromatin immunoprecipitation (ChIP) analysis: collection of 4X 10 under each condition ⁶ The cells were subjected to chip detection using a SimpleChIP Enzymatic Chromatin IP Kit (CST, # 9003) kit, the cells were fixed in a medium containing 1% formaldehyde at room temperature for 10 minutes to crosslink proteins with DNA, the cells were collected, washed, lysed, and then DNA was digested with micrococcus nuclease, sonicated, and then treated with anti-NU at 4 ℃The P98 antibody (C39 A3), anti-P65 (D14E 12) or normal rabbit IgG immunoprecipitated lysates overnight, the drawn chromatin was washed and purified DNA was subjected to quantitative PCR by heating at 65 ℃ for 6h, followed by digestion with proteinase K, using the following primer set:

BRCA1 upstream primer: 5'-ATGGATTGGAGTGTTGTTATGTT-3'; a downstream primer: 5'-TTTCTCCGAGTGTTCGCCAAG-3';

BRCA2 upstream primer: 5'-GGACTCTTAAGGGTCAGCGAG-3'; a downstream primer: 5'-CGCAGCAGTGCCACAGC-3';

EME1 upstream primer: 5'-GGCTCCACCCAGGATGTG-3'; a downstream primer: 5'-CAGTAGTCAGAGCGGGGTG-3';

RAD51 upstream primer: 5'-GCTAGCTCCATTTCCCACTT-3'; a downstream primer: 5'-CTTTTCAACCCGCCACAGCC-3';

BLM upstream primer: 5'-CTTTTCAACCCGCCACAGCC-3'; a downstream primer: 5'-GGAGGGACGCGTATCTCCAA-3';

XRCC2 upstream primer: 5'-CAGACAAGAAAAGCACCAGCTG-3'; a downstream primer: 5'-CGCAGACTCTACGGCCAGT-3';

RAD54L upstream primer: 5'-AGGTTCGATTGACCCGGTCT-3'; a downstream primer: 5'-AGGTGGAAACCAGGATCAGACT-3'.

RNA sequencing and data analysis: total cellular RNA was isolated from the cell pellet using TRIzol reagent (Sigma-Aldrich), purified and analyzed for sequencing.

Example 2:

experimental results:

FIG. 1 analysis reveals specific upregulation of NUP98 in GSCs and GBM. FIG. 1A. MRNA expression profile of NPC gene in TCGA GBM database. Paired analysis of NPC gene RNA sequences of GSC and DGC. C. Quantitative qRT-PCR determination of NUP98 mRNA levels in glioblastoma and normal brain tissues. D. Five pairs of GSCs and DGCs cells of the patient-derived glioblastoma model (T387, T4121, T456, GSC23 and T3028) were subjected to NUP98 protein level determination by immunoblotting, and the turulin protein served as an internal control. E. Protein levels of NUP98 in glioblastoma and normal brain tissues were assessed by immunoblotting, and the turulin protein served as an internal control. F. Immunofluorescent staining of NUP98 in GSCs or DGCs of human glioblastoma samples T387 and 4121.

Figure 2 is NUP98 regulating GSCs growth and self-renewal. FIG. 2A quantitative RT-PCR detection of NUP98 mRNA in 387 and 4121GSCs expressing non-targeted control shRNA (shCONT), shNUP98-1 or shNUP 98-2. Data are expressed as mean ± SEM of six independent experiments, p <0.01.B. Immunoblots of NUP98 levels following GSCs transduction with control non-targeted shRNA sequences (shCONT), shNUP98-1 or shNUP 98-2. C. The cell effect was assessed by direct cell counting using one of the non-targeted control shRNA (shCONT) or two independent, non-overlapping shRNA targeting NUP98 (shNUP 98-1 or shNUP 98-2) to transduce both GSCs (387 and 4121). Data are expressed as mean ± SEM of six independent experiments, p <0.01.D. Two GSC cultures were transduced with either a non-targeted control shRNA (shCONT) or one of two independent, non-overlapping shRNAs targeting NUP98 (shNUP 98-1 or shNUP 98-2) (387 and 4121). Cell effects were assessed by CellTiter-Glo cell viability assay, data are presented as mean ± SEM of six independent experiments, p <0.01.E. Two GSCs were transduced with either a non-targeted control shRNA (shCONT) or one of two independent, non-overlapping shrnas targeting NUP98 (shNUP 98-1 or shNUP 98-2) (387 and 4121). The effect on self-renewal was assessed by an in vitro limiting dilution test (ELDA) of sphere formation. F. Quantitative analysis of the number of spheres (per 1000 cells) formed by the ELDA analyzed GSCs in (E). Data are expressed as mean ± SEM of six independent experiments, p <0.01.G. Representative images of neurospheres derived from GSCs of T387 (left) and T4121 (right) expressing shCont, shNUP98-1 or shNUP 98-2. Scale bar, 100 μm. Each image represents at least 5 replicates.

Figure 3 is NUP98 maintains GSCs stem cell markers, viability and glioblastoma growth. Immunoblots showed Caspase 3 (A) and PARP (B) levels after transduction of GSCs with control non-targeted shRNA sequences (shCONT), shNUP98-1 or shNUP 98-2. Immunofluorescent staining of clear Caspase-3 (green) in GBM tumors expressing shCONT, shNUP98-1 or shNUP98-2 387 and 4121GSCs and quantification of clear Caspase-3 positive cells. Scale bar: 25 μm. Data are expressed as mean ± s.e.m. p <0.01, t-test. E. Expression of NUP98 was inhibited in 387 and 4121GSCs with two non-overlapping shRNAs. Control sequences shRNA not directed against known mammalian genes (shCONT) were used as negative controls. Quantitative RT-PCR was performed on stem cell markers, including SOX2, OLIG2 and MYC. Data are expressed as mean ± SEM of three independent experiments, p <0.01.F. Immunoblots showed stem cell marker (SOX 2, OLIG2, and MYC) levels after transduction of GSCs with control non-targeted shRNA sequences (shCONT), shNUP98-1, or shNUP 98-2. G. Kaplan-Meier survival curves of intracranial immune-compromised mice expressing shCONT, shNUP98-1 or shNUP98-2, 387 or 4121 GSCs. (n=5 animals per group). H. Mouse brain tissue collected on day 18 after 387 or 4121GSCs expressing shCONT, shNUP98-1 or shNUP98-2, hematoxylin and eosin stained sections. Scale bar, 2 mm.

FIG. 4 is a DNA repair pathway regulated by NUP98 in GSCs. FIG. 4A. Analysis of gene enrichment of GO pathways associated with NUP98 expression in TCGA database. B. NUP98 expression was significantly correlated with transcriptional characteristics of DNA repair in TCGA glioblastoma samples. Single sample GSEA scores for TCGA RNA sequence data were calculated using molecular signature databases and NUP98 expression signatures for DNA repair genes. The shading represents the 95% confidence interval. Thermogram analysis of differentially expressed genes between nup98 silenced 387 and 4121GSCs (shNUP 98) and control 387 and 4121GSCs (shCONT). The differentially expressed genes have a 2-fold or greater difference in expression. In the differentially expressed genes, 160 were up-regulated and 185 were down-regulated. D. RNA sequencing (RNA seq) analysis was performed by comparing differentially expressed genes between NUP98 silenced 387 (shNUP 98) and 387GSCs (shCONT), 4121GSCs (shNUP 98) and control 4121GSCs (shCONT). Prioritization is based on the consistency of similar results for each GSC silence. The pictures represent the overlap between shNUP 98-specific genes identified by RNA sequence analysis. GSEA of genes down-regulated after NUP98 knockdown in e.387 and 4121 GSCs. And drawing an enrichment gene characteristic map by using the standardized enrichment score. F. NUP98 knockdown in 387 and 4121GSCs was analyzed for biological repeat RNA-seq and DNA repair pathway genes were selectively analyzed to calculate Z scores from log2 transformed FPKM values for each sample. G. In 387 and 4121GSCs, the expression of NUP98 was inhibited using two non-overlapping shRNA. Control sequences shRNA not directed against known mammalian genes (shCONT) were used as negative controls. Quantitative RT-PCR was performed on DNA repair enzymes, including EME1, RAD54B, RAD, BLM, BRCA1, BRCA2, XRCC2, BRIP1, and RAD54L. Data are presented as mean ± SEM of three independent experiments. * P <0.01.H. Immunoblotting experiments reflect the level of the DNA repair marker gama-H2AX after GSCs are transduced against non-targeted shRNA sequences (shCONT), shNUP98-1 or shNUP 98-2. I. Immunofluorescent staining of gama-H2AX (gama-H2 AX, green) and quantification of gama-H2AX positive cells in GBM tumors expressing shCONT, shNUP98-1 or shNUP98-2, 387 and 4121 GSCs. Scale bar: 25 μm. Data are expressed as mean ± s.e.m. p <0.01, t-test.

FIG. 5 is a DNA repair pathway of NUP98 interacting with P65 to regulate GSCs. Non-specific control IgG or anti-NUP98 antibodies in a 387gsc cell lysates were mass-resolved followed by Immunoprecipitation (IP). Western blotting of Immunoprecipitated (IP) products with anti NUP98 antibodies or anti P65 antibodies in 4121 and 387 GSCs. F. Immunofluorescent staining of NUP98 and P65 in GBM tumors derived from 387 and 4121 GSCs. H. Immunoblots showed levels of DNA repair markers gama-H2AX after transduction of GSCs with control non-targeted shRNA sequences (shCONT), shp65-1 or shp 65-2. I. Immunofluorescent staining of gama-H2AX (gama-H2 AX, green) and quantification of gama-H2AX positive cells in GBM tumors expressing shCONT, shP65-1 or sh65-2 387 and 4121 GSCs. Scale bar: 25 μm. Data are expressed as mean ± s.e.m. p <0.01, t-test.

FIG. 6 is a graph showing that NUP98 and P65 together regulate DNA repair pathways in GSCs. Thermogram analysis of differentially expressed genes between nup98 silenced 387 (shNUP 98) and control 387GSCs (shCONT). The differentially expressed genes have a 2-fold or greater difference in expression. In the differentially expressed genes, 160 were up-regulated and 185 were down-regulated. GSEA down-regulated after P65 knockdown in B.387 and 4121 GSCs. And drawing an enrichment gene characteristic map by using the standardized enrichment score. RNA seq analysis was performed after P65 knockdown in C.387 and 4121GSCs, and DNA repair pathway genes were selectively analyzed to calculate Z scores based on log2 transformed FPKM values for each sample. D. RNA sequencing (RNA seq) analysis was performed by comparing the differentially expressed DNA repair genes in the NUP98 silencing, P65 silencing and JASPAR databases. E. Quantitative RT-PCR was performed on DNA repair enzymes, including BRAC1, BRAC2, RAD51, BLM, RAD54L, XRCC2, EME1, and BLM, using shNUP98, shP65 alone or a combination of shNUP98 and shP. Data are presented as mean ± SEM of three independent experiments. * P <0.01.F. DNA repair enzymes, including BRAC1, BRAC2, RAD51, BLM, RAD54L, XRCC2, EME1, and BLM, were immunoblotted with shNUP98, shP65 alone or in combination with shNUP98 and shP. NUP98 and P65 bind directly to the promoter region of enzymes in the DNA repair pathways of 387 and 4121 GSCs. t-test, P <0.05, P <0.01; n=3 independent experiments per group. Data are expressed as median ± s.e.m.

FIG. 7 is that NUP98 overexpression correlates with low survival in brain tumor patients. (A) mRNA expression of NUP98 in normal brain (n=10) and glioblastoma tissue (n=528) from TCGA glioblastoma database. (B to E) Kaplan-Meier survival analysis of NUP98 by four different glioma databases; (B) TCGA glioblastoma and TCGA glioblastoma LGG RNA sequences; (C) CGGA GBM and CGGA glioblastoma LGG RNA sequences; (D) REMBRANDT (E) Lee Y GBM. (F) ROC curve analysis of NUP98 expression levels as markers for glioblastoma. The diagnostic accuracy of NUP98 as glioblastoma marker was 93%, the diagnostic specificity was 100% and the sensitivity was 85.4%.

FIG. 8 is a correlation analysis of nuclear pore complex family member genes. FIG. 8 shows the correlation of gene expression in TCGA glioblastoma patients. Fig. 9 shows that P65 interacts with NUP98 and its expression is significantly correlated with transcriptional characteristics of DNA repair in TCGA glioblastoma samples.

FIG. 10 is a DNA repair pathway of P65 regulated GSCs. Two non-overlapping shRNA knockdown P65 expression was used in 387 and 4121 GSCs. Control sequences shRNA not directed against known mammalian genes (shCONT) were used as negative controls. Quantitative RT-PCR was performed on DNA repair enzymes, including RAD51, BLM, BRCA2, EME1, BRCA1, BRIP1, XRCC2, BRCC3, RAD54L, and MRE11. Data are presented as mean ± SEM of three independent experiments. * P <0.01.

Taken together, gliobastoma (GBM) is the primary tumor with the highest malignancy of the central nervous system, glioma stem cells (Glioma Stem Cells, GSC) are considered to be critical for GBM treatment tolerance and tumor recurrence, but clinical treatment regimens targeting GSC alone or in combination have not progressed effectively. In the application, the NUP98 gene plays an important role in GSC self-renewal, proliferation and glioma malignant progress by regulating and controlling GSC DNA damage repair. Through analysis of patient databases and cellular levels we found that NUP98 was specifically highly expressed in GSCs. After NUP98 knockdown we found that GSC self-renewal, proliferation, and tumor progression in vivo could be inhibited. Further by transcriptome sequencing and bioinformatic analysis, we found that NUP98 plays a role in GSC self-renewal, proliferation and inhibition of tumor progression in vivo by modulating DNA damage repair. Finally, through protein mass spectrum detection, we find that NUP98 regulates DNA damage repair to be combined with a transcription factor P65 and regulates the transcription activity of the P65, and NUP98 overexpression is related to low survival rate of brain tumor patients. The expression level of NUP98 is used as a glioblastoma marker, the diagnosis accuracy of NUP98 serving as the glioblastoma marker is 93 percent, the diagnosis specificity is 100 percent, and the sensitivity is 85.4 percent.

It should be noted that the above embodiments are only for illustrating the technical solution of the present application and not for limiting the same, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present application may be modified or substituted without departing from the spirit and scope of the technical solution of the present application, which is intended to be covered in the scope of the claims of the present application.

Claims

1. The application of a reagent for detecting NUP98 gene in preparing glioblastoma diagnostic reagent is characterized in that: the NUP98 gene is used as a glioma stem cell specific molecular marker.

2. The use according to claim 1, characterized in that: glioblastoma is diagnosed by detecting the mRNA expression level of NUP98 gene.

3. Use according to claim 1 or 2, characterized in that: the NUP98 gene is combined with the transcription factor P65, and the transcription activity of the P65 is regulated to regulate and control DNA damage repair of glioma stem cells.

4. Use according to claim 1 or 2, characterized in that: NUP98 gene detection primers were, upstream primer: 5'-CTCCACCACTAATTCAGGCTTT-3'; a downstream primer: 5'-GAGGCTGGTAGTCTGCTGATT-3'.