Application of TCF7L2 in treatment of glioblastoma

Abstract

The invention discloses an application of TCF7L2 (T cell factor 7L2) in treatment of glioblastoma. The research shows that the transcription factor TCF7L2 has an important tumor inhibition effect in glioblastoma cells (GBM), and the deletion of the transcription factor TCF7L2 can obviously accelerate the growth and malignant progression of tumor cells, so that the response capability of the GBM cells to exogenous neural signals is enhanced, and the tumor development is further promoted. In mechanism, the TCF7L2 regulates and controls the expression of various neurotransmitter receptors, and inhibitors applying the receptors can effectively reverse tumor promoting phenotypes caused by deletion of the TCF7L2, so that the TCF7L2 plays a key role in inhibiting GBM nerve dependence progression by maintaining the steady state of neural signal related receptors. The invention provides a new diagnostic and prognostic marker and a new therapeutic target for treatment of GBM, and provides a new thought and a new strategy for treatment of GBM.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and relates to application of TCF7L2 in treatment of glioblastoma. BACKGROUND

[0002] Glioblastoma (GBM) is a highly infiltrative and invasive central nervous system (CNS) tumor arising from irregular astrocytic or other glial cells, and is the most common primary brain tumor in adults. The median survival after diagnosis is less than 15 months, and the 5-year survival rate is 6.4%-14%. Despite some progress in glioma research in recent years, including the discovery of molecular biology and genomics, the study of tumor microenvironment, and the attempt of immunotherapy and targeted therapy, there are still significant deficiencies in treatment. Surgery is difficult to completely remove the tumor, radiotherapy and chemotherapy are prone to drug resistance, immunotherapy has limited effect, targeted therapy is difficult to overcome tumor heterogeneity, and the existence of cancer stem cells further exacerbates the complexity of treatment.

[0003] In recent years, many studies have revealed the complex interactions between neurons and glioblastoma (GBM), which have important implications for understanding the occurrence, development and treatment of GBM. Studies have shown that neurons can promote the growth and invasion of GBM through various mechanisms. For example, neurons can activate tumor cell signaling pathways by releasing neurotransmitters such as glutamate, enhancing the proliferation and migration ability of tumor cells. Seifert et al. found that glutamate promotes the proliferation and invasiveness of glioma cells by activating AMPA and NMDA receptors on the surface of glioma cells. In addition, glutamate also indirectly promotes tumor growth by inducing tumor-associated epilepsy and increasing neuronal activity around the tumor. In addition, Venkatesh et al. found that neuronal activity can significantly promote the growth and invasiveness of glioma by secreting neuroligin-3. Neuroligin-3 is a synaptic adhesion molecule mainly expressed in active synapses, and its secretion in the glioma microenvironment can be captured by tumor cells and promote the proliferation and migration of tumor cells by activating the PI3K / AKT signaling pathway.

[0004] In addition, neurons can also directly affect the behavior of glioma cells by secreting various neurotrophic factors. For example, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and glial cell-derived neurotrophic factor (GDNF) all play important roles in the development and functional maintenance of the nervous system. Studies have shown that glioma cells express high levels of TrkB and TrkC receptors on their surface, which enables them to respond to neurotrophic factors, increasing their proliferation and migration ability. Moreover, glioma cells also secrete neurotrophic factors themselves, forming an autocrine loop that further enhances the growth and invasiveness of tumor cells.

[0005] Wnt / β-catenin signaling is involved in many physiological processes, such as cell proliferation, differentiation, organogenesis, tissue regeneration, and tumorigenesis. The canonical Wnt signaling is divided into β-catenin-dependent (canonical, Wnt / β-catenin pathway) and β-catenin-independent (non-canonical, Wnt / planar cell polarity [PCP] and calcium channel) signals. In human cancers, Wnt / β-catenin signaling is highly activated, including colorectal cancer, liver cancer, breast cancer, and glioblastoma. In these cancers, the increased stability of β-catenin leads to its accumulation in the nucleus, which in turn triggers abnormal gene expression, promoting cell proliferation and survival, and enhancing the invasiveness and metastatic ability of tumors. This has led to therapeutic strategies targeting this pathway becoming a research hotspot, with various small molecule inhibitors, antibodies, and nucleic acid drugs being developed to interfere with the binding of Wnt ligands to receptors and the nuclear translocation of β-catenin. However, blocking Wnt signaling can cause side effects such as impaired tissue homeostasis and regeneration. These cancer-specific regulatory processes of Wnt signaling may be the druggable vulnerability of Wnt signaling-related cancers, and currently, various Wnt pathway-targeting drugs have entered the clinical stage.

[0006] TCF7L2, as a key transcriptional effector of the Wnt / β-catenin signaling pathway, is involved in the development of many cancers. Studies have found that mutations in TCF7L2 are often found in gastric cancer and breast cancer, and the loss of function of TCF7L2 in primary colorectal cancer can promote the growth of cells in vitro. In recent years, studies have found that TCF7L2 is expressed in the brain and is involved in neural development and functional regulation, and may play a role in neurodegenerative diseases and mental illnesses, and studies have shown that conditional knockout of TCF7L2 in astrocytes leads to an increase in gap junctions in astrocytes. However, it is not clear whether TCF7L2 is involved in the response between neurons and GBM, and how TCF7L2 regulates this process. SUMMARY

[0007] The purpose of the present application is to solve the above problems, and provide a TCF7L2 application in the treatment of glioblastoma.

[0008] To achieve the object, the present application adopts the following technical scheme:

[0009] The first aspect of the present application provides the use of a reagent for detecting the expression of biomarker TCF7L2 in the preparation of a product for diagnosing or prognosing glioblastoma.

[0010] The method for diagnosis comprises obtaining a test sample from a subject suspected of suffering from glioblastoma, determining the expression level of TCF7L2 in the test sample, and if the expression of TCF7L2 is lower than that of a normal control, it indicates that the subject is a glioblastoma patient.

[0011] The method for prognosis comprises obtaining a test sample from a subject suffering from glioblastoma, determining the expression level of TCF7L2 in the test sample, and if the expression of TCF7L2 is lower than that of a normal control, it indicates that the patient has a poor prognosis of survival.

[0012] The second aspect of the present application provides the use of TCF7L2 as a target in the screening of drugs for treating glioblastoma.

[0013] The drug increases the expression of TCF7L2.

[0014] TCF7L2 regulates the expression of multiple types of neurotransmitter receptors, and its deletion leads to significant up-regulation of genes related to glutamate receptors, acetylcholine receptors, serotonin receptors, adrenergic receptors and GABA receptors.

[0015] The third aspect of the present application provides the use of an expression promoter of TCF7L2 in the preparation of a drug for treating glioblastoma.

[0016] The expression promoter is selected from a nucleic acid molecule, a protein molecule, an overexpression plasmid or a small molecule compound.

[0017] Preferably, the expression promoter is an overexpression plasmid.

[0018] The present application has the following beneficial effects:

[0019] The present application studies the effect of TCF7L2 on the connection between neurons and GBM through in vivo and in vitro experiments, and the important factors that regulate the process. The present application shows that the transcription factor TCF7L2 has an important tumor inhibiting effect in glioblastoma (GBM), and its deletion can significantly accelerate the growth and malignant progression of tumor cells, and enhance the response ability of GBM cells to exogenous nerve signals, thereby further promoting tumor development. Mechanically, TCF7L2 regulates the expression of multiple types of neurotransmitter receptors, and its deletion leads to significant up-regulation of glutamate receptors, acetylcholine receptors, serotonin receptors, adrenergic receptors and GABA receptors related genes. The application of inhibitors of these receptors can effectively reverse the tumor-promoting phenotype caused by the deletion of TCF7L2, suggesting that TCF7L2 plays a key role in inhibiting GBM nerve-dependent progression by maintaining the homeostasis of nerve signal-related receptors. The present application provides a new diagnostic and prognostic marker and a new therapeutic target for the treatment of GBM, and provides a new idea and strategy for the treatment of GBM. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is the expression of TCF7L2 in glioblastoma and clinical correlation analysis results in TCGA and CGGA databases.

[0021] Figure 2 is the TCF7L2 expression detection result after TCF7L2 conditional knockout.

[0022] Figure 3 is the survival time statistics result of mice after TCF7L2 knockout.

[0023] Figure 4 is the cell proliferation EdU staining diagram and cell proliferation statistics result.

[0024] Figure 5 is the TCF7L2 protein level detection result of U87 cells after TCF7L2 overexpression.

[0025] Figure 6 is the survival time statistics result of GBM xenograft tumor mice constructed by TCF7L2 overexpression.

[0026] Figure 7 is the GO enrichment analysis of genes up-regulated after TCF7L2 knockout.

[0027] Figure 8 shows that the invasion and proliferation ability of tumor cells with TCF7L2 deletion is enhanced after treatment with neuron supernatant.

[0028] Figure 9 is the Transwell invasion experiment result.

[0029] Figure 10 This is the result of the transcriptional expression level detection of neurotransmitter receptors after TCF7L2 knockout.

[0030] Figure 11 This is the result of transcriptional level detection of some neurotransmitter receptors after TCF7L2 knockout.

[0031] Figure 12 These are the results of cell proliferation experiments after treatment with GluR, AchR, 5-HTR, AR, and GABAR inhibitors.

[0032] Figure 13 This is a Transwell invasion assay after treatment with GluR, AchR, 5-HTR, AR, and GABAR inhibitors. Detailed Implementation

[0033] Example 1

[0034] I. Data Analysis of TCF7L2 Expression and Clinical Relevance in Glioblastoma

[0035] A search of the TCGA and CGGA databases on the Gliovis website (https: / / gliovis.bioinfo.cnio.es / ) revealed the relationship between TCF7l2 expression in patient tissues and patient prognosis. The data indicated that... Figure 1 TCF7L2 expression decreases in glioma patient tissues with increasing glioma grade and is associated with patient survival.

[0036] II. Effects of TCF7L2 deficiency on the proliferation of primary GBM mouse tumor cells

[0037] 1. Identification of TCF7L2 conditional knockout mice

[0038] Pten mice were generated by crossing Tcf7l2 conditional knockout mice (from Hubrecht Institute for Developmental Biology and Stem Cell Research & University Medical Centre Utrecht, Utrecht, Netherlands) with Pten and P53 conditional knockout mice (from The Jackson Laboratory). fl / fl Trp53 fl / fl Tcf7l2 fl / fl In mice, the DNA-binding region of exon 11 of TCF7L2 was specifically knocked out, and the knockout efficiency was verified by qPCR. The results showed that this model could successfully knock down the functional region of TCF7L2. Figure 2 ).

[0039] The primers for identifying the gene mice are as follows:

[0040] Ptenlox1 (SEQ ID NO. 1): 5'-ATCCCCACCAATGAACAAAC-3';

[0041] Ptenlox2 (SEQ ID NO. 2): 5'-CTCCTCTACTCCATTCTTCCC-3';

[0042] P53_T008 (SEQ ID NO. 3): 5'-CACAAAAACAGGTTAAACCCAG-3';

[0043] P53_T009 (SEQ ID NO. 4): 5'-AGCACATAGGAGGCAGAGAC-3';

[0044] Tcf7l2_f1 (SEQ ID NO. 5): 5'-CCAGATGAGTGTCATCCATAGG-3';

[0045] Tcf7l2_r2 (SEQ ID NO. 6): 5'-GGAATGTGTAGATACTGACAGC-3'.

[0046] Take 2-3 mm of mouse toe or tail tip tissue, add 200 μL of 0.05 mol / L NaOH lysis solution to a metal bath at 95°C for 30 min, then add 20 μL of pH=8.0, 1 mol / L Tris-HCl buffer for neutralization, and take the supernatant as the template. Prepare a system with 25 μL of the system, mix well after preparation, and divide 23 μL / tube into eight PCR tubes. Add 2 μL of sample to each tube and label. Place the eight PCR tubes in a centrifuge and centrifuge slightly to ensure that the liquid is at the bottom of the PCR tube. Place the PCR instrument in the instrument, select the PCR reaction condition program, and start the reaction. Prepare a 2-3% agarose gel and electrophorese at 130V for 20-30 min to ensure that the sample migrates to 2 / 3 of the gel, and then develop on the instrument.

[0047] 2. Construction of GBM primary mouse model

[0048] The PDGFRB-Cre retrovirus is packaged by a retroviral packaging plasmid in 293FT, and the virus titer is usually not less than 1 x 10 8TU / mL. The virus was dissolved in PBS solution, stored at -80°C before use, and avoided repeated freeze-thawing. 6-8 weeks old mice carrying conditional target genes were selected. Anesthesia was performed by 1.5-2% isoflurane inhalation anesthesia. After sufficient anesthesia, the mouse was fixed on the stereotaxic instrument, and the body temperature was kept constant. After anesthesia, the hair on the top of the head was shaved, and iodophor was used for disinfection. The scalp was incised along the midline, the skull surface was exposed, and the bregma was identified. According to the stereotaxic instrument reading, the injection point coordinates were determined: relative to bregma, anterior (AP) +0.5 mm, right (ML) -0.85 mm, and depth (DV) about -2.45 mm. A micro-drill was used to drill a hole at the target position, and the hole diameter was less than 0.5 mm. The glass microneedle containing the virus solution was slowly lowered to the target depth, and 2.0 μL of virus was injected at a speed of 1 μL / min. After injection, it stayed for 2-3 min and then the needle was slowly pulled out to avoid backflow. Then the wound was sutured.

[0049] The survival of 15 mice in the control group and the experimental group was collected Figure 3 , and the survival of mice after TCF7L2 knockout was significantly shortened.

[0050] 3. Tumor primary cell extraction and culture in vitro

[0051] The tumor area of the dying mouse brain was isolated, cut with a sterile blade, resuspended with 3 mL Accutase (containing DNase), digested at 37°C for 20 minutes, and gently blown with a pipette every 5 minutes during the digestion. After digestion, 6 mL of F12 medium was used to terminate the digestion, and a single cell suspension was filtered out using a 70 μm cell screen. After centrifugal collection, the cells were resuspended in a special culture medium and incubated in a 37°C incubator, with a two-day subculture.

[0052] Cell proliferation EdU staining: BeyoClick EdU-488 Cell Proliferation Detection Kit (Bi Yun Tian) was used, and 3x10 TM EdU-488 cell proliferation detection kit (Bi Yun Tian), 3x10 4 Primary tumor cells were plated in 24-well plates pre-incubated with Laminin (10 mM), and after 48 h of adherent culture, EdU working solution (10 mM, 2 μL / mL) was added and incubated for 90 min. After incubation, the medium and non-adherent cells were washed with PBS, fixed with 4% paraformaldehyde for 15 min, blocked, and then stained with corresponding color EdU (488 nm) for 30 min. After incubation, Dapi was incubated and the slide was sealed.

[0053] Figure 4 The results show that the deletion of Tcf7l2 in mouse tumor cells cultured in vitro can significantly increase the proliferation of tumor cells.

[0054] Three, TCF7L2 can inhibit the growth of tumor in xenotransplantation tumor model

[0055] 1. Construction of stable overexpressing TCF7L2 glioma cell line

[0056] (1) Lentivirus plasmid construction: TCF7L2 Gene ID in NCBI: Homo sapiens (human) 6934; Mus musculus (house mouse) 21416.

[0057] (2) Virus packaging:

[0058] Prepare HEK 293T cells and culture at 37°C; when the cells grow to 70% density in a 10 cm culture dish, replace the complete medium with serum-free medium 1 h before transfection. The plasmid transfection system is as follows: pMD2.G 1.875 μg, psPAX2 5.625 μg, TCF7L2-OE 7.5 μg, transfection reagent PEI 45 μL (1 μg / μL). Prepare 500 μL serum-free DMEM medium + 45 μL transfection reagent PEI, mix well, and stand for 5 min; prepare 500 μL serum-free DMEM medium + 7.5 μg target plasmid + 1.875 μg pMD2.G + 5.625 μg psPAX2, mix well, and stand for 5 min. Mix the two prepared liquids together and stand at room temperature for 20 min; add the mixed liquid to the culture dish; after 4-6 h, replace the medium with complete medium containing serum; collect the virus supernatant after 48 h and 72 h of transfection, centrifuge at 3000 rpm for 10 min, and filter with a 0.45 μm filter. Store the filtered virus at -80°C.

[0059] (3) Lentivirus infection steps:

[0060] According to the needs, seed the cells in a six-well plate and place them in an incubator overnight; thaw the virus liquid on ice, remove the medium, and wash with PBS once, then add 1 mL fresh medium + 1 mL virus liquid; after 48 h of infection, add an appropriate concentration of puromycin for stable selection, and use the wild-type cell line with the same concentration of puromycin for selection as a control. When the wild-type cells all die during the selection process, stop the selection.

[0061] (4) Collect part of the selected cells to extract protein: routine method.

[0062] (5) Western Blot detection: routine method.

[0063] Figure 5The results show that: using lentivirus containing TCF7L2 overexpression to infect U87MG cells, and using Western blot experiment to verify the overexpression efficiency. The experimental results show that, compared with the control group, the TCF7L2 protein level of U87 is significantly increased.

[0064] 2. Construct GBM xenograft tumor model

[0065] The U87MG cells overexpressing TCF7L2 were digested to 3x10 4 / 2μL concentration, resuspended with PBS, and injected in situ at the striatal site according to the coordinates (AP) +0.4 mm, right (ML) -1.8 mm, and depth (DV) about -2.48 mm. The tumor cells were transplanted in situ into the brain of mice, and the size of the tumor and the survival period of the mice were analyzed. It was found that after the deletion of TCF7L2, the tumor volume increased and the survival period shortened, but after overexpression, the survival period of the mice was prolonged ( Figure 6 ).

[0066] Four, TCF7L2 knockout upregulates genes and pathways related to neural development, synaptic plasticity, and ion channel

[0067] The rnaseq analysis of mouse tumor cells and tumor tissues was performed, and the genes significantly upregulated after TCF7L2 KO were subjected to GO enrichment analysis ( Figure 7 ), and it was found that genes and pathways related to the nervous system were significantly enriched, including neural development, synaptic plasticity, and ion channel activation.

[0068] Five, TCF7L2-deleted tumor cells have enhanced invasion and proliferation ability after treatment with neuron supernatant

[0069] 1. Extract fetal rat hippocampal neurons and collect neuron supernatant cultured for 3-5 days to treat primary cells and detect EdU expression for cell proliferation. TCF7L2-knockout tumor cells have significantly enhanced proliferation ability under the induction of neuron supernatant ( Figure 8 ).

[0070] 2. In the Transwell invasion experiment ( Figure 9 ), neurons or neuron supernatant were placed in the lower chamber, and primary tumor cells were added to the upper chamber. It was found that TCF7L2-knockout tumor cells have significantly enhanced invasion ability under the induction of neuron supernatant.

[0071] Six, TCF7L2 directly regulates the expression of neurotransmitter receptors in glioma cells

[0072] 1. Quantitative PCR

[0073] The RNA of mouse GBM primary tumor tissue and the RNA of primary cells were extracted, respectively.

[0074] Quantitative PCR: ACTIN as an internal reference, reverse transcription of cDNA as a template, for each sample and each primer, set 3 duplicate holes, each duplicate hole volume is 10 μL, reaction body as follows: 2 x SYBR green 5 μL, cDNA 2 μL, upstream primer 1 μL, downstream primer 1 μL, ddH2O 1 μL. PCR program: 95℃ 30s; 95℃ 10s, 60℃ 30s, 95℃ 15s, 60℃ 1min, 40 cycles; 95℃ 15s. According to the final detection of Ct value results and internal reference gene results, the relative expression of target gene mRNA is calculated according to .

[0075] qPCR results ( Figure 10 ) show that by analyzing the expression of various neurotransmitter receptors, it is found that the transcription level of some neurotransmitter receptors is significantly up-regulated after TCF7L2 knockout.

[0076] Analysis of various neurotransmitter receptors in sequencing data shows that the transcription level of some neurotransmitter receptors is significantly up-regulated after TCF7L2 knockout ( Figure 11 ). 2.

[0078] First, the TCF7L2 knockout primary cells are fixed, and the target protein is recognized and combined with specific antibodies; then the Protein A / G-Tn5 transposase complex combined with the antibody is added to locate near the target protein binding site; then activate the Tn5 transposase to cut the DNA at these sites and insert sequencing adapters at the same time; finally extract the DNA and perform PCR amplification and high-throughput sequencing. The binding map of the target protein on the genome can be accurately drawn through the sequencing results.

[0079] The cut tag experiment was performed using primary cells, and it was found that TCF7L2 directly regulates ionotropic glutamate receptors (NMDA), metabolic glutamate receptors (mGLUR), muscarinic acetylcholine receptors, adrenergic receptors, nicotinic acetylcholine receptors, and GABA receptor genes. Seven、

[0081] GluR (glutamate receptor), AchR (acetylcholine receptor), 5-HTR (5-hydroxytryptamine receptor), AR (adrenaline receptor), and GABAR inhibitor can restore the tumor-promoting phenotype caused by TCF7L2 deletion.

[0082] Cell activity detection ( Figure 12): Using CellTiter-Glo® ATP Assay (cell ATP activity detection) kit, it was found that the partial cell proliferation of Tcf7l2 deleted primary tumor cells was significantly inhibited under the action of GluR, AchR, 5-HTR, AR, GABAR inhibitors (10 μM).

[0083] In Transwell invasion experiment ( Figure 13 ), when containing neuron supernatant, the use of various inhibitors can restore the phenotype of increased cell invasion caused by TCF7L2 deletion.

[0084] Eight, analysis and summary

[0085] In this study, transcription factor TCF7L2 plays an important role in inhibiting glioblastoma (GBM). Its deletion can significantly accelerate the growth and malignant progression of tumor cells, and enhance the response ability of GBM cells to exogenous nerve signals, thereby further promoting tumor development. Mechanistically, TCF7L2 regulates the expression of multiple neurotransmitter receptors, and its deletion leads to significant up-regulation of glutamate receptor, acetylcholine receptor, serotonin receptor, adrenergic receptor and GABA receptor related genes. Notably, the application of inhibitors of these receptors can effectively reverse the tumor-promoting phenotype caused by TCF7L2 deletion, suggesting that TCF7L2 plays a key role in inhibiting GBM nerve-dependent progression by maintaining the homeostasis of nerve signal related receptors.

Claims

1. Application of reagents for detecting the expression of the biomarker TCF7L2 in the preparation of products for the diagnosis or prognosis of glioblastoma patients.

2. The application according to claim 1, characterized in that, The diagnostic method includes: obtaining a test sample from a subject suspected of having glioblastoma, determining the expression level of TCF7L2 in the test sample, and if the expression level is lower than that of normal controls, it suggests that the subject is a glioblastoma patient.

3. The application according to claim 1, characterized in that, The prognostic method includes obtaining test samples from subjects with glioblastoma, determining the expression level of TCF7L2 in the test samples, and indicating a poor prognosis for the patient if the expression level is lower than that of normal controls.

4. Application of TCF7L2 as a target in screening drugs for the treatment of glioblastoma.

5. The application according to claim 4, characterized in that: The drug increases the expression of TCF7L2.

6. The application according to claim 5, characterized in that: TCF7L2 regulates the expression of multiple neurotransmitter receptors, and its absence leads to a significant upregulation of genes related to glutamate receptor, acetylcholine receptor, serotonin receptor, adrenergic receptor, and GABA receptor.

7. Application of TCF7L2 expression promoters in the preparation of drugs for treating glioblastoma.

8. The application according to claim 7, characterized in that: The expression promoter is selected from nucleic acid molecules, protein molecules, overexpression plasmids, or small molecule compounds.

9. The application according to claim 8, characterized in that: The expression promoter is an overexpression plasmid.

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