CN111363820A

CN111363820A - Application of COPZ1 as brain glioma treatment/prognosis judgment target

Info

Publication number: CN111363820A
Application number: CN202010196020.6A
Authority: CN
Inventors: 李新钢; 王剑; 王东海; 倪石磊; 黄斌; 张玉霖
Original assignee: Qilu Hospital of Shandong University
Current assignee: Qilu Hospital of Shandong University
Priority date: 2020-03-19
Filing date: 2020-03-19
Publication date: 2020-07-03
Anticipated expiration: 2040-03-19
Also published as: CN111363820B

Abstract

The invention particularly relates to application of COPZ1 as a brain glioma treatment/prognosis judgment target point. The invention provides the COPZ1 as a therapeutic target, which can remarkably regulate the iron metabolism of GBM cells and participate in the processes of intracellular transport, endosome maturation, lipid homeostasis, autophagy and the like. In glioma patients, overexpression of COPZ1 was associated with increased tumor grade and poor prognosis. The GBM group COPZ1 protein levels were significantly elevated compared to normal brain tissue. The COPZ1 gene knockout can inhibit glioma cell proliferation. The invention proves that the COPZ1 gene knockout directly activates NCOA4, so that ferritin is degraded, the intracellular ferrous level is increased, and a Fenton reaction is triggered, so that glioma cells are killed by iron. These data indicate that COPZ1 plays a key role in iron metabolism, and that the COPZ1/NCOA4/ATG7 axis is a novel therapeutic target for human brain glioma.

Description

Application of COPZ1 as brain glioma treatment/prognosis judgment target

Technical Field

The invention belongs to the technical field of glioma diagnosis markers, and particularly relates to application of COPZ1 as a brain glioma treatment/prognosis judgment target point and application of COPZ1 as a glioma cell iron metabolism, lipid peroxidation or autophagy pathway regulator.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults with an annual incidence of 5.26/10 ten thousand. Median survival of patients was 14.6 months despite maximal surgical resection, radiation therapy and temozolomide treatment. Patients with gliomas generally have a poor prognosis and a poor quality of life. The goal of treatment of GBM is to induce tumor cell death while retaining normal tissue cells. However, tumor cells lead to tumor resistance by activating stress-adaptive responses to genetic or epigenetic changes, and understanding the molecular mechanisms of these protective responses may improve therapeutic efficacy.

Iron death is an iron-dependent regulated cell death that is the result of lethal lipid peroxidation. Although the physiological function of iron death is not known, accumulation of Reactive Oxygen Species (ROS) beyond the redox content maintained by Glutathione (GSH) can induce iron death. Iron is an important cofactor for metabolic enzymes and is closely associated with many biological processes such as neurotransmitter transmission, oxygen transport, cell division, and energy production. Disruption of normal iron transport may result in too high intracellular concentrations of iron, driving intracellular ROS production via Fenton (Fenton) reactions, triggering intracellular lipid peroxidation, and ultimately, cytotoxic effects on the cell. The fenton reaction is a catalytic process that converts ferrous iron and hydrogen peroxide into a highly toxic hydroxyl radical. Studies have shown that iron death is associated with a variety of human diseases, such as ischemic heart disease, brain injury, renal failure, and cancer. However, the role of iron death in GBM is currently poorly understood. A large body of evidence has been obtained over the past 10 years indicating that changes in iron absorption and iron utilization are essential characteristics of tumor cells, and that changes in iron metabolism are key metabolic features of cancer. Cancer cells alter the expression of many iron metabolism-related proteins and the activity of iron-related enzymes compared to normal cells. These changes often contribute to the relatively high utilization of iron by cancer cells and promote the function of iron-dependent proteins that are involved in many physiological processes, such as tumorigenesis, development and metastasis. Strategies based on iron homeostasis remodeling offer promising options for cancer treatment. Furthermore, reducing intracellular iron content, targeting iron-related proteins, or increasing intracellular iron levels by iron chelators is considered a viable approach to the treatment of cancer.

In current studies, several proteins have been shown to be involved in iron uptake, storage and utilization by cancer cells, including iron chaperone poly (rC) binding protein 1(PCBP1), nuclear receptor coactivator (NCOA4), iron response element binding protein 2(IREB2) and heat shock protein β (HSPB 1). Coatomer protein complex zeta 1(COPZ1) belongs to Coatomer protein complex I (COPI) and is involved in intracellular trafficking, endosomal maturation, lipid homeostasis and autophagy.

Disclosure of Invention

Targeting iron-related proteins or increasing intracellular iron levels is considered a viable approach to the treatment of cancer. Therefore, a strategy to remodel iron homeostasis may also be a promising therapeutic strategy for GBM. The present study demonstrates that decreasing expression of COPZ1 leads to iron death by directly increasing NCOA4 protein in human GBM cells and further provides the COPZ1/NCOA4/FTH1 pathway implicated in iron death, these findings not only demonstrate a novel role for COPZ1 in iron death, but also provide a future therapeutic target for treatment of GBM patients by induction of iron death.

According to the technical effects, the invention provides the following technical scheme:

in a first aspect of the invention, the use of COPZ1 as a target for the treatment/prognosis of glioma is provided.

According to the invention, the content of COPZ1 in glioma cells is related to the disease condition and poor prognosis by a statistical method, and the silencing of COPZ1 can reduce the activity of glioma cells and increase the death rate. Further research proves that the COPZ1 induces glioma cell death by regulating approaches such as iron metabolism, lipid peroxidation, autophagy and the like, and fully proves that the COPZ1 can be used as a target point for prognosis judgment and treatment of glioma and can be used for developing corresponding glioma treatment medicines.

In a second aspect of the invention, there is provided the use of COPZ1 as a modulator of target cell iron.

The iron includes target intracellular ferric ions (including ferrous and ferric ions in particular), Transferrin (TF) bound to iron, transferrin receptor (TFR) which transfers the transferrin iron complex into cells, ferritin which regulates ferrous iron (FTH 1).

Iron plays an important role in the body in oxygen storage, transport, maintenance of hematopoietic function, and the like, and is involved in many physiological processes such as cancer cell death, neurotoxicity, ischemia-reperfusion injury, and T cell immunity. The research of the invention shows that the COPZ1 participates in regulating the iron metabolism level in glioma cells by promoting the transport of intracellular iron, not only provides a basis for the development of antitumor drugs, but also is expected to apply the result to the development of related reagents for diseases such as neurotoxicity, ischemia-reperfusion injury, T cell immunity and the like.

In a fourth aspect of the invention, there is provided the use of an inhibitor of COPZ1 as an inducer of lipid peroxidation or as an agonist of malondialdehyde.

In a fifth aspect of the invention, there is provided the use of an inhibitor of COPZ1 as an agonist of reactive oxygen species.

In a sixth aspect of the invention, there is provided the use of an inhibitor of COPZ1 as an autophagy inducer.

In a seventh aspect of the invention, there is provided the use of an inhibitor of COPZ1 as an agonist of NCOA 4.

The beneficial effects of one or more technical schemes are as follows:

the CoPZ1 gene knockout can directly activate NCOA4, so that ferritin is degraded, the intracellular ferrous level is increased, and Fenton reaction is triggered, so that glioma cells are driven to enter an iron death state, a unique mechanism of iron metabolism induced glioma cell iron-dependent reaction is determined, and detailed evidence and treatment basis are provided for development of related drugs of glioma.

2. The above protocol strongly demonstrates that autophagy plays an important role in regulating iron death by increasing intracellular iron metabolism and cellular ROS accumulation. Iron death is activated when deletion of COPZ1 induces ferritin degradation, degrading intracellular iron storage and ferritin via NCOA 4-mediated pathways. Thus, elevated ferrous levels can cause a dramatic increase in ROS by triggering the Fenton reaction, leading to the accumulation of lipid peroxidation and further iron death. Therefore, the COPZ1/NCOA4/FTH1 axis and the resulting iron up-regulation may be a new target for the treatment of glioma.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description, serve to explain the invention and not to limit the invention.

FIG. 1 is a graph showing the correlation between the expression of COPZ1 in human glioma patients and glioma cells in example 1;

wherein (a) is COPZ1 RNA expression (log2) obtained from TCGA database classification based on 2016 WHO;

(B) Kaplan-Meier survival analysis of patient global survival data based on high and low expression of the TCGA dataset COPZ 1;

(C) (D) is representative Western blotting analysis of the protein level of COPZ1 in lysates (20 microgram) prepared from human brain glioma tissue (n ═ 9) and non-tumorous brain tissue (n ═ 3) in sequence, and the Western blotting statistical results of 3 independent experiments;

(E) (F) representative images of IHC staining in human brain glioma and non-tumorous brain tissue samples in sequence (normal, n-6; WHOII, n-18; WHOIII, n-18; WHOIV, n-18), scale bar, 50 μm and 100 μm; the graphical results show IHC scores;

(G) (H) sequentially carrying out representative western blotting analysis on the protein levels of the COPZ1 in a normal human astrocyte and a human brain glioma cell line, wherein the western blotting statistical results of 3 independent experiments are shown in the figure;

(I) immunofluorescent staining of U87MG and U251 cells for COPZ1, analysis under fluorescent microscope, nuclear staining with DAPI, scale bar, 10 μm, single-factor analysis of variance for multiple group comparisons: p <0.05, p <0.01, p < 0.001; log rank test: p < 0.05.

FIG. 2 is the Lonberg dataset described in example 1;

wherein (a) is COPZ1 RNA expression (log2) classified based on the 2016 world health organization lunbergan database;

(B) overall survival data for patients were analyzed for Kaplan-Meier survival curves based on expression of the Rembrandt dataset COPZ1, data shown as mean-Standard Error of Mean (SEM) for each group, one-way analysis of variance for multiple group comparisons: NS is not significant, p < 0.01; log rank test: p < 0.05.

FIG. 3 is a graph of the results of suppression of the GBM malignant phenotype and induction of cell death by COPZ1 silencing in example 2;

wherein (A) mRNA levels of COPZ1 in U87MG, U251 and P3# GBM cells infected with two independent COPZ1 siRNAs, si-COPZ1#1 and si-COPZ1#2 are detected by qRT-PCR;

(B) western blotting analysis of the protein levels of U87MG, U251 and P3-GBM cells COPZ1 transfected with si-COPZ1 and si-COPZ 1-2;

(C) the activity of the U87MG, U251 and P3# GBM cells is detected by a CCK8 method;

(D) (E) graphical representation of the proliferation rates of U87MG and U251 cells infected with si-COPZ1#1, followed by the EdU method, the cell nuclei stained with DAPI (scale bar, 100 μm), and the EdU-positive cell rates in U87MG and U251 cells transfected with si-COPZ1# 1;

(F) (G) representative images of the colony formation assay for U251 and P3# GBM cells transfected with si-COPZ1#1 in that order;

(H) lactate dehydrogenase release test results;

(I) (J) in turn are representative images of live/dead assays performed on U251 and P3# GBM cells transfected with si-COPZ1#1 to assess cell mortality, graphical representation of U251 and P3# GBM cell mortality of transfected si-COPZ1#1 (scale bar, 100 μm), two comparative sets of samples t-test: p <0.05, p <0.01, p < 0.001; one-way analysis of variance for multiple comparisons: p <0.05, p <0.01, p < 0.001.

FIG. 4 shows the levels of U87MG, U251 and P3-GBM cell COPZ1 protein transfected with sh-COPZ1 and sh-COPZ1 analyzed by Western blotting in example 2.

FIG. 5 is a graph showing the results of the change in iron metabolism caused by the deletion of the COPZ1 gene in example 3;

wherein (A) deletion of COPZ1 increases the total iron content in U87MG, U251 and P3-GBM glioma cells 24 hours after transfection of si-COPZ1 cells;

(B) the iron content determination kit displays the result;

(C) (D) representative fluorescence images (scale bar, 25 μm) of U87MG and U251 cells, in order, stained with JC-1 probe; (E) transmission electron microscopy images showed an increase in cell membrane density of U87MG infecting si-COPZ1 cells (white arrows), and a decrease in mitochondrial and autophagosome (black arrows) for 24 hours;

(F) western blot analysis showed that the COPZ1 gene knockout up-regulated the expression of TFR, TF, FTH1 in U87MG, U251 and P3# GBM glioma cells; and (4) t test: p <0.05, p <0.01, p < 0.001.

FIG. 6 is a graph showing the results of fluorescence imaging for detecting intracellular TFR in example 3;

wherein, (A) fluorescent image detects TFR protein and content histogram of U87MG and U251 cells, DAPI stains cell nucleus, phalodiin stains actin, and GSH and GSSG kit detects 25 μm;

(B) intracellular GSH levels, two sets of t-test comparisons: p <0.05, p <0.01, p < 0.001.

FIG. 7 is a graph of the results of glioma cell death induced by the upregulation of iron in the cells of example 4 by causing ROS and lipid peroxidation leading to the knockout of the COPZ1 gene;

(A) and (3) measuring the content of malonaldehyde: deletion of COPZ1 increased malondialdehyde in Si-COPZ1 transfected U87MG, U251 and P3-GBM glioma cells at 48 h;

(B) and (3) measuring the content of malonaldehyde: pretreatment with DFO, Fer-1 or GSH prevented overproduction of malondialdehyde induced by the COPZ1 gene knockout, which was exacerbated by FAC;

(C) iron analysis results: in the presence of DFO, the ferrous content decreases;

(D) LDH release test results: DFO and Fer-1 can reduce the death rate of glioma cells caused by the deletion of COPZ1, and FAC can aggravate the death rate;

(E) a representative dihydroethyl ether (DHE) image shows: the red fluorescence detected by the superoxide probe was significantly enhanced after 48h, statistical analysis of the fluorescence intensity of U87MG and U251 glioma cell lines transfected with si-COPZ1#1 compared to the respective control cells, 2 cell lines per group, 3 triplicate experimental images (scale bar, 75 μm.) were counted;

(F) histogram of superoxide content in the Dihydroether (DHE) image;

(G) hydrogen peroxide analysis showed hydrogen peroxide accumulation in P3# GBM cells, two comparative Student t-tests:

p <0.05, p <0.01, p < 0.001; one-way analysis of variance for multiple comparisons: NS is not significant, p <0.05, p <0.01, p < 0.001.

FIG. 8 is a graph showing the effect of FAC and FO treatments on glioma cells in example 4;

(A) (B) sequentially measuring the levels of U87MG and U251 malondialdehyde: pretreatment with FO, Fer-1 or GSH prevented overproduction of malondialdehyde by the COPZ1 gene knockout, while FAC increased malondialdehyde overproduction in U87MG and U251 cells;

(C) (D) sequentially detecting the results of the U87MG and U251 cell lines by using an iron kit: when DFO is present, the ferrous level decreases;

(E) (F) results of the lactate dehydrogenase release test for U87MG and U251 in this order: DFO reduces glioma cell mortality due to COPZ1 depletion, FAC exacerbates it, and multiple comparative single-factor analysis of variance: NS is not significant in the sense that,

*p<0.05，**p<0.01，**p<0.001。

FIG. 9 is a graph showing the results of in vitro GBM autophagy induced by depletion of COPZ1 in example 6;

(A) (B) GFP/mCherry-LC3B punctate fluorescence images and content histograms (scale bar, 25 μm) in U87MG cells are sequentially obtained;

(C) western blottings showed protein levels of LC3B, SQSTM1(P62), ATG7 and ACTB (load control) in U87MG, U251 and P3-GBM cells following COPZ1 depletion;

(D) western blotting showed protein levels of FTH1, NCOA4 and ACTB in U87MG, U251 and P3# GBM cells following deletion of COPZ 1;

(E) fluorescence images show intracellular localization of NCOA4 and COPZ1, Alexa-NCOA4 is extensively localized to the cytoplasm in normal U87MG and U251 cells and is largely co-localized with FITC-COPZ1 (scale bar, 10 μm, magnified image scale bar, 1 μm);

(F) western blottings showed levels of NCOA4 and LC3B in U87MG-sh-COPZ1 cells after 1h of pretreatment with 3-MA (10mM) and CQ (3. mu.M), the data representing 3 independent experiments;

(G) relative iron levels after 1h of pretreatment with 3-MA (10mM) and CQ (3. mu.M) in U87MG-sh-COPZ1 cells;

(H) cell mortality of U87MG-sh-COPZ1 cells after 1h of pretreatment with 3-MA (10mM) and CQ (3. mu.M);

(I) changes in MDA levels after 1h of pretreatment of U87MG-sh-COPZ1 cells with 3-MA (10mM) and CQ (3. mu.M), two comparative t-tests: p < 0.01; one-way analysis of variance for multiple comparisons: NS is not significant, p <0.01, p < 0.001.

FIG. 10 is a graph of the results of the western and autophagy related studies described in example 6;

(A) (B) NCOA4 and LC3B levels in U87MG-sh-COPZ1#1 and U251-sh-COPZ1#1 cells in sequence;

(C) is the ferrous level in U251 cells;

(D) is the ferrous level in P3# GBM cells;

(E) is U251 cell mortality;

(F) is the cell death rate of P3# GBM;

(G) is Malondialdehyde (MDA) level in U251 cells;

(H) is the Malondialdehyde (MDA) level in P3# GBM cells;

one-way analysis of variance for multiple comparisons: NS is not significant, p <0.05, p <0.01, p < 0.001.

FIG. 11 is a graph of the results of the NCOA4 mediated autophagy transmission of ferritin and control of iron homeostasis described in example 7;

(A) western blotting showed levels of NCOA4 and ACTB in U87MG, U251 and P3# GBM glioma cells infected with si-NCOA4#1 and si-NCOA4# 248 hours;

(B) representative western blotting showed NCOA4, FTH1 and ACTB levels of P3# GBM cells stably expressing sh-COPZ1#1 and two independent si-NCOA4(si-NCOA4#1 and si-NCOA4# 2);

(C) sub-iron levels following NCOA4 gene knockout in U87MG, U251 and P3# GBM cells stably expressing sh-COPZ 1;

(D) typical Western blots of FTH1 and ACTB in U87MG-sh-COPZ1#1, U251-sh-COPZ1#1 and P3# GBM-sh-COPZ1#1 cells transfected with si-NCOA4# 1;

(E) effect of siRNAs knock-out NCOA4 on viability of sh-COPZ1 stably expressing U87MG, U251 and P3-GBM glioma cells;

(F) effect of siRNAs knock-out NCOA4 on MDA levels of U87MG, U251 and P3-GBM glioma cells stably expressing sh-COPZ 1;

(G) effect of si-NCOA4#1 knock-out NCOA4 on U87MG-sh-COPZ1 cell DHE, t-test: p < 0.01; multi-group comparison one-way analysis of variance: p <0.05, p <0.01, p < 0.001.

FIG. 12 is a graph showing the results of iron metabolism-related targets in glioma cells described in example 7;

(A) western blotting showed ATG7 and ACTB levels in U87MG, U251 and P3# GBM glioma cells transfected with si-ATG7#1 and si-ATG7# 248 h;

(B) ferrous levels detected following ATG7 knock-out of U87MG, U251 and P3# GBM cells stably expressing sh-COPZ1# 1;

(C) (D) sequentially knocking out the activity level of ATG7 to U87MG and P3-GBM glioma cells stably expressing sh-COPZ1 by siRNAs;

(E) (F) sequentially, the MDA level of the siRNAs knocking out ATG7 in U87MG and P3-GBM glioma cells stably expressing sh-COPZ1, and two groups of comparative t tests: p < 0.01; one-way analysis of variance for multiple comparisons: NS is not significant, p <0.05, p <0.01, p < 0.001.

FIG. 13 is a graph of the results of the downregulation of COPZ1 in vivo tumor growth in example 8;

(A) imaging intracranial tumor growth of luciferase-expressing U87MG-sh-COPZ1#1 cells or U87MG-NC cells monitored on

days

7, 14 and 21 post-implantation using the IVIS-200 imaging system;

(B) quantification of tumor bioluminescence signals in situ in mice implanted with U87MG-sh-COPZ1#1 cells or U87MG-NC cells on

days

7, 14 and 21;

(C) determining the total survival rate by using a Kaplan-Meier survival curve, and evaluating the statistical significance of the difference by using log-rank test;

(D) representative images of hematoxylin and eosin stained sections (scale bar, 100 μm) after implantation of U87MG-sh-COPZ1#1 cells or U87MG-NC cells in the brains of nude mice;

(E) representative images of immunohistochemical staining of NCOA4 and Ki67 in brain sections of mice injected with U87MG-sh-COPZ1 cells or U87MG-NC cells, (scale bar first and third columns, 100 μm, scale bar second and fourth columns, 25 μm)

(F) Comparison of ferrous levels in mice injected with U87MG-sh-COPZ1 cells or U87MG-NC cells in orthotopic xenograft specimens;

(G) comparison of malondialdehyde levels in mouse xenograft orthotopic specimens injected with U87MG-sh-COPZ1#1 cells or U87MG-NC cells;

(H) scheme for the intracellular effect of GBM on iron metabolism:

knock-out of COPZ1 leads to autophagic degradation of ferritin by activation of the selective receptor NCOA4, resulting in intracellular iron, particularly ferrous (Fe)²⁺) Excess of, unstable ferrous ironFenton reaction is triggered, cytotoxic lipid peroxidation is accumulated, and the main characteristic of iron sag is; and (4) t test: p<0.05，**p<0.01，**p<0.001; log rank test: p is a radical of<0.05。

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, 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 belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

As described in the background, aiming at the defects in the prior art, in order to solve the technical problems, the invention provides the application of COPZ1 as a target for treatment/prognosis judgment of glioma.

Preferably, the application of the therapeutic target comprises the application of the COPZ1 inhibitor as an anti-glioma active ingredient or the application of the COPZ1 inhibitor in the preparation of anti-glioma drugs.

Preferably, the application of the prognosis judgment target comprises the application of a reagent for detecting the content of COPZ1 in a brain glioma prognosis judgment reagent or the application of a preparation of a prognosis judgment kit.

Preferably, the use comprises the use of an inhibitor of COPZ1 as an iron ion (including in particular ferrous and ferric ions), Transferrin (TF) bound to iron, a transferrin receptor (TFR) agonist to transfer the transferrin iron complex into cells.

Further preferred is the use of said COPZ1 inhibitor as a ferrous ion agonist.

Preferably, the use of a COPZ1 inhibitor as a ferrosoferrin modulating (FTH1) agonist is described.

Preferably, the use comprises the use of a COPZ1 inhibitor as an agonist of hydrogen peroxide and superoxide, including superoxide radicals (O)₂ ^-) And highly cytotoxic hydroxyl radicals (. OH).

Preferably, the use comprises the use of a COPZ1 inhibitor as an agonist of LC3B-II, or an inhibitor of the autophagy substrate SQSTM1(P62), or an agonist of autophagy-related protein 7(ATG 7).

In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific examples and comparative examples. The materials and methods used in the following examples are illustrated below:

statement of ethics

All experiments with human samples were approved by the university of Shandong (Shandong) research ethics Committee. Human glioma tumor specimens (WHO ii-iv grade, n 60) were obtained from the neurosurgery of qilu hospital. Normal brain tissue samples (n ═ 6) were taken from patients with brain trauma who received reduced pressure treatment of severe craniocerebral injury in the same hospital. All patients received written informed consent. All animal procedures were approved by the university of Shandong animal protection and use Committee (IACUC).

Cell lines and cultures

Human glioma cell lines U87MG, U251, A172, LN229, and T98 were all purchased from the Chinese academy of sciences cell Bank (Shanghai). Professor ralf Bjerkvig, department of biologies, university of alpine, norway, provided tumor cells P3# GBM proliferated by biopsy of Normal Human Astrocytes (NHA) and primary human GBM. U87MG, U251, A172, LN229 and T98 cells were cultured in Dulbecco's modified Medium (DMEM; Thermo Fisher Scientific; Waltham, MA, USA), supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific) and cultured in a humidified incubator at 37 ℃ in 5% carbon dioxide. P3-GBM cells were cultured in Neurobase TM medium (Gibco, Thermo Fisher Scientific), supplemented with 2% B27 neural mixture (Thermo Fisher Scientific), 20ng/mL epidermal growth factor (EGF; Thermo Fisher Scientific), and 10ng/mL basic fibroblast growth factor (bFGF; PeproTech; Rocky Hill, N.J.) in a humidified incubator with 5% carbon dioxide at 37 ℃. Accutase (thermo Fisher scientific) was used to digest tumors for expansion of GSC spheres.

Transfection of SiRNA

SiRNA transfection genes (specific SiRNA and negative control SiRNA) were synthesized by the Genepharmacy company (Shanghai, China) and U87MG, U251 and P3# GBM cells were transfected with liposomes 2000(Thermo Fisher Scientific) for 48h according to the manufacturer's protocol 3 × 10⁵The density of cells/well was seeded in 6-well plates. When the cell fusion rate is 70-80%, siRNA double strand is transfected into the cell.

The following siRNA sequences were used to target a as indicated:

COPZ1#1and COPZ1#2：5’-CCAUCGGACUGACAGUGAAATT-3’and5’-CCGGCCTGTATACTGTCAAAGCCAT-3’；

NCOA4#1 and NCOA4# 2: 5'-ACTCTTGTTTATCGAAGTATA-3' and

5’-CTCTTATTCCAGTCCTATAAT-3’；

ATG7#1 and ATG7# 2: 5'-GGAGTCACAGCTCTTCCTT-3' and

5’-CAGCTATTGGAACACTGTA-3’。

western blotting was used to evaluate siRNA knockdown efficiency. Short hairpin transfection with ShRNA: sh-COPZ1#1 and sh-COPZ1#2 (5'-ccauccggacugagugaaatt-3' and 5'-ccggcctgtatactcaagcat-3') were ligated to pLKO.1 lentiviral vectors having a puromycin resistance region (genefia; Shanghai, China). Luciferase-stable U87MG, U251 and P3# GBM cells were infected, respectively. After 48h, U87MG, U251 and P3# GBM cells were exposed to puromycin (2. mu.g/mL; Thermo Fisher Scientific) for 2 weeks to enrich the cells containing the structure. The knockout efficiency was verified by Western blotting and the cells were analyzed differently. And detecting shRNA gene knockout efficiency by Western blotting.

Immunohistochemistry

All specimens were fixed in 4% paraformaldehyde Phosphate Buffered Saline (PBS), embedded in paraffin, and cut into 4 μm sections. The sections were dewaxed and rehydrated, and then incubated with 0.01M citrate buffer at 95 ℃ for 20 minutes to obtain the antigen. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide (ZSGB-Bio; Beijing, China), nonspecific antigen with 10% normal goat serum (ZSGB-Bio), followed by incubation with the original antibody at 4 ℃ for 12h, rabbit anti-COPZ 1(20440-1-AP, 1: 100; Proteintech, Wuhan, China) and rabbit anti-Ki 67(ab15580,1: 500; Abcam, Cambridge, UK). Sections were washed with PBS3X5min, treated with goat anti-rabbit secondary antibody (ZSGB-Bio), then developed with 3, 3' -diaminobenzidine (DAB, ZSGB-Bio), slides counterstained with hematoxylin (Beyotime; Shanghai, China) at 25 ℃ for 2min, negative control, and sections incubated with normal goat serum instead of the original antibody. Cancer cell staining is divided into: 0, no dyeing; 1, < 50% weak staining of cells; 2, -weak staining of 50% cells; 3, < 50% strong staining of cells; 4, -50% of the cells were strongly stained. Staining was assessed independently by two experienced neurologists.

Cell viability and proliferation assays

Analysis using cell counting kit-8 (CCK-8) (D) according to the manufacturer's protocolojindo; kumamoto, Japan) to assess cell viability. In 96-well plates at 2-10³Cells were seeded at a rate of one cell/well and incubated in a humidified incubator containing 5% carbon dioxide at 37 ℃ for 24, 48 and 72 hours. CCK-8 solution (10. mu.L) was added to each well and the dishes were incubated at 37 ℃ for 1 h. The absorbance of light per well was read at 450 nm (OD450) with a microplate reader (Bio-Rad; Hercules, Calif., USA). Proliferation was assessed using the EdU test according to the manufacturer's protocol (Rib bio; Guangzhou, China). Briefly, eDU bound to proliferating cells and was detected by a catalyzed reaction with fluorescently labeled azide. The labeled cells were observed under a fluorescent microscope and the number of EdU positive cells was counted from 500 cells in three independent experiments.

Western blotting analysis

Cells and tissues were collected, lysed with RIPA lysis buffer (Thermo Fisher Scientific) and protease inhibitor PMSF (Solarbio, Beijing, China) at a ratio of 100:1 (v/v.) and protein concentration was determined with BCA protein assay kit (Beyotime). equal amounts of protein extract were separated by 10% SDSPAGE and transferred to PVDF membrane (Merck Millipore; Billerica, MA, USA). the membrane was blocked with skim milk for 1h and then incubated overnight at 4 ℃ with primary antibodies rabbit anti- β -actin (ab8226, 1:2000, Abcam), rabbit anti-COPZ 1(20440-1-AP, 1:1000, Proteintetech), rabbit anti-NCOA 4 (86707, 1:2000, Abcam), rabbit anti-ferritin (75972, 1:1000, Abcam), rabbit anti-SQ 1923 (SQ 19232, SQ) for cell lysis using a Bioluminescence assay protocol (Bioluminescence) and staining protocol for detecting the secondary antibody using a Bioluminescence assay protocol of Bioluminescence (Beckia) for cell lysis in Bioluminescence buffer (Biotech; Szem) for detecting the antibody binding to Bioluminescence medium for cell binding to the Bioluminescence assay kit (SQ 3932, Biotech) for detecting the following Biotech (Biotech).

Lactate dehydrogenase releasing cell death assay

Cell mortality was measured using the lactate dehydrogenase cytotoxicity assay kit (beyosine) according to the manufacturer's instructions. After the treatment is finished, theThe cell culture plate was centrifuged at 400g for 5min to remove the supernatant as much as possible, and 150. mu.l of a lactate dehydrogenase-releasing reagent provided in the kit was added, diluted 10-fold with PBS, shaken up and then cultured in a cell culture chamber for 1 hour. The absorbance at 490nm (OD490) was measured in a microplate reader (Bio-Rad) and all experiments were performed in triplicate. The calculation results are as follows: cell death rate = (processed sample absorbance-sample control well absorbance)/(maximum enzyme activity absorbance of cell-sample control well absorbance)

Determination of iron content

Ferrous iron concentration was analyzed using an iron colorimetric assay kit (Abcam; California, USA) according to the manufacturer's instructions. Briefly, cells were added to iron assay buffer on ice and centrifuged at 16000g for 10min at 4 ℃ to obtain a supernatant. A50 microliter sample was incubated with 50 microliter assay buffer in a 96 well microplate at 25 ℃ for 30 minutes, then with 100 microliter iron probe at 25 ℃ for 60 minutes and protected from light. The absorbance at 593nm was measured with a microplate reader (Bio-Rad). Experiments were performed in triplicate. Iron concentration (Sa/Sv) d.sa: iron content in the sample well calculated from the standard curve (nmol), Sv: sample volume added to the reaction well (L), D: sample dilution factor. The iron (III) content of the sample can be calculated as: iron (III) ═ total iron (II III) -iron (II).

Lipid peroxidation assessment

Lipid peroxidation level was detected using Malondialdehyde (MDA) detection kit (beyotine). According to the manufacturer's protocol, the collected cells are added to a lysis buffer homogenized on ice, centrifuged at 1600g for 10min at 4 ℃, the supernatant is collected after centrifugation, 100. mu.l of the supernatant is incubated with 100. mu.l of the test solution for 15min at 100 ℃, and then cooled to room temperature. The mixture was centrifuged at 1000g for 10min to obtain a supernatant, and the absorbance was read at 530nm with a microplate reader (Bio-Rad). Malondialdehyde (MDA) content is directly proportional to the absorbance value of control cells.

Superoxide anion detection

The superoxide anion content of U87MG, U251 and P3 cells was measured with dihydroethylamine (DHE, Beyotime) and after 24 h incubation with a. mu. -Slide 8Well (Ibidi; Martinsried, Germany), the cells were washed three times in PBS and then loaded with DHE (10mmol/L) for 30 min at 37 ℃ in fresh medium. Fluorescence was measured using a Leica SP8 confocal microscope (Leica microsystems; Wetzlar, Germany) at an excitation wavelength of 485nm and an emission wavelength of 530 nm. The amount of superoxide anion is proportional to the absorbance of control cells. The fluorescence intensity was calculated with ImageJ software.

Mitochondrial membrane potential determination

Mitochondrial membrane potential was detected with JC-1 kit (Beyotime). Cells were analyzed by confocal microscopy (Leica SP8) after staining with JC-1 according to the manufacturer's protocol. The excitation wavelength of JC-1 is 488nm, and the emission wavelengths of the monomers and J-aggregates are 529 and 590nm, respectively. The intensities of red fluorescence (excitation light 530nm, emission light 590nm) and green fluorescence (excitation light 485nm, emission light 528nm) were measured. The test results were repeated three times. Fluorescence intensity was calculated with ImageJ software.

Live/dead staining live/dead cell staining

The use of live/dead cell double staining kit (Sigma Aldrich, missouri, usa) was performed according to the manufacturer's protocol. Briefly, calcein AM and disodium Propyliodide (PI) working solutions were prepared at appropriate dilutions in PBS. The staining solution and cells were mixed in a ratio of 1:2(v/v) to a working solution, incubated at 37 ℃ for 15 minutes, and live/dead cell images were taken with a Leica SP8 confocal microscope.

Animal experiments

Intracranial xenograft studies were performed in 4-week-old female nude mice (n-20; Shanghai SLAC laboratory animals Co., Ltd.) implanted with U87MG-NC and U87MG-sh-COPZ1#1 glioma cells. Mice were divided into two groups (10 per group) and injected intraperitoneally with 80 microliters ketamine hydrochloride (25mg/ml), xylene (2.5mg/ml) and 14.25% ethanol (diluted 1:3 in 0.9% NaCl). U87MG-NC and U87MG-sh-COPZ1#1 glioma cells (106 cells/cell, 10. mu.l/cell) were implanted into the right frontal lobe of mice by intracranial injection (1 mm anterior to bregma, 2.5mm lateral, 2mm deep). Animals exhibiting symptoms (e.g., severe hunchback, apathy, reduced exercise or activity, dragging legs, bloating hair, or rapid weight loss) are sacrificed by cervical dislocation. The mice were then perfused with saline and 4% Paraformaldehyde (PFA). Brain tissue was taken and further fixed in 4% PFA before paraffin embedding. The tumor tissue was further examined.

Statistical analysis

For determining the relationship between COPZ1 expression and clinical pathology factors. The log-rank test was used to compare the Kaplan-Meier survival curves of the two groups to assess the difference in survival between the two groups. Statistical analysis was performed using GraphPad Prism version 7.00 software (GraphPad; La Jolla, Calif., USA). All experiments were repeated at least three times, three times each, unless otherwise indicated. Data for each treatment group are presented by mean scanning electron microscopy. All tests were two-sided, and P values <0.05 were considered statistically significant.

Example 1 correlation of COPZ1 with glioma and poor prognosis

COPZ1 is highly expressed in human brain gliomas and predicts poor prognosis. This example first examined the level of COPZ1 in human brain glioma samples by analyzing published data for a cancer genomic map (TCGA). The results show elevated COPZ1 mRNA levels for low grade gliomas (WHO ii; n 226) and high grade gliomas (WHO iii, n 244; WHO iv, n 150; p <0.0001) compared to normal brain tissue (n ═ 4) (fig. 1A). In addition, high CPOZ1 expression predicted a shorter overall survival based on Kaplan-Meier analysis of TCGA datasets (fig. 1B). The published luneberg dataset was analyzed in the same way (fig. 2). Next, this example used the Western blotting method to detect the levels of COPZ1 protein in primary human brain glioma and normal brain tissue. There was a clear correlation between elevated levels of COPZ1 protein and malignancy (fig. 1c.d), and to determine whether expression of COPZ1 was associated with aggressive clinical pathology, 60 paraffin-embedded clinical specimens were subjected to Immunohistochemical (IHC) staining, including grade ii (n-18), grade three (n-18), grade four (n-18), and normal brain tissue (n-6) (fig. 1E). Quantitative analysis of the COPZ1-IHC staining score showed that COPZ1 levels increased with disease stratification (fig. 1F). Next, this example was analyzed by Western blotting to determine the level of COPZ1 protein in Normal Human Astrocytes (NHA), the glioma cell line U87MG, U251, a172, LN229, T98 and glioma primary cells (P3# GBM, fig. 1G). All tumor cell lines showed elevated levels of COPZ1 protein compared to NHA (fig. 1H). To determine the intracellular localization of COPZ1, an Immunofluorescence (IF) assay was performed on COPZ1 in U87MG and U251 glioma cell lines. COPZ1 was mainly localized within the cytoplasm (fig. 1I). Taken together, COPZ1 may play an important role in glioma progression and may serve as a novel diagnostic marker.

Example 2 silencing COPZ1 inhibits glioma cell proliferation and induces cell death

As the expression of COPZ1 protein was elevated in glioma clinical specimens, GBM cell lines and glioma primary cells, this example examined its biological effects by inhibiting the expression of COPZ1 in U87MG, U251 and P3-GMB cells. Knockdown in U87MG, U251 and P3# GMB cells using two small interfering RNAs (si-COPZ1#1 and si-COPZ1#2) decreased by about 80% and about 60% at mRNA level (FIG. 3A) and protein level (FIG. 3B), respectively. Knock-out experiments were performed with two different shRNAs and showed effective knock-out (fig. 4). As shown by CCK8 analysis, si-COPZ1#1 and si-COPZ1#2 gene knockouts also inhibited proliferation of U87MG, U251 and P3# GBM cells (fig. 3C). EdU assays were performed in U87MG and U251 cells using si-COPZ1#1 (fig. 3D, E), and in colony formation assays in U251 and P3# GBM cells using si-COPZ1#1 (fig. 3F, G). We also observed an increase in the mortality of U87MG, U251 and P3# GBM cells when COPZ1 was down-regulated by using the LDH cytotoxicity test kit (fig. 3H). Viable/dead cell viability assays showed an increase in the number of dead U251 and P3# GBM cells compared to the number of viable cells following a si-COPZ1#1 knock-out (fig. 3I, J). Taken together, these data indicate that the COPZ1 gene knockout inhibits cell proliferation and induces cell death in GBM cell lines.

Example 3CoPZ1 Gene knockdown increases intracellular iron levels

Since COPZ1 is involved in iron metabolism, this example next investigated whether COPZ1 gene knock-down induces GBM cellsIron dies. First, si-COPZ1#1 gene was used to knock down U87MG, U251 and P3# GBM cells, and compared to NC cells. After inhibition of COPZ1, intracellular iron content increased by about 70% (fig. 5A). Notably, the ferrous level (Fe)²⁺) Increased over the ferric (Fe) level³⁺Fig. 5B). This indicates that intracellular ferrous iron content increases when COPZ1 is knocked down. Elevated intracellular iron levels lead to iron death, a characteristic of which is altered mitochondrial morphology. Thus, this example uses the JC-1 probe to detect changes in mitochondrial membrane potential. JC-1 exists in the cytoplasm in a monomeric form, aggregates in normal mitochondria and emits red fluorescence, and emits green fluorescence when the mitochondrial membrane depolarizes. Observations using confocal microscopy showed that downregulation of COPZ1 decreased red fluorescence and increased green fluorescence, indicating a decrease in mitochondrial membrane potential (fig. 5C, D). This was also confirmed by transmission electron microscopy, with U87MG glioma cells having reduced mitochondria and increased membrane density following COPZ1 gene knock-out compared to control cells (fig. 5E). Next, Western blotting was performed in this example to reveal the effect of the COPZ1 gene knock-out in increasing intracellular iron. Upon downregulation of COPZ1, Transferrin (TF), which binds iron, and transferrin receptor (TFR), which transfers the transferrin iron complex into cells, are both upregulated. In addition, ferritin (FTH1), which regulates ferrous iron, was down-regulated in cells (fig. 5F). Fluorescence imaging detected intracellular TFR, indicating that TFR was elevated when COPZ1 was inhibited (fig. 6). Taken together, the results of the study indicate that knockdown COPZ1 induces iron accumulation in cells by promoting intracellular iron transport.

Example 4 upregulation of intracellular iron glioma cell death by lipid peroxidation induced knockdown of the COPZ1 gene

Iron death is characterized by lipid peroxidation, the final product of which is Malondialdehyde (MDA). Thus, this example examined COPZ 1-induced changes in malondialdehyde levels. MDA levels in U87MG, U251 and P3# GBM knockdown glioma cells were significantly increased after 48h incubation of si-COPZ1#1 compared to control cells (fig. 7A). Knock-down COPZ 1-induced malondialdehyde production was inhibited by the iron chelator Desferrioxamine (DFO). However, when Ferric Ammonium Citrate (FAC) was added, malondialdehyde levels increased (fig. 7B, fig. 8A, B). In addition, DFO-pretreated GBM cells showed a decrease in ferrous content (fig. 7C, fig. 8C, D). In addition, P3# GBM cells pretreated with GSH for 1h inhibited MDA levels (fig. 7B). This suggests that intracellular iron upregulated by knock-out of COPZ1 can induce lipid peroxidation.

Ferrostatin-1 (Ferrostatin-1) is a specific lipid peroxidation scavenger that inhibits the levels of Malondialdehyde (MDA) (fig. 7B). Following pretreatment of P3# GBM cells with Fer-1, cell mortality decreased using LDH release assay (FIG. 7D). When COPZ1 expression was down-regulated, the presence of FAC increased cell death, however, pretreatment with DFO reduced cell death (fig. 7D, fig. 8E, F). Taken together, these results suggest that lipid peroxidation may be involved in si-COPZ 1-induced glioma cell death.

Example 5 elevated intracellular iron levels increase reactive oxygen species production

Reactive Oxygen Species (ROS) such as hydrogen peroxide (H)₂O₂) Superoxide radical (O)₂ ^-) And highly cytotoxic hydroxyl radicals (. OH) are the main cause of intracellular oxidative stress. Iron is increased by using hydrogen peroxide and ferrous iron as substrates, releasing iron, superoxide radicals and hydroxyl radicals, triggering the intracellular Fenton reaction. Lipid peroxidation may in turn be generated by hydroxyl radicals. This example investigated the potential difference in hydrogen peroxide levels in si-COPZ1 cells and NC cells using a catalase detection kit. As shown in fig. 7G, hydrogen peroxide accumulated in COPZ1 knockout cells compared to control cells. As mentioned previously, hydrogen peroxide is produced from superoxide, and therefore this example studies the production of superoxide. The red fluorescence monitored by the superoxide probe dihydroethyl ether (DHE) was significantly stronger in COPZ1 knock-out cells compared to NC cells (fig. 7E, F). In summary, an increase in iron content increases the production of hydrogen peroxide and superoxide within the cell.

Example 6 deletion of COPZ1 induces autophagy in GBM cells

Autophagy is a conserved biodegradation pathway that maintains cellular homeostasis. However, excessive autophagy promotes cell death, rather than cell survival. Deletion of COPZ1 can induce lethal autophagy, leading to tumor cell death. To determine whether COPZ1 depletion induces GBM autophagy, confocal microscopy studies were performed to detect autophagic vesicles. U87MG cells were transfected with GFP/mCherry-LC3 lentivirus for 48 hours, followed by 124 hour knock-down of the COPZ. As shown in FIG. 9A, the GFP-LC3 fluorescence spot was elevated in si-COPZ1 cells and the mCherry-LC3 fluorescence spot was also elevated compared to the NC group. The green fluorescence intensity of si-COPZ1#1 cells was about 2 times that of NC cells, and the red fluorescence intensity was about 1.6 times that of NC cells (FIG. 9B). These data are consistent with TEM results of RNAi-mediated COPZ1 gene knock-out of U87MG cells, showing increased numbers of autophagosomes (fig. 9E). Western blotting analysis of cell lysates of the absence and presence of the COPZ1 gene knockout also indicated that the COPZ1 gene knockout induced autophagosome formation. When COPZ1 was depleted, U87MG, U251 and P3# GBM glioma cell autophagy marker LC3B-II increased (fig. 9C). Protein levels of the autophagy substrate SQSTM1(P62) were down-regulated when COPZ1 was depleted (fig. 9C). Autophagy-related protein 7(ATG7) plays a central role in the regulation of autophagy, being up-regulated in the si-COPZ1#1 group (fig. 9C), these results support the promotion of autophagy in COPZ 1-deficient GBM cells.

NCOA4 plays a central role in COPZ1 deacetylation-mediated autophagy

Ferritin is the major intracellular iron storage protein complex and is composed of FTL1 (ferritin light polypeptide 1) and FTH1 (ferritin heavy polypeptide 1). It has been shown that increased autophagy promotes ferritin degradation, increases intracellular iron content, leads to a Fenton reaction, and subsequent iron death. In line with this, FTH1 levels were reduced in COPZ1 depleted U87MG, U251 and P3# GBM cells (fig. 9D). Considering that NCOA4 is a selective receptor for autophagic degradation of ferritin, this example first analyzed whether expression of NCOA4 was associated with iron death induced by COPZ1 gene knockout. NCOA4 protein levels increased after COPZ1 depletion (fig. 9D).

Next, this example examined the intracellular localization of NCOA 4. Alexa-NCOA4 was extensively localized in the cytoplasm of normal U87MG and U251 cells, aggregated in cytoplasmic punctate structures, and co-localized with FITC-COPZ1 punctate structures (fig. 9E). To further demonstrate that depletion of COPZ1 increased autophagy flux, U87MG-sh-COPZ1#1 cells were treated with 3-MA or Chloroquine (CQ). The western blotting result shows that 3-MA has an inhibiting effect on the conversion of LC3B-I to LC 3B-II. However, treatment with 3. mu.M CQ for 48h inhibited autophagy by reducing the autophagosome-lysosomal fusion process, resulting in accumulation of LC3B-II (FIG. 9F). NCOA4 levels were significantly elevated when autophagosome degradation was blocked by 3-MA and CQ (fig. 9F, fig. 10A, B). When 3-MA or CQ inhibited the ferritin digestion process, ferrous levels decreased (fig. 9G, fig. 10C, D), cell mortality decreased (fig. 9H, fig. 10E.F) and malondialdehyde levels were also inhibited (fig. 9I, fig. 10G, H).

Taken together, COPZ1 appears to interact with NCOA4 and modulate its function, whereas knock-out of COPZ1 induces NCOA 4-mediated ferritin degradation, suggesting that NCOA 4-mediated ferritin degradation plays a key role in COPZ 1-mediated iron death. Example 7NCOA 4-mediated ferritin degradation leads to COPZ 1-deficient induced iron death

Since the COPZ1 gene knockout increased the level of NCOA4 leading to ferritin degradation, this example next investigated the effect of NCOA4 gene knockout on iron death. NCOA4 gene knockouts were performed on U87MG, U251 and P3# GBM cells with two independent siRNAs. Western blotting experiments showed significant reduction in NCOA4 protein levels (FIG. 11A). Deletion of NCOA4 increased the basal ferritin level (FTH1) in P3# GBM cells stably transfected with sh-COPZ1#1 (fig. 11B). This indicates that NCOA4 deficiency counteracts the upregulation of iron caused by COPZ1 deficiency, resulting in reduced bioavailability of iron. Thus, this example detected ferrous iron (Fe) in U87MG, U251 and P3# GBM cells transfected with sh-COPZ1#1²⁺) The level of (c). NCOA4 down-regulated the decrease in ferrous levels in cells when stably transfected sh-COPZ1#1 (fig. 11C). Furthermore, FTH1 levels were reduced by iron loading (FAC) or chelation (DFO, fig. 11D), indicating that iron metabolism can be regulated by the NCOA4-FTH1 pathway when COPZ1 is knocked out. When NCOA4 was knocked out, cell mortality and malondialdehyde levels in COPZ1 deficient GBM cells were reduced (fig. 11E, F), and DHE levels were reduced in U87MG-sh-COPZ1#1 cells (fig. 11G). Since ATG7 is involved in autophagic degradation of ferritin, an ATG7 depletion assay was performed. Two independent siRNAs were used to suppress the expression level of ATG7 protein. With NC cell phaseIn contrast, these two sirnas significantly down-regulated the protein levels of ATG7 (fig. 12A), ferrous iron levels (fig. 12B), cell mortality (fig. 12C, D), and malondialdehyde levels (fig. 12E, F) in U87MG, U251, and P3# GBM cells. This resulted in a reduction in the levels of the above substances in U87MG and P3# GBM cells stably expressing sh-COPZ1#1 compared to the corresponding NC cells. These data indicate that COPZ1 deletion can induce iron death in glioma cells by increasing levels of NCOA4 and ATG7, and therefore the COPZ1/NCOA4/FTH1 axis may be a new target for glioma therapy.

Example 8CoPZ1 Down-Regulation of growth of GBM cells in vivo

To determine the potential effect of COPZ1 on cell growth in vivo, luciferase-tagged U87MG-sh-COPZ1#1 cells or U87MG-NC cells were implanted into the brains of nude mice (10 per group). Tumor growth was monitored using bioluminescence values. The results showed that the bioluminescence signal was significantly weaker in the COPZ1 knock-out group compared to the control group (fig. 13A). The mean total flux of the 14d, U87MG-sh-COPZ1#1 tumors after glioma cell injection was significantly different from that of the normal control group. By day 21, the total flow rate was about 60% lower in group U87MG-sh-COPZ1#1 than in the NC group (P < 0.001; FIG. 13B). Kaplan-Meier analysis of survival data showed an increase in overall survival from 20.8 days (control) to 27.8 days (knock-out, P < 0.05; FIG. 13C). Histological examination showed that the U87MG-sh-COPZ1#1 tumor was smaller than the NC tumor (FIG. 13D). Immunohistochemistry of two groups of animal sections showed that the downregulation of COPZ1 expression inhibited Ki67 expression, Ki67 being a marker of proliferation, while NCOA4 expression was increased (fig. 13E). Furthermore, in U87MG-sh-COPZ1#1 tumors, levels of both ferrous and malondialdehyde were elevated (FIGS. 13F, 13G). Taken together, these findings indicate that COPZ1 deletion inhibits GBM tumor growth and leads to tumor cell death through iron death.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Qilu Hospital of Shandong university

Application of <120> COPZ1 as brain glioma treatment/prognosis judgment target

<130>2010

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<170>PatentIn version 3.3

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Claims

The application of COPZ1 as a target for treatment/prognosis judgment of brain glioma.
2. The use of COPZ1 as a target for the treatment/prognosis of brain glioma according to claim 1, wherein the use of the target for treatment comprises the use of a COPZ1 inhibitor as an active ingredient against brain glioma, or the use of a COPZ1 inhibitor for the preparation of a medicament against brain glioma; or the application of the prognosis judgment target comprises the application of applying the detection reagent of the content of COPZ1 to the brain glioma prognosis judgment reagent or the application of preparing a prognosis judgment kit.
Use of COPZ1 as a modulator of target cell iron.
4. The use of COPZ1 as a regulator of target cellular iron according to claim 3, wherein the iron comprises target intracellular ferric ions, transferrin which binds to iron, transferrin receptor which transfers transferrin iron complexes to cells, ferritin which regulates ferrous iron.
5. The use of COPZ1 as a modulator of target cell iron according to claim 3, which comprises the use of a COPZ1 inhibitor as the iron ion, transferrin which binds iron, a transferrin receptor agonist which transfers transferrin iron complexes to the inside of cells; preferably, the use of said COPZ1 inhibitor as a ferrous ion agonist.
6. The use of COPZ1 as a modulator of target cell iron according to claim 3, wherein the use of a COPZ1 inhibitor is as a ferroporphyrin agonist for the modulation of ferrous iron.
Use of an inhibitor of COPZ1 as an inducer of lipid peroxidation or as an agonist of malondialdehyde.
Use of an inhibitor of COPZ1 as an agonist of reactive oxygen species; preferably, the use comprises the use of a COPZ1 inhibitor as an agonist of hydrogen peroxide and superoxide, including superoxide radicals and highly cytotoxic hydroxyl radicals.
Use of a COPZ1 inhibitor as an autophagy inducer; preferably, the use comprises the use of a COPZ1 inhibitor as an agonist of LC3B-II, or an inhibitor of the autophagy substrate SQSTM1, or an agonist of autophagy-related protein 7.
Use of a COPZ1 inhibitor as an agonist of NCOA 4.