CN117248020A - Application of HMGCL as glioma diagnosis/prognosis marker and treatment target - Google Patents
Application of HMGCL as glioma diagnosis/prognosis marker and treatment target Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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Abstract
The invention belongs to the technical fields of disease diagnosis and treatment and molecular biology, and particularly relates to application of HMGCL as a glioma diagnosis/prognosis marker and a treatment target. The invention demonstrates for the first time that HMGCL expression increases with increasing glioma malignancy and is inversely related to survival. Meanwhile, the HMGCL lentivirus can obviously reduce the expression level of the HMGCL of glioma, and the knock-down of the HMGCL inhibits the proliferation and colony formation of glioma cells, so that the HMGCL lentivirus can be used as an effective target spot for preventing and/or treating glioma. Furthermore, the present invention demonstrates that specific knockdown of HMGCL can lead to a significant down-regulation of histone acetylation modification levels in GBM cells. In a word, the HMGCL and the molecular mechanism thereof provided by the invention are beneficial to understanding the pathogenesis of glioma in depth, and are also beneficial to providing potential biomarkers and therapeutic targets for clinical diagnosis, prognosis evaluation, prevention and treatment of glioma, so that the HMGCL and the molecular mechanism thereof have good practical application value.
Description
Technical Field
The invention belongs to the technical fields of disease diagnosis and treatment and molecular biology, and particularly relates to application of HMGCL as a glioma diagnosis/prognosis marker and a treatment target.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Gliomas are the most common primary malignancy of the central nervous system (Central Nerve System, CNS), with 50% of patients appearing as the most aggressive type, gliobastoma (GBM). Despite recent advances in chemotherapy and surgery, the Overall Survival (OS) of GBM has not been significantly increased over the last decades, with median Survival still generally lower. Molecular etiology studies against gliomas have elucidated various genetic alterations involving cell survival and DNA repair pathways, however targeted therapies against these pathways are not very effective. Recent studies revealed 14 features of tumors, and the disclosure of these tumor features summarizes the course of tumor studies and also represents a new direction for tumor treatment strategies. Wherein, new targets are developed based on metabolism reprogramming of tumors, new targeted drugs are designed, and a new idea is provided for drug treatment of tumors.
Compared to normal cells, gliomas have abnormal lipid metabolism, and expression of lipid-related genes such as SREBP1 and FAS is changed, resulting in a change in lipid composition and lipogenesis to keep up with energy demand. GBM tumors also accumulate more fatty acids than surrounding normal brain tissue. These lipid stores can be used as energy stores, can provide support for GBM cell proliferation, and can also affect pathways associated with malignant progression of tumors. Targeting glioma lipid metabolism-related molecules facilitates glioma treatment. The applicant has screened the lipid metabolism gene specifically expressed in glioma through database letter analysis in the early stage, wherein HMGCL is taken as a candidate gene, and the expression difference of glioma in different levels is larger. Furthermore, few studies of HMGCL have been reported, and no report has yet been made in gliomas.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention provides application of HMGCL as a glioma diagnosis/prognosis marker and a treatment target. The invention proves that the HMGCL expression increases along with the malignant degree of glioma for the first time and is inversely related to the survival rate of glioma patients, thereby showing that the HMGCL can be used for diagnosis and prognosis evaluation of glioma, and simultaneously, the malignant growth and invasion capacity of glioma cells can be controlled by regulating and controlling the HMGCL expression, thereby showing that the HMGCL can be used as a glioma treatment target. Based on the above results, the present invention has been completed.
Specifically, the invention relates to the following technical scheme:
in a first aspect of the invention, the use of a reagent for detecting HMGCL encoding gene and its expression products for the preparation of a product for glioma diagnosis and/or prognosis is provided.
The product is capable of diagnosing, detecting, monitoring or predicting (early or assisted) the progression of glioma by detecting the expression level of the HMGCL encoding gene and/or the HMGCL encoding gene expression product; experiments prove that the HMGCL has correlation with glioma grade, IDH mutation condition and the like, the expression quantity of the HMGCL is increased along with the increase of glioma grade (malignancy degree), and the high expression can indicate poor prognosis; meanwhile, the expression level of HMGCL in glioma cell lines and glioma stem cell lines is higher than that of normal astrocytes. Thus, the HMGCL encoding gene and its expression products may be used as glioma diagnostic and/or prognostic biomarkers.
Wherein, the HMGCL encoding gene and the expression product thereof can be human; the expression product of the HMGCL encoding gene may obviously be the HMGCL protein, i.e.3-hydroxy-3-methylglutaryl CoA lyase.
In a second aspect of the present invention, there is provided a system for glioma diagnosis and/or prognosis evaluation, the system comprising at least:
an acquisition module configured to: obtaining the expression level of a biomarker in a subject;
an evaluation module configured to: assessing the disease condition of the subject based on the expression level of the biomarker obtained by the obtaining module;
wherein the biomarker is HMGCL encoding gene and/or HMGCL encoding gene expression product (such as hydroxymethyl glutaryl coenzyme A lyase).
In a third aspect of the invention, there is provided a computer readable storage medium having stored thereon a program which when executed by a processor performs the functions of the system according to the second aspect of the invention.
In a fourth aspect the invention provides an electronic device comprising a memory, a processor and a program stored on the memory and executable on the processor, the processor implementing the functions of the system according to the second aspect of the invention when executing the program.
In a fifth aspect of the invention, there is provided the use of HMGCL as a target in glioma control and/or screening of glioma-related drugs.
In a sixth aspect of the present invention, there is provided the use of a substance which inhibits the expression and/or reduces the activity of an HMGCL encoding gene and its expression product in the preparation of a product;
the function of the product is any one or more of the following:
(a1) Inhibit proliferation of glioma cells;
(a2) Inhibiting invasive migration of glioma cells;
(a3) Promoting glioma cell cycle arrest;
(a4) Inhibiting glioma cell colony formation;
(a5) Inhibiting the acetylation modification expression of glioma cell histone;
(a6) Inhibiting the expression and transcription regulation functions of glioma cell cycle related molecules FoxM 1;
(a7) Preventing and/or treating glioma.
The product can be a medicine or an experimental reagent, and the experimental reagent can be used for basic research, so that a guarantee is provided for basic research related to glioma.
The beneficial technical effects of one or more of the technical schemes are as follows:
the technical scheme proves that the HMGCL expression increases with the increase of the malignant degree of glioma for the first time and is inversely related to the survival rate. Meanwhile, the HMGCL lentivirus can obviously reduce the expression level of the HMGCL of glioma, and the knock-down of the HMGCL inhibits the proliferation and colony formation of glioma cells, so that the HMGCL lentivirus can be used as an effective target spot for preventing and/or treating glioma.
Furthermore, the technical scheme proves that the specific knock-down of HMGCL can lead to the obvious down regulation of histone acetylation modification level in GBM cells, which suggests the possibility of regulating epigenetic modification through metabolic means and promotes the related research of metabolic joint epigenetic science.
In conclusion, the HMGCL and the molecular mechanism thereof discovered in the technical scheme are helpful for understanding the pathogenesis of glioma in depth, and are also helpful for providing potential biomarkers and therapeutic targets for clinical diagnosis, prognosis evaluation, prevention and treatment of glioma, so that the HMGCL and the molecular mechanism thereof have good practical application value.
Drawings
The accompanying drawings, which 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.
FIG. 1 shows the expression level and prognostic significance of HMGCL in glioma tissue in an example of the present invention. Wherein, figure a is the correlation of HMGCL with glioma grade, IDH mutation status; FIG. B, C is the expression of HMGCL in the IDH wild type group and the 1p/19q non-co-deleted group; panel D is the prognostic significance of HMGCL in three databases; panel E is the expression of HMGCL in the glioma cases collected (n=20); panel F is the expression of HMGCL in glioma cell lines, glioma stem cells, normal astrocytes.
Fig. 2 is a graph of inhibition of glioma cell line malignant phenotype by HMGCL specifically interfered with in the examples of the present invention. Wherein, panel a is a graph of CCK-8 detection after transfection of cells with lentivirus, LN229, U251, and gbm#p3 cell growth profile based on OD450, and panel B is a further proliferation potency detection based on EdU assay; FIG. C, D shows the change in tumor invasion migration capacity after lentivirus transfection of cells; panel E shows the change in cell colony forming ability after lentivirus transfection of cells; FIG. F, G, H is a graph showing the results of in vivo experiments performed after cells were transfected with lentiviruses, and FIG. I is a graph showing in vivo experiments involving immunohistochemistry.
Fig. 3 is a graph showing the down-regulation of the acetylation appearance modification of glioma histone by HMGCL knockdown in the examples of the present invention. Panel a is the alteration of the downstream metabolite of HMGCL after knockdown; panel B shows histone acetylation modification changes after HMGCL knockdown; FIG. C, D, E shows that histone acetylation is modified by TSA to change the concentration of glucose and acetic acid in the medium.
FIG. 4 shows that HMGCL can regulate the transcription of cycle-related molecule FoxM1 in an embodiment of the present invention. Panel A shows RNA-seq sequencing of cell samples after HMGCL knockdown; panel B is a sequencing-related GSEA enrichment analysis; FIG. C shows the intersection of the down-regulating gene, the gene associated with the cell proliferation gene set and the gene associated with glioma prognosis; panel D is a validation at RNA level after knockdown of HMGCL; panel E is a validation at protein level after knock-down of HMGCL; FIG. F, G shows changes in FoxM1 downstream transcript levels.
FIG. 5 is a graph showing the effect of HMGCL on FoxM1 expression by inducing hyperacetylation around the promoter in the examples of the present invention. FIG. A is a diagram showing the acetylation modification of FoxM1 promoter histone in UCSC; FIG. B, C is a ChIP verification based on histone acetylation modification sites; FIG. D, E shows that FoxM1 levels were saved after varying the concentration of glucose and acetic acid in the medium.
FIG. 6 shows that HMGCL/FoxM1 can regulate beta-catenin translocation and function in the embodiment of the invention. Panel A shows the beta-catenin nuclear translocation after HMGCL knockdown; panel B shows immunofluorescent staining for β -catenin; panel C shows the detection of β -catenin RNA levels; panel D shows altered transcription levels of the β -catenin downstream molecule.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. 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 application 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 present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof. Experimental methods in the following embodiments, unless specific conditions are noted, are generally in accordance with conventional methods and conditions of molecular biology within the skill of the art, and are fully explained in the literature. See, e.g., sambrook et al, molecular cloning: the techniques and conditions described in the handbook, or as recommended by the manufacturer.
The invention will be further illustrated with reference to specific examples, which are given for the purpose of illustration only and are not to be construed as limiting the invention. If experimental details are not specified in the examples, it is usually the case that the conditions are conventional or recommended by the sales company; materials, reagents and the like used in the examples were commercially available unless otherwise specified.
In a typical embodiment of the present invention, the use of reagents for detecting HMGCL encoding genes and their expression products for the preparation of a product for glioma diagnosis and/or prognosis is provided.
The product is capable of diagnosing, detecting, monitoring or predicting (early or assisted) the progression of glioma by detecting the expression level of the HMGCL encoding gene and/or the HMGCL encoding gene expression product; experiments prove that the HMGCL has correlation with glioma grade, IDH mutation condition and the like, the expression quantity of the HMGCL is increased along with the increase of glioma grade (malignancy degree), and the high expression can indicate poor prognosis; meanwhile, the expression level of HMGCL in glioma cell lines and glioma stem cell lines is higher than that of normal astrocytes. Thus, the HMGCL encoding gene and its expression products may be used as glioma diagnostic and/or prognostic biomarkers.
Wherein, the HMGCL encoding gene and the expression product thereof can be human; the expression product of the HMGCL encoding gene may obviously be HMGCL protein, i.e. hydroxymethylglutaryl coa lyase.
Wherein, the reagent for detecting the HMGCL encoding gene and the expression product thereof comprises a substance for detecting the transcription of the HMGCL encoding gene based on RT-PCR, real-time quantitative PCR, in situ hybridization, a gene chip and gene sequencing, and/or a substance for detecting the condition of the HMGCL expression product (such as hydroxymethyl glutaryl coenzyme A lyase) based on an immunodetection method.
Such products include, but are not limited to, primers, probes, (gene or protein) chips, nucleic acid membrane strips, detection kits, detection devices or equipment for detecting the expression level of HMGCL in a test sample.
The sample to be tested is a human sample including, but not limited to, glioma cells and glioma tissue of a subject.
In yet another embodiment of the present invention, there is provided a system for glioma diagnosis and/or prognosis evaluation, the system comprising at least:
an acquisition module configured to: obtaining the expression level of a biomarker in a subject;
an evaluation module configured to: assessing the disease condition of the subject based on the expression level of the biomarker obtained by the obtaining module;
wherein the biomarker is HMGCL encoding gene and/or HMGCL encoding gene expression product (such as hydroxymethyl glutaryl coenzyme A lyase).
The assessment of the disease condition of the subject includes assessment of the diagnosis of glioma, the malignancy of glioma, and the survival of glioma patients in the subject.
It should be noted that, the system for diagnosing or assisting in diagnosing glioma according to the present invention may be a virtual device, as long as the functions of the analysis module and the evaluation module can be implemented. The analysis module can comprise various detection reagent materials and/or detection instrument devices and the like; the evaluation module may be any operation instrument, module or virtual device capable of analyzing the detection result of the analysis module to obtain the disease risk evaluation status of the glioma, for example, a corresponding data chart may be formulated in advance for each possible detection result and the corresponding disease risk status, and the detection result of the detection module is compared with the data chart to obtain the disease risk evaluation status of the glioma.
In yet another embodiment of the present invention, a computer-readable storage medium is provided, on which a program is stored which, when executed by a processor, performs the functions of the system.
In yet another embodiment of the present invention, an electronic device is provided that includes a memory, a processor, and a program stored on the memory and executable on the processor, the processor implementing the functions of the system when executing the program.
In yet another embodiment of the invention, there is provided the use of HMGCL as a target in glioma control and/or screening of glioma-related drugs.
In still another embodiment of the present invention, the method for screening glioma-associated drugs comprises:
1) Treating the expressed and/or HMGCL-containing system with a candidate substance; setting a parallel control without candidate substance treatment;
2) After step 1) is completed, detecting the expression level of HMGCL in the system; the candidate substance may be a candidate glioma drug if the expression level of HMGCL is significantly reduced in a system treated with the candidate substance compared to a parallel control.
In yet another embodiment of the present invention, the system may be a cellular system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system.
In a further embodiment of the present invention, there is provided the use of a substance that inhibits the expression and/or reduces the activity of an HMGCL encoding gene and its expression products in the preparation of a product;
the function of the product is any one or more of the following:
(a1) Inhibit proliferation of glioma cells;
(a2) Inhibiting invasive migration of glioma cells;
(a3) Promoting glioma cell cycle arrest;
(a4) Inhibiting glioma cell colony formation;
(a5) Inhibiting the acetylation modification expression of glioma cell histone;
(a6) Inhibiting the expression and transcription regulation functions of glioma cell cycle related molecules FoxM 1;
(a7) Preventing and/or treating glioma.
In yet another embodiment of the present invention, the substance that inhibits the expression level of HMGCL may take HMGCL as a target sequence and is capable of inhibiting an interfering molecule expressed by HMGCL, and may specifically include shRNA (small hairpin RNA), small interfering RNA (siRNA), dsRNA, microrna, antisense nucleic acid, or a construct (e.g., lentivirus) capable of expressing or forming the shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid; and antibodies directed against hydroxymethylglutaryl-CoA lyase, may also include inhibitors of the class of compounds.
In yet another embodiment of the invention, the above-mentioned product may be a pharmaceutical or an experimental reagent, which can be used for basic research.
When the above-mentioned product is a medicament, the medicament may further comprise one or more pharmaceutically or dietetically acceptable excipients. The adjuvant can be solid or liquid. Solid-state forms of the formulation include powders, tablets, dispersible granules, capsules, pills, and suppositories. Powders and tablets may contain from about 0.1% to about 99.9% of the active ingredient. Suitable solid excipients may be magnesium carbonate, magnesium stearate, talc, sugar or lactose. Tablets, powders, pills and capsules are solid dosage forms suitable for oral administration. Formulations in liquid form include solutions, suspensions and emulsions, examples of which are aqueous solutions for parenteral injection or water-propylene glycol solutions, or oral solutions with the addition of sweeteners and contrast agents. In addition, the injection can be made into small water injection, freeze-dried powder injection for injection, large transfusion or small transfusion.
In yet another embodiment of the present invention, there is provided a method for preventing and/or treating glioma, the method comprising: administering to the subject a therapeutically effective dose of the above-described drug.
The subject is an animal that has been the subject of treatment, observation or experiment, and may be human or non-human mammals such as mice, rats, guinea pigs, rabbits, dogs, monkeys, chimpanzees, etc., most preferably humans. By "therapeutically effective amount" is meant that amount of active compound or pharmaceutical agent, including a compound of the present invention, which causes a biological or medical response in a tissue system, animal or human that is sought by a researcher, veterinarian, medical doctor or other medical personnel, which includes alleviation or partial alleviation of the symptoms of the disease, syndrome, condition or disorder being treated. It must be recognized that the optimal dosage and spacing of the active ingredients of the present invention is determined by its nature and external conditions such as the form, route and site of administration and the particular mammal being treated, and that such optimal dosage may be determined by conventional techniques. It must also be appreciated that the optimal course of treatment, i.e. the daily dosage of the simultaneous compounds over the nominal time period, can be determined by methods well known in the art.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
Examples
1. Materials and methods
1. Ethical statement and clinical glioma tumor specimen
The protocol in this study was approved by the ethical committee of the Qilu hospital at Shandong university (DWLL-2021-096). The study was conducted with full compliance with relevant regulations and guidelines. Human glioma tissue samples were from the operations performed by the zilu hospital on patients. Non-neoplastic brain tissue samples are from patients requiring surgery due to brain trauma events. All enrolled patients provided written informed consent. Clinical information and expression data for glioma samples of the public database were taken from the TCGA, CGGA and Rembrandt databases.
2. Cell culture and reagents
Human glioblastoma cells were purchased from cell banks of the national academy of sciences. All glioma cell lines were cultured using Dulbecco's modified Eagle's medium (Thermo Fisher Scientific; waltham, mass., USA) with 10% fetal bovine serum (FBS; thermo Fisher Scientific) and incubated at 37℃in a 5% carbon dioxide cell incubator. Patient-derived GBM Stem Cells (GSCs) P3 were previously isolated from GBM surgical specimens and characterized. GSCs were cultured in Neurobasal medium (Gibco/Thermo Fisher Scientific) containing 2% B-27 Neuromix (Thermo Fisher Scientific), 20ng/mL epidermal growth factor (EGF; peproTech; eastWindsor, NJ, USA) and 10ng/mL basic fibroblast growth factor (bFGF; peproTech). Normal Human Astrocytes (NHA) and NHA transfected with human papillomavirus 16E6/E7 and human TERT (immortalized NHA-ET) were obtained from Lonza (Walkersville, MD, USA) and cultured in astrocyte medium (scientific) supplemented with astrocyte growth medium BulletKit (scientific; carlsbad, CA, USA).
HMGCL stable knock-down virus construction
Both transient and stable transfection were performed with Lipofectamine 2000 reagent (ThermoFisher Scientific) according to the manufacturer's instructions. For siRNA experiments, cells were transfected with 100pmol of siRNA (GenePharma; shanghai, china) for 48 hours. After 48 hours of transfection of HEK293T cells with lentiviral packaging plasmids psPAX2 and pCMV-VSV-G and lentiviral expression constructs, stably expressed lentiviral supernatants were harvested. Target cells were incubated with supernatant for 24 hours and after 48 hours selection with puromycin was initiated. The siRNA sequence against HMGCL is as follows: siHMGCL-1:5'-CCAGCUUUGUGUCUCCUAAGU-3'; siHMGCL-2:5'-ACCAAGAAGUUCUACUCAAUG-3'; siNC 5'-UUCUCCGAACGUGUCACGUTT-3'. The expression structures of shNC, shHMGCL were purchased from Obio Technology (Shanghai, china).
4. Real-time quantitative PCR
RNA was extracted from glioma cells using Trizol reagent (Invitrogen, life Technologies). And reverse transcription is performed. The primers of HMGCL are forward primers: 5'-GCTCTTGGCTGCCCTTATGA-3'; reverse primer: 5'-TTACAGGTAGCCTGAGCCAC-3'.
5.Western blot
The harvested cells were lysed with thermal denaturation in RIPA cell lysis buffer. Protein lysates (20. Mu.g) were analyzed and the proteins were transferred to a polyethylene difluoride film (PVDF). The primary antibodies HMGCL (Proteintech), H3K27ac (CST), foxM1 (CST), beta-Catenin (CST) were incubated. Specific proteins were detected by enhanced chemiluminescence (ECL, millipore, bredford, USA).
6. Analysis of cell proliferation Capacity
Glioma cells were taken in 96-well cell culture dishes at a density of 3000 cells/well. Cell proliferation was analyzed by cell count Kit-8 (CCK-8) 24, 48, 72', 96h after transfection. mu.L of CCK-8 solvent was added to each well and incubated for 1h in a cell incubator. The optical density was then measured at 450nm using Ensight (PerkinElmer) and analyzed to plot cell proliferation curves.
7. Cell colony generation assay
Cells were seeded into 6-well plates at a density of 1000 cells/well. DMEM containing 10% fetal bovine serum was replaced every three days. After 15 days, methanol was fixed, stained with crystal violet for 15 minutes and photographed. Each test was repeated 3 times.
8. Cell balling experiment
Cells were seeded into 12-well plates at a density of 1000 cells/well and cultured using Neurobasal stem cell culture broth. After 10 days, the number of balls was detected by a microscope and counted by photographing. Each test was repeated 3 times.
9. Cell invasion Capacity detection experiment
Glioma balling is the incubation of cells in a balling matrix for 72 hours, implantation of spheres >2mm in diameter in 96-well plates, and addition of invasive glue (Trevigen; gaithersburg, MD, USA). Glioma spheroids were photographed every 24 hours with a microscope. Ellipsoids at 0h served as reference points for measuring the area of invasion of the invading cells.
GBM-brain organoid co-culture ex vivo system GFP-transfected GBM cells were cultured into glioma spheroids and then co-cultured with mature brain organs for 72 hours. Images of GBM cell invasion were captured under confocal microscopy (Leica TCS SP8; wetzlar, germany).
10. Flow cytometry
For cell cycle analysis, cells were harvested, fixed with 75% ethanol for 48 hours, 75% ethanol for 48 hours at 4℃and propidium iodide (PI; BD Biosciences; franklin Lakes, N.J.) for 15 minutes. To detect apoptosis of cells. Cells were rinsed with PBS and resuspended in staining for 15 min with the attachments V-FITC and PI (BD Biosciences). (BD Biosciences) for 15 min. All cells were on a C6 flow cytometer (BD Biosciences) and the data was analyzed using FlowJo software (V10, BD Biosciences).
11. Chromatin immunoprecipitation (ChIP) assay
ChIP detection was performed using the EZ-ChIP immunoprecipitation kit (Millipore; billerica, mass., USA). The following antibodies were used: anti-H3K 27ac (ab 4729,1:100, abcam) and normal rabbit IgG (# 2729,1:100,Cell Signaling Technology). The primer sequences for the H3K27ac binding site of the FoxM1 promoter are as follows:
| FOXM1(H3K27ac)-88F | TAAGCAGTGAGAAGGCCACG |
| FOXM1(H3K27ac)-88R | TGGAGATTTGGGTCACACGG |
| FOXM1(H3K27ac)-183F | GGAGCAGGGGAGTGTGTATG |
| FOXM1(H3K27ac)-183R | CGTGGCCTTCTCACTGCTTA |
briefly, GBM cells or NHA were crosslinked with 1% formaldehyde solution for 10 min and quenched with 0.125M glycine. The cells are spun down, washed, resuspended, lysed and sonicated. The broken chromatin extracts were pre-cleared with agarose beads in the ChIP kit and incubated overnight with antibodies or normal rabbit IgG as a control. After washing, elution and reverse cross-linking, the DNA was analyzed by qPCR.
12. In vivo experiments in mice
Intracranial gliomas were established, and gbm#p3 fluorescent cells (1×106) were transfected with HMGCL stable knockdown virus, and then stereotactically implanted into the mouse brain. The growth of intracranial tumors was examined on days 4 and 20 using bioluminescence imaging. Kaplan-Meier survival curves were used to describe time to survival and body weight.
13. Statistical analysis
ANOVA or t-test was applied using GraphPad Prism 7 software. All experiments were repeated 3 times and mean ± standard error was taken. The Kaplan-Meier survival curve was analyzed using the log-rank test. The chi-square test and fisher's deterministic analysis were applied to determine the relationship between HMGCL expression and clinical pathology. P <0.05 is statistically significant for the differences.
2. Experimental results
HMGCL gene is abnormally high expressed in GBM and is related to tumor grading and prognosis
The analysis result of TCGA bioinformatics shows that HMGCL has correlation with glioma grade, IDH mutation condition and the like (figure 1A), and HMGCL is highly expressed in IDH wild type group and 1p/19q non-co-deletion group (figures 1B and 1C); kaplan-Meier survival curve, log-rank test hint: of the three databases, HMGCL expression levels were indicative of overall glioma post-operative survival, high expression could be predictive of poor prognosis (fig. 1D), and our immunohistochemical staining in the collected clinical cases (n=20) suggested that HMGCL expression levels increased with increasing glioma levels (fig. 1E); meanwhile, HMGCL was expressed in higher amounts in glioma cell lines and glioma stem cell lines than normal astrocytes (fig. 1F). The information shows that the HMGCL has important clinical indication significance in glioma tissues, can be used as a novel biological marker, and provides basis for glioma patient prognosis evaluation.
2. Specific interference HMGCL significantly inhibits glioma malignant proliferation
To assess HMGCL function in GBM, we constructed lentiviruses to specifically knock down HMGCL protein expression in GBM cells LN229, U251MG and gbm#p3. Subsequent CCK-8 and EdU experiments showed that the proliferation of all three GBM cell lines of the HMGCL knockdown group was reduced after 4 days (FIGS. 2A, 2B). Our 3D sphere invasion experiments and brain-like invasion experiments suggest that knockdown of HMGCL results in reduced tumor cell invasion activity (fig. 2C, 2D), while knockdown of HMGCL also causes G1/S phase arrest and results in reduced colony formation (fig. 2E). Our subsequent in vivo experiments suggest that HMGCL knockdown can inhibit tumor growth (fig. 2F, 2G) and can extend tumor-bearing nude mice survival (fig. 2H). Immunohistochemical staining suggested a decrease in Ki-67 levels following HMGCL inhibition (fig. 2I).
Hmgcl knockdown results in down-regulation of glioma histone acetylation appearance modification
Since HMGCL is a key metabolizing enzyme, its downstream metabolites include acetyl-coa, acetoacetate, and β -hydroxybutyrate, which acetoacetate is further metabolized, to examine which metabolite in particular works, we examined several metabolites and found that only acetyl-coa was significantly reduced after HMGCL knockdown (fig. 2A). Since acetyl-coa is a key raw material for histone acetylation, we examined the histone acetylation level and found that the histone acetylation modification level was reduced after HMGCL knockdown (fig. 2B), and to further demonstrate that the reduced histone acetylation level was saved back after TSA was used to change the concentration of glucose and acetic acid in the medium (fig. 2C, 2D, 2E).
mRNA-seq sequencing suggests that HMGCL can regulate cycle-related molecule FoxM1 transcription
Histone acetylation modifications can exert a carcinomatous effect by activating transcription of multiple classical oncogenes downstream. Based on this, we first performed RNA-seq sequencing of HMGCL knockdown cell samples (completed by the company bicuchong, fig. 4A). Enrichment analysis showed that the cycle-related pathway in HMGCL-KD cells was significantly altered (fig. 4B), and two possible downstream molecules, foxM1 and CASP2, were finally determined by crossing down-regulated genes, cell proliferation gene set-related genes and glioma prognosis-related genes (fig. 4C). Later validation experiments showed that FoxM1 decreased on average at RNA level and protein level after HMGCL knockdown (fig. 4D, 4E). Fluorescent quantitative PCR suggested that the expression level of FoxM1 downstream was reduced after HMGCL knockdown, suggesting that FoxM1 transcriptional regulatory function was inhibited (fig. 4F, 4G). At the same time, the p21 and p27 expression levels increased, correlating with G1/S phase block.
Hmgcl affects FoxM1 expression by inducing hyperacetylation around the promoter.
From the UCSC database, we determined that the FoxM1 promoter region was regulated by histone H3K27ac modification (fig. 5A), and we designed primers at selection sites upstream and downstream of the FoxM1 transcription initiation site to perform ChIP experimental verification, which suggested that the level of H3K27ac modification in the FoxM1 promoter region was reduced (fig. 5B, 5C). Rescue experiments further demonstrated that FoxM1 levels could be rescued by H3K27ac rescue conditions (fig. 5D, 5E).
HMGCL/FoxM1 can regulate beta-catenin translocation and functions.
Since FoxM1 can interfere with the β -catenin function, we further validated the β -catenin function. Following HMGCL knockdown, β -catenin decreased at protein levels (fig. 6A) while distribution was decreased in the nucleus (fig. 6B). We examined RNA levels to find that FoxM1 levels were reduced, while the changes in β -catenin levels were not significant (FIG. 6C), so we speculate that HMGCL affects β -catenin by affecting FoxM1 levels. At the same time, the transcription level of the beta-catenin downstream molecule was also reduced (FIG. 6D).
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The application of a reagent for detecting HMGCL encoding genes and expression products thereof in preparing glioma diagnosis and/or prognosis products.
2. Use according to claim 1, wherein the HMGCL encoding gene and its expression product are all of human origin; the expression product of the HMGCL encoding gene is HMGCL protein, namely hydroxymethyl glutaryl coenzyme A lyase;
the prognosis includes an assessment of the subject's survival.
3. Use according to claim 1, wherein said reagent for detecting HMGCL encoding gene and its expression product comprises a substance for detecting HMGCL encoding gene transcription based on RT-PCR, real-time quantitative PCR, in situ hybridization, gene chip and gene sequencing, and/or a substance for detecting HMGCL expression product condition based on immunodetection method;
the product comprises a primer, a probe, (gene or protein) chip, a nucleic acid membrane strip, a detection kit, a detection device or equipment for detecting the expression level of the HMGCL in a sample to be detected;
the sample to be tested is a human sample and comprises glioma cells and glioma tissues of a subject.
4. A system for glioma diagnosis and/or prognosis evaluation, characterized in that it comprises at least:
an acquisition module configured to: obtaining the expression level of a biomarker in a subject;
an evaluation module configured to: assessing the disease condition of the subject based on the expression level of the biomarker obtained by the obtaining module;
wherein the biomarker is an HMGCL encoding gene and/or an HMGCL encoding gene expression product.
5. The system of claim 4, wherein the assessment of the disease condition in the subject comprises assessing the diagnosis of glioma, the malignancy of glioma, and the survival of glioma patients in the subject.
6. A computer readable storage medium having stored thereon a program, which when executed by a processor, performs the functions of the system of claim 4 or 5.
7. An electronic device comprising a memory, a processor and a program stored on the memory and executable on the processor, characterized in that the processor implements the functions of the system of claim 4 or 5 when executing the program.
8. Use of a substance that inhibits expression of and/or reduces activity of an HMGCL encoding gene and its expression product in the preparation of a product;
the function of the product is any one or more of the following:
(a1) Inhibit proliferation of glioma cells;
(a2) Inhibiting invasive migration of glioma cells;
(a3) Promoting glioma cell cycle arrest;
(a4) Inhibiting glioma cell colony formation;
(a5) Inhibiting the acetylation modification expression of glioma cell histone;
(a6) Inhibiting the expression and transcription regulation functions of glioma cell cycle related molecules FoxM 1;
(a7) Preventing and/or treating glioma.
9. The use according to claim 8, wherein the product is a pharmaceutical or experimental agent for use in basic research.
10. Use according to claim 8, wherein the substance inhibiting the expression level of HMGCL comprises shRNA, siRNA, dsRNA, microrna, antisense nucleic acid, or a construct capable of expressing or forming said shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid; antibodies to hydroxymethylglutaryl-CoA lyase and inhibitors of compounds.
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