CN117491634A - LDHA + Application of EVs as biomarker in prediction, detection and evaluation of recurrence risk after brain glioma operation - Google Patents
LDHA + Application of EVs as biomarker in prediction, detection and evaluation of recurrence risk after brain glioma operation Download PDFInfo
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Abstract
The invention belongs to the technical field of biological medicine, and particularly discloses LDHA + The application of EVs as a biomarker in prediction, detection and evaluation of recurrence risk after brain glioma operation. The invention is based on the phenomenon that LDHA protein specificity exists in plasma/serum extracellular vesicles obtained from brain glioma patients in vitro, and LDHA does not exist in normal people and brain glioma non-recurrent patients + EVs high expression phenomenon, proposes LDHA + EVs can be used as biomarkers for predicting recurrence risk after glioma operation; through LDHA + EVs detection kit for detecting plasma/serum LDHA of target patient + EVs, the number, concentration and proportion of which are evaluated by the evaluation method can rapidly, accurately and clearly determine the postoperative recurrence risk probability of the glioma.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to LDHA + The application of EVs as a biomarker in prediction, detection and evaluation of recurrence risk after brain glioma operation.
Background
Gliomas, also known as Gliomas (GBMs), are one of the most deadly central nervous system tumors, with an incidence of about 70% of brain tumors, and have the characteristics of high heterogeneity and blurred tumor tissue. The etiology of the glioma has the characteristics of hidden disease, high growth speed, short average life time, poor postoperative prognosis and the like. The current treatment regimen for gliomas is mainly surgical excision, chemotherapy-combined therapy of temozolomide TMZ, and palliative treatment. However, due to the highly heterogeneous nature and invasive growth of gliomas, surgery is often not completely resected, patient prognosis is poor, with median survival (Medium Overall survival, mOS) of only 12-18 months, and due to the presence of glioma stem cells (Glioma stem cells, GSCs), glioma postoperative recurrence rates are extremely high, usually within 1-5 years, and result in poor prognosis. The current glioma recurrence detection means mainly comprise imaging means such as Magnetic Resonance (MRI) examination and electronic Computed Tomography (CT), but the detection means depend on clinical experience of doctors, and have the factors of expensive equipment, poor convenience and the like, so that the glioma recurrence detection means are not beneficial to long-term monitoring and management after operation.
In recent years, liquid biopsy has the characteristics of convenience, rapidness, noninvasive property and good repeatability, and plays an important role in cancer diagnosis and prognosis management. Tumors can be prepared by releasing components with biological information, such as free tumor cells, free tumor DNA, RNA, proteins and the like, into body fluid, and liquid biopsy technology can be used for diagnosing and prognosis management of diseases by detecting body fluid components including blood, cerebrospinal fluid, ascites, urine and the like to obtain key information of tumor release. At present, common molecular pathological diagnosis markers of glioma comprise IDH1, ATRX, 1p/19q, EGFR amplification and the like, but the related markers are usually obtained by a pathological biopsy mode, the detection method is complex and relatively high in cost, such as immunohistochemical staining, methylation sequencing and the like, and the prognosis effect of the related markers is poor, and the sensitivity and the specificity are limited. In addition, in brain glioma diseases, key information of brain tumors is difficult to obtain due to the existence of blood-brain barrier in the brain.
Disclosure of Invention
In view of the above-described drawbacks of the prior art, an object of the present invention is to provide an LDHA + Application of EVs as biomarker in prediction, detection and evaluation of postoperative recurrence risk of glioma, namely postoperative recurrence risk of gliomaPrediction provides a new technical means.
To achieve the above and other related objects, a first aspect of the present invention provides lactate dehydrogenase a-positive extracellular vesicles (LDHA) + EVs) as biomarkers in the preparation of reagents for predicting, detecting and evaluating the risk of recurrence after brain glioma surgery.
In some embodiments, the lactate dehydrogenase a-positive extracellular vesicles are derived from plasma or serum or other body fluids.
In a second aspect, the invention provides a kit for evaluating risk of recurrence after brain glioma surgery, which is used for detecting lactate dehydrogenase A positive extracellular vesicles (LDHA) in a biological sample + EVs)。
In some embodiments, the biological sample is a glioma ex vivo plasma or serum or other body fluid sample.
In some embodiments, the kit comprises at least one of the following components: the kit comprises an LDHA protein antibody, magnetic beads combined with an exosome marker, a detection chip for detecting the exosome, a buffer solution, a standard substance and a reference substance, but is not limited to the following.
In some embodiments, the exosome marker is selected from CD63 or CD9.
In some embodiments, the detection chip is selected from the group consisting of optical discs used in ExoCounter exosomes analyzers.
In some embodiments, the kit is used to detect lactate dehydrogenase a-positive extracellular vesicles (LDHA) in a biological sample + EVs) to determine the risk of recurrence after a glioma surgery.
In some embodiments, when lactate dehydrogenase a-positive extracellular vesicles (LDHA + EVs) is higher than Yu Yushe reference intervals, the risk of postoperative recurrence of glioma patients is high, and the prognosis is poor; when lactate dehydrogenase a-positive extracellular vesicles (LDHA) + EVs) is lower than the preset reference interval, the postoperative recurrence risk of the glioma patient is low, and the prognosis is good; the preset reference interval is the number, concentration and proportion of healthy subjects/patients with the brain glioma without recurrenceWithin a numerical range.
In a third aspect, the invention provides a method for assessing risk of recurrence after brain glioma surgery by determining lactate dehydrogenase A-positive extracellular vesicles (LDHA) + EVs) are evaluated for the risk of postoperative recurrence in glioma patients.
As described above, the LDHA of the present invention + The application of EVs as a biomarker in prediction of recurrence risk after brain glioma operation has the following beneficial effects:
(1) The invention provides the LDHA positive extracellular vesicles which can be used as biomarkers for predicting the postoperative recurrence risk of glioma for the first time. The invention provides the in vitro plasma extracellular vesicle LDHA of a patient based on the phenomenon that the specificity of the LDHA protein is up-regulated in the in vitro obtained plasma extracellular vesicle of a patient with brain glioma, and the phenomenon that the LDHA protein of the in vitro obtained plasma extracellular vesicle of a normal person and other tumor patients is low or not expressed + The pl-EVs can be used as tumor markers for evaluating the postoperative recurrence risk of the glioma, and the LDHA expression level in the extracellular vesicles of the plasma obtained by detecting a target patient in vitro through mass spectrometry can rapidly, accurately and clearly determine the recurrence risk of the glioma.
(2) Clinical verification shows that compared with an MRI method, the invention detects the extracellular vesicle LDHA of the blood plasma through the developed kit + The quantity, concentration and proportion of pl-EVs can be used for prognosis evaluation of brain glioma patients, and meanwhile, the detection has the characteristics of simplicity and convenience in operation, sensitivity in detection, low cost, high repeatability and the like.
Drawings
Fig. 1 shows clinical cohort information incorporated in an embodiment of the invention, where a is the clinical cohort group and b is the pathology statistics of patients with recurrent glioma.
FIG. 2 shows an electron microscope image and an immunoblot analysis image of extracellular vesicles after enrichment in the embodiment of the invention, wherein a is an electron microscope image and b is an immunoanalysis image.
FIG. 3 shows protein mass spectrometry of brain glioma recurrent patients, non-recurrent patients, healthy control plasma extracellular vesicles in an example of the invention, wherein panels a-b are protein cluster analysis of Heatmap of proteomics analysis of three sets of extracellular vesicles of origin, c is Venn diagram of EV protein crossover analysis, d is the main pathway of KEGG pathway analysis of differential proteins in HD-EVs, NR-EVs and R-EVs, and e is a list of KEGG pathway enriched in EVs.
FIG. 4 shows LDHA in plasma of patients with recurrent GBM and non-recurrent GBM according to an embodiment of the invention + Detection results of pl-EVs.
FIG. 5 shows an embodiment of the invention based on plasma extracellular vesicles LDHA + prediction of risk of recurrence of brain glioma by pl-EVs, wherein a is a representative nmr image of preoperative, postoperative and postoperative recurrence of brain glioma patient, and b is plasma LDHA of preoperative, postoperative patients of brain glioma calculated by the evaluation method + The number of pl-EV and c is the LDHA of the patient after the brain glioma operation and recurrence calculated by the evaluation method + pl-EV number, d is clinical information of the glioma patient, e is plasma LDHA of the glioma patient obtained by the evaluation method + Absolute number of pl-EV.
Detailed Description
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.
In recent years, related researches show that extracellular vesicles (Extracellular Vesicles, EVs) as a microvesicle structure secreted by cells and having a size of 30-2000nm comprise biological macromolecular components such as proteins, nucleic acids, lipids and the like from donor cells, can be used as a brand-new intercellular information transmission substance, can penetrate through blood brain barrier and play an important role in the occurrence, development, invasion, metastasis and immune escape of central nervous system diseases including gliomas. Proteins serve as the main contributors to vital activities, and the biological information they contain plays an important role in the diagnosis of clinical disease. Nowadays, more and more researches indicate that extracellular vesicle protein components have the effects of promoting tumor proliferation, promoting angiogenesis, enhancing tumor resistance and the like in the tumor development process, and for example, the latest researches of the university of kannel team show that: the results of the exosome proteomic analysis of the 426 person source sample suggest that the contents of the plasma exosomes can be effectively differentiated for tumor status. Thus, plasma-derived exosome content has value in potential biomarker mining. Based on the method, the effective postoperative risk biomarker of the brain glioma is sought from the viewpoint of extracellular vesicles and is used for warning the recurrence risk of the brain glioma, and the extracellular vesicle protein markers based on the plasma and the detection kit thereof are developed through mass spectrometry technical analysis of the extracellular vesicles of clinical samples, so that a novel technical means is provided for postoperative recurrence risk assessment of patients with the brain glioma, and the survival time of GBM patients is prolonged.
According to the invention, the lactic dehydrogenase A (LDHA) is found to be significantly up-regulated in plasma extracellular vesicles of patients with recurrent glioma by mass spectrometry relative to healthy donors and patients without recurrent glioma. Human LDHA is located on chromosome 11p15.4, the transcribed protein has 332 amino acids and the predicted molecular weight is 37kDa, and the LDHA protein encoded by the LDHA gene is a cytoplasmic enzyme mainly involved in anaerobic and aerobic glycolysis (Warburg effect). In brain tissue, LDHA is widely present and provides energy to brain tissue, and studies have shown that cancer cells in brain tumors utilize LDHA to increase glycolysis, ATP and lactate production rate compared to normal cells to promote brain tumor progression, but there is currently no clear study on how LDHA regulates the brain glioma progression mechanism and no LDHA is a biomarker of brain disease.
Specifically, the invention firstly collects plasma samples of 265 brain glioma patients and 50 healthy controls, identifies proteins in extracellular vesicles of two groups of queues through mass spectrometry, screens differential expression proteins, and discovers that the 'pyruvic acid-lactic acid conversion' pathway is obviously up-regulated in EVs derived from plasma of recurrent GBM patients, and LDHA protein is one of the most obvious up-regulated proteins.
Then, the invention performs staged quantitative analysis on the screened plasma extracellular vesicles by periodically collecting post-operation brain glioma patients and contemporaneous healthy control plasma samples, and performs staged quantitative analysis on the screened plasma extracellular vesicles in the plasma extracellular vesicles of patients in a queue, and compares nuclear Magnetic Resonance (MRI) imaging results to evaluate the potential of the screened plasma extracellular vesicles as biomarkers for postoperative recurrence risk assessment of brain glioma.
The specific implementation process is as follows:
as shown in fig. 1a, plasma of 265 patients and 50 healthy controls included in the study was approved and supported by the south hospital ethics committee of the university of south medical science.
Wherein the clinical queue inclusion criteria include: 1) Successful surgery to treat the patient, 2) pathologically confirmed glioma; clinical queue exclusion criteria included: 1) Unknown histological grade, 2) no visit group.
Matched healthy control inclusion criteria included: 1) The matched age and sex, 2) no abnormal result of physical examination index; 3) The same medical institution; matched healthy control exclusion criteria included: people suffering from diseases.
According to the pathology grade, age, sex, histology type, IDH gene mutation and cancer recurrence of the glioma patients, the patients in the clinical queue are subjected to clinical information arrangement, and a pathology result statistical table shown in figure 1b is obtained.
The specific experimental process is as follows:
patient whole blood was taken from healthy individuals and patients with recurrent or primary GBM of glioma (hereinafter abbreviated as GBM). Collecting whole blood of a patient by using a blood collection tube containing an anticoagulant; centrifuging at 4deg.C and 3000rpm for 10 min, and collecting supernatant; centrifuging at 4deg.C for 15min at 1500g, removing cell debris, collecting supernatant, and preserving at-80deg.C for a long period.
1. Characterization of plasma extracellular vesicles of GBM relapse/non-relapse/primary and healthy controls
1.1 Experimental methods
(1) Obtaining plasma EVs of healthy subjects and GBM postoperative patients (including postoperative recurrent patients and postoperative non-recurrent patients) by a super-high-speed centrifugation method, centrifuging plasma or cell supernatant at 800g/3min, and collecting the supernatant into a new centrifuge tube; high-speed centrifuge: collecting supernatant into new centrifuge tube at 4deg.C and 10000g/30min, centrifuging at 135,000g for 2 hr, removing supernatant, re-suspending with PBS, centrifuging at 135,000 for 2 hr, and re-suspending the exosome in 60-80 microliter PBS.
(2) Immunoblotting experiments: EV lysates were prepared using RIPA (10 mM Tris-HCl, pH 7.4, 150mM NaCl, 1mM EDTA, 0.1% SDS, 1% Triton X-100 and 1% sodium deoxycholate) with protease inhibitors added. EVs concentrations were determined by using BCA kit and manufacturer's protocol. Equal amounts of EVs were separated by electrophoresis (SDS-PAGE) using FD bioscience one-step gel kit (12% gel, FD 346), transferred to polyvinylidene fluoride (PVDF) membrane (0.45 μm, millipore), blocked with 5% Bovine Serum Albumin (BSA) in TBST, incubated with primary antibody for 1 hour at room temperature overnight, then washed 3 times with TBST, incubated with HRP-labeled anti-mouse or rabbit secondary antibody for 1 hour, then washed with TBST at room temperature. For protein visualization, ECL-HRP substrate kit (Advansta inc., CA, USA) was used. Chemiluminescent images were acquired using a chemiluminescent imaging system (BioRad).
1.2 experimental results:
plasma EV, GBM post-operative recurrent plasma EV, post-operative non-recurrent plasma EVs were examined by Transmission Electron Microscopy (TEM) and the results showed that three groups of plasma EVs had a distinct teacup-like structural morphology (fig. 2 a); the EVs were found to have positive markers TSG101, CD9 and CD63, and negative marker Alb, without negative marker calnexin by western blot detection (fig. 2 b).
2. Protein mass spectrometry analysis of plasma EVs of GBM recurrent/non-recurrent/healthy control patients
2.1 experimental method:
EV was cleaved with 100. Mu.L of lysis buffer (2% SDS, 25Mm HEPES pH 7.6, 1mM DTT, 1 Xprotease inhibitor) for 10 min at room temperature; the lysis buffer was heated at 95℃for 5 minutes and sonicated for 1 minute, and the supernatant was collected after centrifugation at 14,000g for 10 minutes. Protein concentration was measured by BCA assay, with 200 μl of 8M urea buffer (8M urea, 25mM HEPES,pH 7.6) added per 30 μl protein sample. The sample was then transferred to a 10kDa cut-off ultrafiltration tube (PALL) and centrifuged at 14000g for 15 minutes. Thereafter, 50mM iodoacetamide was added and incubated for 10 minutes, followed by centrifugation again at 14,000g for 15 minutes. Iodoacetamide was eluted with 8M urea buffer and the 8M urea solution was replaced with 100mM HEPES (pH 8.5) solution. The samples were incubated with trypsin overnight at 37 ℃. Enzymolysis was obtained by centrifugation at 14,000g for 15 min. Following tryptic digestion, the peptide fragments were labeled according to the TMT kit instructions. Peptides were captured on a C18 trap column and separated with 8% -32% acetonitrile, pH 1.0. A gradient from 5% to 26% B, 0-65 min, a gradient from 26% to 35% B, 82-83 min, from 35% -90% B,83-90 min, and 90% (solvent A:0.2% formic acid, solvent B:98% acetonitrile, 0.2% formic acid) were used at a flow rate of 300 nl/min. The ion source voltage is set to 2.0kV, peptide precursor ions are detected by high-resolution Orbitrap, and secondary fragments are detected and analyzed by LTQ. Protein identification and quantification was performed using Protein Discoverer (v 2.3).
2.2 experimental results:
this section of the experiment performed a mass spectrometric analysis of 11 extra-plasma vesicles (pl-EV) from healthy donors or GBM patients by proteomics, including: healthy donors (group #1-3, healthy donor, HD); GBM recurrent groups (groups #1-4, recurrent, R) and non-recurrent groups (groups #1-4, non-recurrent, NR) post-operatively; plasma-derived exosome proteomics quantitative experiments were performed using 11-label TMT labeling technique and high resolution liquid chromatography-mass spectrometry quantitative proteomics research strategy, and the following results were obtained:
(1) Together 395 proteins, 3100 peptides and 18,212 matching spectra were identified. The amount of protein quantified was 372, where 212 protein was subcellular localization, including membrane localization.
(2) Based on plasma membrane localization screening, 61 proteins in the R vs NR comparison group were up-regulated and 29 proteins were down-regulated in quantitative proteins, with a 1.2 fold change threshold as a standard. Proteins that did not change in NR and Ctrl and R and NR were selected, as well as 34 proteins with subcellular localization, including membrane localization, with a significant difference in 3 proteins (p < 0.05) (FIGS. 3 a-b).
(3) The results of the protein mass spectrum showed: 21 proteins were significantly up-regulated in group R, but not in the HD and NR groups (FIG. 3 c-d), where a total of 9 signal pathways were observed to be up-regulated by KEGG pathway enrichment analysis, as described in the table (FIG. 3 d); among them, LDHA is one of the proteins with the highest up-regulation rate (FIG. 3 e).
3. LDHA in plasma of GBM recurrent patients and non-recurrent patients + Detection of EVs
3.1 experimental method:
developing a brain glioma postoperative recurrence risk assessment kit, wherein the kit comprises an LDHA protein antibody, magnetic beads (CD 63/CD9 coupled magnetic beads), a detection chip (an ExoCounter exosome analyzer compact disc, reference document Development of a Highly Sensitive Device for Counting the Number of Disease-Specific Exosomes in Human Sera, DOI 10.1373/clinchem.2018.29963), a buffer solution (a washing buffer solution: PBST, PBS containing 0.05% Tween 20) and deionized water; sealing liquid: PBST containing 1% casein; dilution buffer: PBS), standard/reference (CD 63/CD 9), etc., and detecting LDHA in the blood plasma of a subject by labeling an LDHA protein antibody on a detection chip in advance, and combining the detection chip with an antibody affinity technology by a size exclusion technology + EVs, and then the quantity, proportion and concentration are determined by evaluation.
3.2 experimental results:
plasma from 15 GBM patients was tested using the kit, with LDHA as the coating antibody, and the results showed: comparing the plasma of the patients in the non-recurrence group after GBM operation with the LDHA in the plasma of the patients in the recurrence group after GBM operation + The number of EVs was significantly increased (fig. 4) (. Times., p<0.01；***,p<0.001；****p<0.0001)。
4. Plasma LDHA-positive extracellular vesicles (LDHA) + EVs) can predict the recurrence risk of brain glioma
To determine LDHA + Whether pl-EVs have the potential to predict GBM and GBM recurrence, whether have the potential for further clinical application, and further will relapse LDHA in GBM patient plasma + The number of EVs is compared to their MRI detection images. In FIG. 1, 12 GBM relapsed patients were shownMRI detection was performed after blood withdrawal by comparison of LDHA in blood samples from these 12 patients + Number of EVs, found that 5/12 of patients had postoperative LDHA + Reduced number of EV (FIG. 5 b), LDHA at recurrence + Phenomenon of EV rise (fig. 5 c). Meanwhile, by comparing the imaging data and detection data (FIGS. 5a, d, e) of 2 patients with recurrent GBM, which were typical, after MRI, at the time of recurrence, it was found that LDHA was present at the time of recurrent GBM + The detection data of EVs are respectively 1-1.5 months earlier than the MRI data.
The experimental data and results show that by monitoring LDHA + The number of pl-EVs predicts recurrence of GBM, indicating LDHA + pl-EVs have a potential as biomarkers for monitoring GBM recurrence.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Claims (10)
1. Lactate dehydrogenase A positive extracellular vesicle LDHA + Application of EVs as biomarker in preparation of reagents for predicting, detecting and evaluating recurrence risk after brain glioma operation.
2. The use according to claim 1, characterized in that: the lactate dehydrogenase a-positive extracellular vesicles are derived from plasma, serum or other body fluid samples.
3. A brain glioma postoperative recurrence risk assessment kit, which is characterized in that: the kit is used for detecting lactate dehydrogenase A positive extracellular vesicle LDHA in biological samples + EVs。
4. The brain glioma postoperative recurrence risk assessment kit according to claim 3, wherein: the biological sample is brain glioma in-vitro plasma or serum or other body fluid samples.
5. The brain glioma postoperative recurrence risk assessment kit according to claim 3, wherein: the kit comprises at least one of the following components: the kit comprises an LDHA protein antibody, magnetic beads combined with exosome markers, a detection chip for detecting exosomes and a buffer solution.
6. The brain glioma postoperative recurrence risk assessment kit according to claim 5, wherein: the exosome marker is selected from CD63 or CD9.
7. The brain glioma postoperative recurrence risk assessment kit according to claim 5, wherein: the detection chip is selected from optical discs used by an ExoCounter exosome analyzer.
8. The brain glioma postoperative recurrence risk assessment kit according to claim 3, wherein: the kit detects lactate dehydrogenase A positive extracellular vesicle LDHA in biological samples + The number, concentration and/or proportion of EVs are used to determine the risk of recurrence after glioma surgery.
9. The brain glioma postoperative recurrence risk assessment kit according to claim 8, wherein: extracellular vesicle LDHA when lactate dehydrogenase A-positive + When the number, concentration and/or proportion of EVs are high in Yu Yushe reference intervals, the postoperative recurrence risk of patients with brain glioma is high, and the prognosis is poor; extracellular vesicle LDHA when lactate dehydrogenase A-positive + When the number, concentration and proportion of EVs are lower than the preset reference interval, the postoperative recurrence risk of the brain glioma patient is low, and the prognosis is good; the preset reference interval is a numerical range of the number, concentration and proportion of healthy subjects/patients with the brain glioma without recurrence.
10. Postoperative recovery of brain gliomaA method of risk assessment for non-disease detection or treatment purposes, characterized by: the method is carried out by determining lactate dehydrogenase A positive extracellular vesicle LDHA + The number, concentration and/or proportion of EVs are evaluated for the risk of postoperative recurrence in glioma patients.
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