CN114182009B - Application of plasma exosome CircOGDH as acute ischemic stroke diagnostic biomarker - Google Patents

Abstract

The invention provides application of a plasma exosome CircOGDH as a diagnostic biomarker for acute ischemic stroke, and particularly relates to application of the plasma exosome CircOGDH as the diagnostic biomarker in preparation of a detection reagent for acute ischemic stroke and a diagnostic kit or a diagnostic preparation. After acute ischemic stroke occurs, the circular RNA CircOGDH is highly expressed in neurons with ischemia and hypoxia and is transported to peripheral blood from brain tissues through exosomes, so that the expression of the exosome CircOGDH in plasma is increased, and the circular RNA CircOGDH can be used as a diagnostic biomarker for detecting the acute ischemic stroke. In addition, the plasma exosome CircOGDH provided by the invention has higher sensitivity and specificity as a biomarker for diagnosing AIS, and has clinical popularization value.

Description

Application of plasma exosome CircOGDH as acute ischemic stroke diagnostic biomarker

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of a plasma exosome CircOGDH as an acute ischemic stroke diagnostic biomarker.

Background

Acute Ischemic Stroke (AIS), also called cerebral infarction, is a clinical syndrome with corresponding neurological deficit due to ischemia and hypoxic necrosis of local brain tissue caused by sudden interruption of blood supply to the brain caused by various reasons, has high morbidity, and is a main cause of human death and disability. The key point of the acute ischemic stroke treatment is to open blocked blood vessels as soon as possible and save ischemic penumbra. At present, the main treatment means of the venous thrombolysis treatment and the arterial thrombus removal treatment for acute cerebral infarction has more benefits the earlier the treatment time is, but the venous thrombolysis treatment and the arterial thrombus removal treatment both have time windows, and patients beyond the time windows cannot receive the two effective treatment methods. Taking intravenous thrombolytic therapy as an example, current clinical treatment guidelines recommend that intravenous thrombolytic therapy be administered based on clinical and craniocerebral CT options for patients who meet the indications within 4.5 hours of onset of cerebral infarction, but craniocerebral CT is not able to identify cerebral infarction within 24 hours and can only rule out cerebral hemorrhage. Currently, when a clinician diagnoses the condition of a patient according to clinical diagnosis and craniocerebral CT, hysteria, transient Ischemic Attacks (TIA), todd paralysis of epilepsy and the like are often misdiagnosed as cerebral infarction, so that intravenous thrombolysis treatment is selected, and the risk of bleeding and the economic burden of the patient are increased. And in many remote hospitals, intravenous thrombolytic therapy cannot be performed due to the lack of imaging equipment needed to diagnose cerebral infarction. Therefore, there is a need for a convenient and rapid biomarker that can assist in diagnosing AIS in clinical practice.

Circular RNA (circRNA) is a class of non-coding RNA molecules with a special closed circular structure. Due to its closed covalent structure and resistance to exonuclease, circRNA is stably expressed and less susceptible to degradation by exonuclease than other linear RNAs. In addition, the base sequence and expression of circRNAs are rather conserved among species, and these characteristics make circular RNAs have significant advantages as novel clinical diagnostic markers. circRNAs are highly enriched in brain synapses and are now found to be potentially useful as biomarkers in a variety of neurological disorders. Exosomes refer to nanoscale extracellular vesicles released from various cells, generally in the form of cup-shaped circular structures with diameters of about 40-150nm. Exosomes are present in almost all body fluids, including plasma, cerebrospinal fluid, etc., and are rich in many biologically active molecules, such as non-coding RNAs, proteins, lipids, etc. It has been reported that circRNA is present in high amounts in exosomes, that it is at least twice enriched in exosomes compared to circRNA retained in cells, and that exosomes can cross the blood brain barrier. These lines of evidence allow circular RNA in exosomes to serve as a biomarker with great potential for central nervous system or other diseases. In the previous experiments, cyclic RNA OGDH (circular RNA OGDH) generated by transcription of ketoglutarate Dehydrogenase (OGDH) gene is obtained by performing combined reanalysis on MCAO mouse brain tissue secondary sequencing data and blood chip data and screening. And further experiments show that the CircOGDH is mainly highly expressed in the cytoplasm of an ischemic penumbra neuron and can be transferred to peripheral blood through an exosome pathway, so that the high expression of the CircOGDH in a plasma exosome is caused. At present, the correlation between exosome CircOGDH and acute ischemic stroke pathological changes and the application thereof are not reported.

Disclosure of Invention

In view of the problem that the existing acute ischemic stroke lacks a biomarker to assist diagnosis, the invention provides application of a plasma exosome CircOGDH as a diagnostic biomarker of acute ischemic stroke.

One of the objects of the present invention is the use of plasma exosome CircOGDH for the preparation of a diagnostic biomarker of acute ischemic stroke.

The invention also aims to provide application of plasma exosome CircOGDH as a diagnosis biomarker in preparation of a detection reagent for acute ischemic stroke.

Furthermore, the plasma exosome CircOGDH is circular RNA CircOGDH, and comprises a nucleotide sequence shown in SEQ ID NO.1 of murine CircOGDH mmu _ circ _0000231, a corresponding parent gene of the murine CircOGDH is positioned at chr11:6213790-6217083, and a nucleotide sequence shown in SEQ ID NO. 2 of human CircOGDH hsa _ circ _0003340, and a corresponding parent gene of the murine CircOGDH hsa _ circ _0003340 is positioned at chr7:44684925-44687358. The CircOGDH is the code number of RNA in a related database (such as a circBase database), and can be searched in the database.

The invention also aims to provide a diagnostic kit or a diagnostic preparation for acute ischemic stroke, which comprises a reagent for measuring the expression level of plasma exosome CircOGDH.

Further, the method also comprises a reagent for extracting the exosome by a polymer precipitation method or a reagent for other methods for separating the exosome.

Further, the primers of the reagent comprise a primer for detecting murine CircOGDH mmu _ circ _ 0000231:

an upstream primer: 5 'AACTCGTGGAGGACCATTG-3' as shown in SEQ ID NO. 3;

a downstream primer of 5 'GAGCTTCGACTCAGGGAAAG-3', which is shown as SEQ ID NO. 4;

the internal reference used for detection is actin, and primers for detecting actin are as follows:

an upstream primer: 5 'ACGGCCAGGTCATCACTATTG-3' as shown in SEQ ID NO. 5;

a downstream primer: 5 'CAAGAAGGAAGGCTGGAAAAGA-3' as shown in SEQ ID NO. 6.

Further, the primers of the reagent comprise a primer for detecting human-derived CircOGDH has _ circ _ 0003340:

an upstream primer: 5 'GTCGCTCATCAGGGCATATATC-3', as shown in SEQ ID NO. 7;

a downstream primer: 5 'GCTTCTACCAGGGACTGTGC-3', as shown in SEQ ID NO. 8;

the internal reference used for detection is actin, and primers for detecting actin are as follows:

an upstream primer: 5 'AAGGATTCCTATGTGGGCGAC-3', as shown in SEQ ID NO. 9;

a downstream primer: 5 'CGTACAGGGGATAGCACAGCC-3' as shown in SEQ ID NO. 10.

The diagnostic kit or diagnostic preparation of the invention also comprises common reagents for PCR reaction, such as Taq enzyme, reverse transcriptase, buffer solution, dNTPs, mgCl2, DEPC water and the like; may also contain standard substance and/or positive and negative control substance; the invention can also select beta-Actin or GAPDH as internal reference.

The diagnostic kit or diagnostic formulation of the invention may also contain other supplementary consumables, which are well known to those skilled in the art. Such reagents or consumables include, but are not limited to: fluorescent quantitative PCR reaction plate, sealing film of PCR reaction plate, etc.

After acute ischemic stroke happens, the circular RNA CircOGDH has high expression in neurons with ischemia and hypoxia, and is transported to peripheral blood from brain tissues through exosomes, so that the expression of the exosomes CircOGDH in plasma is increased, and the circular RNA CircOGDH can be used as a diagnostic biomarker for detecting the acute ischemic stroke. In addition, the plasma exosome CircOGDH provided by the invention has higher sensitivity and specificity as a biomarker for diagnosing acute ischemic stroke, and has clinical popularization value.

Drawings

FIG. 1 shows that knocking down MCAO model mouse brain tissue CircOGDH leads to the down-regulation of serum CircOGDH expression. Wherein: a) Mouse experiment design flow chart. b) Mice were divided into SHAM + sh _ CircCtrl group, MCAO + shExperiments were performed in the _ CircCtrl group and the MCAO + sh _ CircoGDH group, and the expression level of CircoGDH in serum of each group of mice was analyzed by RT-qPCR. Data are expressed as mean ± sd, and the number of mice in the three groups is 4, 5, and 6. ^** P<Comparing the 0.01MCAO + sh CircCtrl group and the SHAM + sh _ CircCtrl group, ^&&& P<comparing the 0.001MCAO + sh circOGDH group and the MCAO + sh _ circCtrl group, and carrying out single-factor analysis and variance analysis test;

fig. 2 is an identification of primary neuronal exosomes. Wherein: a) And observing the form of the primary neuron exosomes under a transmission electron microscope. The scale is 100nm. b) The size of the distribution of exosomes in normoxic group and ischemia-hypoxia reperfusion group was evaluated by nanoparticle tracking analysis. c) Western blot images showed the relative expression levels of the exosome protein markers (CD 63, TSG101 and HSP 90) and the endoplasmic reticulum marker protein Calnexin in neuronal cells and neuronal exosomes;

FIG. 3 shows high expression of the neuron exosome, circOGDH. And analyzing the expression level of the CircOGDH in the control neuron exosome and the hypoxia reperfusion neuron exosome by using an RT-qPCR method. Data are presented as mean ± standard deviation of 3 independent experiments. ^** P<0.01, double-tail t test;

FIG. 4 shows the extraction and identification of plasma exosomes. Wherein: a) And observing the plasma exosome form of the control group of subjects under a transmission electron microscope. The scale is 100nm. b) Assessing the size of the distribution of exosomes of NCD control subjects by nanoparticle tracking analysis;

FIG. 5 shows increased expression of CircoGDH in plasma exosomes of AIS patients. Wherein: a) Detecting the expression level of CircOGDH by an RT-qPCR method in an Acute Ischemic Stroke (AIS) group and a non-cerebrovascular disease control group (NCD) plasma exosome, wherein the non-cerebrovascular disease control group (NCD) n =30, the AIS group n =31, ^** P<0.01, two-tailed t-test. b) ROC curve of plasma exosome CircOGDH expression levels within 24 hours of AIS onset, NCD control n =30, AIS group n =31. The area under the CircOGDH curve (AUC) was 0.8817, sensitivity was 80.65%, and specificity was 86.67%.

Detailed Description

The following will further illustrate the design, positive sample verification and result analysis of the present invention with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

Example 1 ischemic-hypoxic neurons transport CircOGDH to peripheral blood via exosomes

To verify whether CircOGDH can be transported from ischemic brain tissue to peripheral blood via exosomes, we constructed adenovirus-packaged shRNA that targets the reverse splice site of CircOGDH to knock down its expression and carries GFP fluorescent protein, abbreviated english as ADV _ GFP _ shRNA _ CircOGDH, sh _ CircOGDH for short; negative Control (NC) adenovirus, abbreviated as ADV _ GFP _ shRNA _ NC in English, and abbreviated as sh _ CircCtrl, and experiments prove that the sh _ CircOGDH adenovirus can knock down mouse brain tissue CircOGDH. In addition, we further designed the following experiments: dividing mice into a SHAM (pseudo surgery) + sh _ CircCtrl group, a middle cerebral artery occlusion Model (MCAO) + sh _ CircCtrl group and a MCAO + sh _ CircOGDH group, performing stereotactic injection on the brain tissue of the mice on the 1 st day of an experiment by sh _ CircCtrl or sh _ CircOGDH adenovirus, manufacturing an MCAO model on the 6 th day after virus transfection, collecting the serum of the mice after 3 hours of the model, and detecting the expression level of the CircOGDH in the serum of the MCAO mice by using real-time fluorescent quantitative PCR (RT-qPCR) (figure 1 a), wherein the result shows that the expression level of the CircOGDH in the MCAO + sh _ CircCtrl group is remarkably increased (P < 0.01) compared with that in the SHAM + sh _ CircCtrl group; in the MCAO + sh _ CircOGDH group (CircOGDH knock-down group), the level of CircOGDH in serum was significantly lower (P < 0.001) than in the MCAO + sh _ CircCtrl group (fig. 1 b). The experiment shows that the knockout of the MCAO model mouse brain tissue CircOGDH can reduce the expression quantity of the CircOGDH in serum, and combined with the previous experimental result, the results prove that the CircOGDH is transported from ischemic brain tissue to peripheral blood through an exosome pathway.

Example 2 high expression of exosome, circOGDH, in ischemia hypoxic neurons

2.1 extraction and identification of Primary neuronal exosomes

After culturing the primary neurons to the 5 th day, molding is carried out by dividing the primary neurons into a normoxic group and an ischemia-ischemia (AI)/reperfusion group, and after molding is finished, exosomes are purified from the primary neuron culture medium by using an ultra-high speed centrifugation method. Transmission electron microscopy showed that the average size of exosomes collected from primary neuronal medium was about 100nm (fig. 2 a). The sizes of two groups of primary neuron exosomes were further confirmed by Nanoparticle Tracking Analysis (NTA), with the peak of the normoxic group corresponding to particle size 143nm and the peak of the ischemia-hypoxia reperfusion group corresponding to particle size 150nm, both of which substantially fit the sizes of exosomes (fig. 2 b). Western blot experiments confirmed the presence of CD63, TSG101 and HSP70 exosome protein markers in primary neuronal exosomes (fig. 2 c).

2.2 high expression of ischemia-hypoxia reperfusion Primary neuron exosome CircOGDH

After neuron exosomes are extracted by an ultra-high-speed centrifugation method, the expression level of the neuron exosomes CircOGDH of an normoxic group and an ischemia-hypoxia reperfusion group is detected by real-time fluorescent quantitative PCR (RT-RT-qPCR), and the result shows that the expression level of the neuron exosomes CircOGDH of the ischemia-hypoxia reperfusion group is remarkably improved (P < 0.01) (figure 3).

Example 3 Acute Ischemic Stroke (AIS) patients exosome plasma CircoGDH high expression

3.1 extraction and identification of plasma exosomes

We purified exosomes from plasma of non-cerebrovascular disease control (NCD) subjects using polymer precipitation. Transmission electron microscopy showed that the average size of exosomes collected from patient plasma was approximately 100nm (fig. 4 a). The plasma exosome size from the NCD control subjects was further confirmed by Nanoparticle Tracking Analysis (NTA), and the peak of the NCD control corresponded to a particle size of 126nm, all substantially consistent with the exosome particle size (fig. 4 b).

3.2 increased expression of CircOGDH in plasma exosomes of AIS patients

Exosomes were extracted from the plasma of the study subjects and the expression level of CircOGDH was measured in 31 patients with ischemic stroke and 30 NCD control groups. We used RT-qPCR method to detect the expression level of CircoGDH of plasma exosomes of AIS group and control group, and the result shows that the expression level of the CircoGDH of plasma exosomes of AIS group is obviously higher than that of the control group (P < 0.05) (FIG. 5 a). We performed ROC curves to evaluate the value of plasma exosome CircOGDH expression levels as diagnostic AIS biomarkers within 24 hours of AIS onset, and the results show: area under CircOGDH curve (AUC) 0.8817; at the maximum of the jotan index, the cut-off value was 1.6732, the sensitivity was 80.65% and the specificity was 86.67% (FIG. 5 b).

In review, the patent of the invention shows that after ischemic stroke, the CircOGDH is highly expressed in neurons with ischemia and hypoxia, and can be transported from brain tissues to peripheral blood through exosomes, so that the expression of the CircOGDH in plasma is increased. In addition, the patent of the invention also shows that the plasma exosome CircOGDH has higher sensitivity and specificity as a biomarker for diagnosing AIS.

While the preferred embodiments and examples of the present invention have been described in detail, the present invention is not limited to the embodiments and examples, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art.

Sequence listing

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Claims (1)

1. The application of a reagent for detecting the expression level of human-derived CircOGDH hsa _ circ _0003340 in preparing a detection reagent for acute ischemic stroke is characterized in that: the nucleotide sequence of the humanized CircOGDH hsa _ circ _0003340 is shown as SEQ ID NO. 2, and the corresponding parent gene is positioned at chr7:44684925-44687358;

the primers of the reagent comprise a primer for detecting human-derived CircOGDH hsa _ circ _ 0003340:

an upstream primer: 5 'GTCGCTCATCAGGGCATATATC-3' as shown in SEQ ID NO. 7;

a downstream primer: 5 'GCTTCTACCAGGGACTGTGC-3', as shown in SEQ ID NO. 8;

the internal reference used for detection is actin, and primers for detecting actin are as follows:

an upstream primer: 5 'AAGGATTCCTATGTGGGCGAC-3', as shown in SEQ ID NO. 9;

a downstream primer: 5 'CGTACAGGGGATAGCACAGCC-3' as shown in SEQ ID NO. 10.

Images