CN116904573A - Plasma exosome marker of OSA, screening method and application thereof - Google Patents
Plasma exosome marker of OSA, screening method and application thereof Download PDFInfo
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- 210000001808 exosome Anatomy 0.000 title claims abstract description 30
- 239000003550 marker Substances 0.000 title claims abstract description 21
- 238000012216 screening Methods 0.000 title claims abstract description 16
- 238000000034 method Methods 0.000 title claims abstract description 12
- 239000000090 biomarker Substances 0.000 claims abstract description 17
- 230000014509 gene expression Effects 0.000 claims abstract description 11
- 238000011156 evaluation Methods 0.000 claims abstract description 7
- 102000017011 Glycated Hemoglobin A Human genes 0.000 claims abstract description 6
- 108010014663 Glycated Hemoglobin A Proteins 0.000 claims abstract description 6
- 238000012165 high-throughput sequencing Methods 0.000 claims abstract description 4
- 238000012163 sequencing technique Methods 0.000 claims description 9
- 238000010219 correlation analysis Methods 0.000 claims description 4
- 239000000091 biomarker candidate Substances 0.000 claims description 3
- 230000009274 differential gene expression Effects 0.000 claims description 3
- 238000010195 expression analysis Methods 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims 1
- 238000003745 diagnosis Methods 0.000 abstract description 10
- 208000001797 obstructive sleep apnea Diseases 0.000 description 33
- 238000004458 analytical method Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 102000039446 nucleic acids Human genes 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 206010041349 Somnolence Diseases 0.000 description 2
- 208000008784 apnea Diseases 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000013211 curve analysis Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 208000007590 Disorders of Excessive Somnolence Diseases 0.000 description 1
- 206010020591 Hypercapnia Diseases 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 206010021079 Hypopnoea Diseases 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 206010041235 Snoring Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 206010003119 arrhythmia Diseases 0.000 description 1
- 230000006793 arrhythmia Effects 0.000 description 1
- 230000006399 behavior Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 208000035850 clinical syndrome Diseases 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000000032 diagnostic agent Substances 0.000 description 1
- 229940039227 diagnostic agent Drugs 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 230000002008 hemorrhagic effect Effects 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000000302 ischemic effect Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
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- 238000004393 prognosis Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 201000002859 sleep apnea Diseases 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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Abstract
The invention discloses a plasma exosome marker of OSA, wherein the marker is ENST00000592016, and a screening method of the plasma exosome marker of OSA comprises the steps of high-throughput sequencing and marker screening and the step of biomarker clinical evaluation. The plasma exosome marker of the OSA screened by the invention has the advantages of stability, high reliability and high association degree, and ENST00000592016 has better diagnosis efficiency when the OSA sample is identified, and the expression of the marker in the OSA is remarkable; the diagnostic value of ENST00000592016 for OSA is superior to the combined diagnosis of CPR and HbA1 c.
Description
Technical Field
The invention relates to the field of medical molecular biology, in particular to a plasma exosome marker of OSA, a screening method and application thereof.
Background
Obstructive Sleep Apnea (OSA), a clinically more common sleep-related respiratory disease, refers to a clinical syndrome in which apnea or hypopnea occurs repeatedly in a sleep state mainly caused by an obstructive cause, and hypoxia, hypercapnia and sleep interruption are caused, so that a body is caused to generate a plurality of pathophysiological changes. Symptoms of OSA patients are daytime sleepiness, drowsiness, inattention, and the like, and nighttime snoring, apnea, abnormal sleep behavior, and the like may occur, and in addition, hypertension, coronary heart disease, arrhythmia, ischemic or hemorrhagic cerebrovascular disease may occur.
Currently, the diagnosis of obstructive sleep apnea relies mainly on Polysomnography (PSG) as a diagnostic tool. However, the complexity of the PSG inspection process greatly restricts the accessibility of the PSG due to factors such as the implementation site and the professional requirements of operators. Therefore, a diagnosis kit is developed, and the diagnosis of the body fluid sample or the blood sample of the patient in vitro by using the kit can greatly shorten the diagnosis of the obstructive sleep apnea syndrome. Currently, medical workers focus on the research work of small extracellular vesicles, and because of the wide sources of exosomes, strong specificity, rich content and unique mechanisms, they can be used for diagnosis, disease progress monitoring and prognosis prediction, and as carriers for certain drugs.
However, the small extracellular vesicles of human body are rich in nucleic acids, which nucleic acid or nucleic acids can be used as biomarkers for OSA diagnosis and how to screen reliable, stable, highly specific OSA biomarkers is a current urgent problem to be solved.
Disclosure of Invention
In order to solve the problems, the invention provides a reliable, stable and high-specificity plasma exosome marker for diagnosing obstructive sleep apnea, which has high association degree with OSA, can be used as a marker for diagnosing OSA, and can be used for early diagnosis of OSA.
A first object of the present invention is to provide plasma exosome markers for OSA.
The plasma exosome marker of the OSA provided by the invention is ENST00000592016.
A second object of the present invention is to provide a method for screening plasma exosome markers for OSA.
A method for screening plasma exosome markers for OSA comprising the steps of:
s1, high-throughput sequencing and marker screening:
s11, dividing the patient into an OSA group, an OSA-HTN group and an NC group;
s12, extracting and identifying sample small extracellular vesicles of 3 groups of patients by a UC method;
s13, performing LncRNA-seq sequencing on the extracted plasma exosomes, and performing differential gene expression analysis on a sequencing result;
s14, verifying a sequencing result by using dd-PCR;
s15, screening out candidate biomarkers;
s16, determining a biomarker for clinical value evaluation of the next stage;
s2, clinical evaluation of biomarkers:
s21, further grouping the patients into an OSA-Mlid group, an OSA-Moderate group, an OSA-quality group and an NC group;
s22, extracting the grouped plasma exosomes, and detecting with CPR and HbA1 c;
s23, comparing the diagnostic abilities of the biomarker and CRP, hbA 1;
s24, correlation analysis of clinical stage and correlation analysis of various clinical indexes.
A third object of the present invention is the use of biomarker ENST00000592016 for the protection of OSA in the preparation of a diagnostic agent or kit for OSA.
The beneficial effects of the invention are as follows: the plasma exosome marker of the OSA screened by the invention has the advantages of stability, high reliability and high association degree, and ENST00000592016 has better diagnosis efficiency when the OSA sample is identified, and the expression of the marker in the OSA is obvious; the diagnostic value of ENST00000592016 for OSA is superior to the combined diagnosis of CPR and HbA1 c.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flow chart of screening for plasma exosome markers for OSA provided by the invention;
FIG. 2 is a graph showing particle size and concentration of plasma exosomes from three groups of patients in step S12 under NTA assay;
FIG. 3 shows the morphology and size of plasma exosomes of three groups of patients under electron microscopy in step S12;
FIG. 4 is a graph showing the expression of plasma exosome marker proteins in WB assays in three groups of patients in step S12;
FIG. 5 is a graph showing the results of statistical analysis of plasma exosome particle concentrations in three groups of patients in step S12;
FIG. 6 is a graph showing comparison of ddPCR detection of three sets of plasma exosome differences lncRANs in step S14;
FIG. 7 is a ROC graph of ENST 00000592016;
FIG. 8 is a graph of clinical indices of the group of patients in step S21;
FIG. 9 is a graph of statistical comparisons of the expression levels of biomarkers of different clinical stages of OSA;
FIG. 10 is a ROC analysis of ENST00000592016 in combination with CPR and HbA1 c;
FIG. 11 is a graph of ENST00000592016 expression correlation with clinical features;
FIG. 12 is an ENST00000592016 stability analysis chart.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Examples
The plasma exosome marker of OSA in this example was ENST00000592016.
Examples
This example combines the screening method of plasma exosome markers that specifically elucidate OSA in clinic, comprising the steps of:
the first step: high throughput sequencing and marker screening:
s11, dividing the selected patients into an OSA group, an OSA-HTN group and an NC group; OSA group 23, OSA-HTN group 15, NC group 25.
S12, the UC method is used for extracting and identifying the small extracellular vesicles of the samples of the 3 groups of patients.
S13, performing LncRNA-seq sequencing on the extracted plasma exosomes, and performing differential gene expression analysis on a sequencing result.
S14, verifying the sequencing result by using dd-PCR, wherein the verification result is shown in FIG. 6.
S15, screening a candidate biomarker ENST00000592016, and performing ROC curve analysis on the biomarker, wherein the effectiveness of ENST00000592016 is 77.27% and the specificity reaches 94.4% after analysis, as shown in FIG. 7.
S16 determined that ENST00000592016 is a biomarker for the next stage of clinical value assessment.
Second step, clinical evaluation of plasma exosome biomarker ENST00000592016 of OSA:
s21, patients are further grouped into an OSA-Mlid group 10 person, an OSA-Moderate group 11 person, an OSA-quality group 10 person and an NC group 10 person, and clinical indexes of the grouped patients are shown in figure 8.
S22, extracting the grouped plasma exosomes, and detecting with CPR and HbA1 c.
S23, dd-PCR measures biomarker expression of OSA.
S24, correlation analysis of clinical stage is shown in FIG. 9.
S25, diagnostic ability evaluation and comparison, ROC curve analysis was performed on ENST00000592016, cpr+hba1c, and ENST00000592016+cpr+hba1c, respectively, and as shown in fig. 10, diagnostic efficacy of ENST00000592016 is auc=0.916 under ROC curve, 95% [ CI:0.823-1], and diagnostic efficacy of cpr+hba1c is auc=0.781, 95% [ CI under ROC curve: 0.597-0.964], ENST00000592016+cpr+hba1c is diagnostic efficacy of auc=0.984, 95% [ CI:0.000-01.000], it is shown that the diagnostic efficacy of ENST00000592016 in combination with CPR+HbA1c is effectively improved.
As shown in fig. 11, expression of ENST00000592016 and BMI (r=0.5222, p<0.01)、ODI(r=0.5047,P<0.01)、AHI (r=0.5636,P<0.05)、TS90(r=0.4598,P<0.01 Positively correlated with LSaO) 2 (r=0.4862,P<0.05 Negative correlation, with the most AHI correlation. From this, it can be seen that ENST00000592016 is BMI, DOI, TS and LSaO 2 The AHI is obviously relevant, and has reference value for OSA risk management.
As shown in fig. 12, stability analysis of ENST 00000592016: the expression level of ENST00000592016 in 10 supernatant and plasma samples was measured and it was found that ENST00000592016 in the supernatant was significantly lower than in the plasma samples, considering that it was mainly encapsulated in the extracted svvs. The reason for the stable presence of ENST00000592016 in plasma may depend on what form it is primarily present in. The expression level of ENST00000592016 was measured in plasma from which the svvs have not been extracted and in the supernatant from which the svvs have been extracted (plasma without svvs), and it was found that the expression was very small in the supernatant, and only trace amounts were detected by ddPCR. It is therefore speculated that ENST00000592016 may exist mainly in plasma mainly in the form of svvs, which also provides a more sufficient basis for supporting the conclusion that it has clinical application value as a biomarker.
In the description of the present specification, the descriptions of the terms "one embodiment," "example," "specific example," and the like, mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
Claims (3)
- A plasma exosome marker of osa, characterized in that the marker is ENST00000592016.
- A method of screening for plasma exosome markers of osa comprising the steps of:s1, high-throughput sequencing and marker screening:s11, dividing the patient into an OSA group, an OSA-HTN group and an NC group;s12, extracting and identifying sample small extracellular vesicles of 3 groups of patients by a UC method;s13, performing LncRNA-seq sequencing on the extracted plasma exosomes, and performing differential gene expression analysis on a sequencing result;s14, verifying a sequencing result by using dd-PCR;s15, screening out candidate biomarkers;s16, determining a biomarker for clinical value evaluation of the next stage;s2, clinical evaluation of biomarkers:s21, further grouping the patients into an OSA-Mlid group, an OSA-Moderate group, an OSA-quality group and an NC group;s22, extracting the grouped plasma exosomes, and detecting with CPR and HbA1 c;s23, dd-PCR is used for determining biomarker expression of OSA;s24, correlation analysis of clinical stage;s25, evaluating and comparing diagnostic capability.
- Use of biomarker ENST00000592016 of OSA in the preparation of an OSA diagnostic reagent or kit.
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