CN114231615A - Application of miR-5112 as acute ischemic stroke diagnostic biomarker - Google Patents
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Abstract
The invention provides application of miR-5112 as an acute ischemic stroke diagnostic biomarker, and particularly relates to application of miR-5112 as a diagnostic biomarker in preparation of a detection reagent for acute ischemic stroke and a diagnostic kit or a diagnostic preparation. The miR-5112 disclosed by the invention is highly expressed in plasma and exosomes of patients suffering from acute ischemic stroke after the acute ischemic stroke is developed. In addition, the ROC curve analysis shows that the expression level of miR-5112 has high sensitivity and specificity on acute ischemic stroke diagnosis. Therefore, miR-5112 can be used as a biomarker for diagnosing and identifying acute ischemic stroke, has the characteristics of good specificity and high sensitivity, and has good clinical application value for acute ischemic stroke diagnosis.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of miR-5112 as a diagnostic biomarker of acute ischemic stroke.
Background
Acute Ischemic Stroke (AIS) refers to a type of clinical syndrome with corresponding neurological deficit caused by ischemia and hypoxic necrosis of local brain tissues due to 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. Current clinical treatment guidelines recommend that intravenous thrombolytic therapy be administered based on clinical and cerebral CT options for patients who meet the indications within 4.5 hours of the onset of cerebral infarction, but cerebral CT is not able to identify cerebral infarction within 24 hours and can only rule out cerebral hemorrhage. Although the diagnosis of acute ischemic stroke by cranial magnetic resonance is accurate, the equipment and examination cost is high, the examination time is long, and the treatment time is delayed. At present, biomarkers for diagnosing acute ischemic stroke are lacked, which brings great obstacle to early diagnosis of acute ischemic stroke.
MicroRNA (miRNA) is a non-coding single-stranded RNA molecule of about 20-22 nucleotides in length encoded by an endogenous gene. MicroRNA is rich in content and widely expressed in various tissue cells, and mediates a plurality of important pathophysiological functions such as gene silencing and the like through combination with circular RNA, long-chain non-coding RNA and the like. In earlier experiments, circular RNA OGDH (circular RNA OGDH) generated by transcription of ketoglutarate Dehydrogenase (OGDH) gene is obtained by performing combined reanalysis on MCAO mouse brain tissue second-generation sequencing data and blood chip data and screening, and further, experiments show that the circular RNA OGDH can be transferred to peripheral blood by combining miR-5112. Studies have now demonstrated that mirnas have immeasurable potential value as diagnostic biomarkers for acute ischemic stroke. Therefore, the search of the plasma miRNA with good specificity and high sensitivity as the diagnostic marker of the acute ischemic stroke has great significance.
Disclosure of Invention
In view of the problem that the existing acute ischemic stroke lacks biomarkers to assist diagnosis, the invention provides application of miR-5112 as a biomarker for acute ischemic stroke diagnosis.
One of the purposes of the invention is the use of miR-5112 in the preparation of a diagnostic biomarker for acute ischemic stroke.
The invention also aims to provide application of miR-5112 as a diagnostic biomarker in preparation of a detection reagent for acute ischemic stroke.
Further, the miR-5112 is miR-5112 in serum or plasma.
Further, the nucleotide sequence of the miR-5112 which is murine mmu _ miR-5112 is shown in SEQ ID NO. 1. The miR-5112 is the code number of a miRbase database, the specific code number is mmu _ miR _5112, the length of the nucleotide sequence is 19bp, and the specific sequence is SEQ ID NO 15 '-: UAGCUCAGCGGGAGAGCAC-3'. miR-5112 can be detected in human and murine samples and has better conservation, but the human miR-5112 is not recorded in a 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 miR-5112.
Further, the primer of the reagent is a primer for detecting the murine miR-5112 or a primer for detecting the human miR-5112, and the primer of the murine miR-5112 and the primer of the human miR-5112 are the same primer:
an upstream primer miR _ 5112-F5'-TAGCTCAGCGGGAGAGCAC-3', which is shown in SEQ ID NO. 2;
and (3) detecting the used internal reference primers:
an upstream primer U6(human-F) 5'-CTCGCTTCGGCAGCACA-3' shown as SEQ ID NO. 3;
a downstream primer U6(human-R) 5'-AACGCTTCACGAATTTGCGT-3' shown as SEQ ID NO. 4;
an upstream primer U6(mouse-F) 5'-CGCTTCGGCAGCACATATAC-3' shown as SEQ ID NO. 5;
the downstream primer U6(mouse-R) 5'-AAATATGGAACGCTTCACGA-3' is shown as SEQ ID NO. 6.
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 Actin, beta-Actin or GAPDH as internal reference.
The diagnostic kit or diagnostic formulation of the invention may also contain other auxiliary 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.
The miR-5112 disclosed by the invention is highly expressed in plasma and exosomes of patients suffering from acute ischemic stroke after the acute ischemic stroke is developed. In addition, the ROC curve analysis shows that the expression level of miR-5112 has high sensitivity and specificity on acute ischemic stroke diagnosis. Therefore, miR-5112 can be used as a biomarker for diagnosing and identifying acute ischemic stroke, has the characteristics of good specificity and high sensitivity, and has good clinical application value for acute ischemic stroke diagnosis.
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FIG. 1 shows agarose electrophoresis detection of miR-5112 expression level in human neuron and patient plasma. miR-5112 is expressed in human neuronal cells, as well as in plasma of non-cerebrovascular disease control (NCD) and Acute Ischemic Stroke (AIS) patients;
FIG. 2 shows the expression level of miR-5112 in plasma of patients with acute ischemic stroke. Wherein: A) expression levels of miR-5112 in NCD (n ═ 8) and AIS patients (n ═ 25). Data are expressed as mean ± standard deviation, n ═ 3.*P<0.05, two-tailed t-test. B) The ROC curve of miR-5112 in acute ischemic stroke diagnosis has the area AUC under the curve being 0.925, the sensitivity being 80% and the specificity being 87.5%;
FIG. 3 is an identification of plasma exosomes. Wherein: A) schematic diagram of plasma exosome extraction by polymer precipitation method. B) Detecting the plasma exosome particle size of an acute ischemic stroke patient. C) Detecting the expression levels of plasma exosome protein markers CD63, CD9, TSG101 and HSP70 of a patient by a Western blot experiment, and taking a HEK293T cell lysate as a control;
FIG. 4 shows the expression level of miR-5112 in plasma exosomes of patients with acute ischemic stroke. Expression levels of miR-5112 in plasma exosomes from non-cerebrovascular disease control (NCD, n ═ 7) and acute ischemic stroke (AIS, n ═ 9) patients. Data are expressed as mean ± standard deviation.*P<0.05, two-tailed t-test.
Detailed Description
The design, positive sample validation and result analysis of the present invention are further illustrated below 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 detection of expression level of miR-5112 in plasma of acute ischemic stroke patient
1. Extracting sample RNA and verifying miR-5112 expression quantity in sample by PCR
(1) Extracting plasma sample RNA of human neuron, non-cerebrovascular disease (NCD) and Acute Ischemic Stroke (AIS) patients by using Trizol method;
(2) performing reverse transcription reaction on a plasma RNA sample by using a miRcute enhanced miRNA cDNA first strand synthesis kit (TIANGEN, product number KR211-02) to obtain cDNA after reaction;
(3) carrying out PCR reaction according to the instruction of a MIRcute enhanced miRNA fluorescent quantitative detection kit (TIANGEN, product number FP 411-02);
(4) PCR products are obtained through PCR reaction, and the expression level of miR-5112 in human neuron and patient plasma is detected through agarose electrophoresis (figure 1).
2. Detecting expression level of miR-5112 in plasma of acute ischemic stroke patient
And carrying out PCR reaction according to a MIRcute enhanced miRNA fluorescent quantitative detection kit (TIANGEN, product number FP 411-02).
If the real-time fluorescence quantitative PCR amplification product dissolution curve is a single peak, the amplification product is unique and is specifically amplified; further calculate relative quantification of PCR for comparison between groups, formula: expression Fold Change 2-ΔΔCT，2-ΔΔCT＝(CT miR-5112–CT U6)Sample A-(CT miR-5112–CT U6)Sample B.
Expression levels of miR-5112 in plasma samples after onset of 8 non-cerebrovascular disease control (NCD) and 25 Acute Ischemic Stroke (AIS) patients were examined by RT-qPCR, and miR-5112 was found to be highly expressed (P <0.05) (FIG. 2A). We further evaluated miR-5112 as a ROC curve for its diagnostic value in acute ischemic stroke, with an area under the curve, AUC, of 0.925, a sensitivity of 80% and a specificity of 87.5% at the maximum of the john index (fig. 2B).
3. Detecting the level of miR-5112 of plasma exosome of acute ischemic stroke patient
3.1. Plasma exosome of the patients was extracted using plasma exosome extraction kit (SBI), and exosome particle size was extracted by nanosight assay, and exosome was identified using exosome protein markers CD63, CD9, TSG101, HSP70 (fig. 3A-C).
3.2. And detecting the expression level of miR-5112 in the plasma exosomes of the patient by using a MIRcute enhanced miRNA cDNA first strand synthesis kit and a MIRcute enhanced miRNA fluorescence quantitative detection kit. miR-5112 was found to be significantly highly expressed in plasma exosomes of AIS patients (FIG. 4).
According to the invention, miR-5112 is highly expressed in plasma and exosome of an acute ischemic stroke patient after ischemic stroke is caused. In addition, the ROC curve analysis shows that the expression level of miR-5112 has high sensitivity and specificity on acute ischemic stroke diagnosis. Therefore, miR-5112 can be used as a biomarker for diagnosing and identifying acute ischemic stroke, has the characteristics of good specificity and high sensitivity, and has good clinical application value for acute ischemic stroke diagnosis.
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
<110> river south university subsidiary first hospital (Guangzhou Chinese hospital)
<120> application of miR-5112 as acute ischemic stroke diagnostic biomarker
<141> 2021-12-29
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
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uagcucagcg ggagagcac 19
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<213> Artificial sequence (Artificial sequence)
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tagctcagcg ggagagcac 19
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<213> Artificial sequence (Artificial sequence)
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ctcgcttcgg cagcaca 17
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<213> Artificial sequence (Artificial sequence)
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<213> Artificial sequence (Artificial sequence)
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Claims (6)
- Use of miR-5112 in the preparation of a diagnostic biomarker for acute ischemic stroke.
- Application of miR-5112 as a diagnostic biomarker in preparation of a detection reagent for acute ischemic stroke.
- 3. Use according to claim 1 or 2, characterized in that: the miR-5112 is miR-5112 in serum or plasma.
- 4. Use according to claim 1 or 2, characterized in that: the nucleotide sequence of the miR-5112 which is murine mmu _ miR-5112 is shown in SEQ ID NO 1.
- 5. A diagnostic kit or diagnostic preparation for acute ischemic stroke, characterized in that: comprises a reagent for measuring the expression quantity of miR-5112.
- 6. The diagnostic kit or diagnostic formulation of claim 5, wherein: the primer of the reagent is a primer for detecting the mouse miR-5112 or a primer for detecting the human miR-5112, and the primer of the mouse miR-5112 and the primer of the human miR-5112 are the same primer:an upstream primer miR _ 5112-F5'-TAGCTCAGCGGGAGAGCAC-3', which is shown in SEQ ID NO. 2;and (3) detecting the used internal reference primers:an upstream primer U6(human-F) 5'-CTCGCTTCGGCAGCACA-3' shown as SEQ ID NO. 3;a downstream primer U6(human-R) 5'-AACGCTTCACGAATTTGCGT-3' shown as SEQ ID NO. 4;an upstream primer U6(mouse-F) 5'-CGCTTCGGCAGCACATATAC-3' shown as SEQ ID NO. 5;the downstream primer U6(mouse-R) 5'-AAATATGGAACGCTTCACGA-3' is shown as SEQ ID NO. 6.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105441550A (en) * | 2015-12-25 | 2016-03-30 | 南昌大学第一附属医院 | Early biomarker for auxiliary diagnosis of acute ischemic stroke, and gene chip and real-time fluorescent quantitative PCR (polymerase chain reaction) evaluation methods of early biomarker |
| CN111534584A (en) * | 2020-06-10 | 2020-08-14 | 南通大学 | Application of serum exosome miR-410-3p as acute cerebral infarction diagnosis marker and detection method thereof |
| CN112111570A (en) * | 2020-09-11 | 2020-12-22 | 武汉科技大学 | Serum miRNA marker related to auxiliary diagnosis of ischemic stroke and application thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105441550A (en) * | 2015-12-25 | 2016-03-30 | 南昌大学第一附属医院 | Early biomarker for auxiliary diagnosis of acute ischemic stroke, and gene chip and real-time fluorescent quantitative PCR (polymerase chain reaction) evaluation methods of early biomarker |
| CN111534584A (en) * | 2020-06-10 | 2020-08-14 | 南通大学 | Application of serum exosome miR-410-3p as acute cerebral infarction diagnosis marker and detection method thereof |
| CN112111570A (en) * | 2020-09-11 | 2020-12-22 | 武汉科技大学 | Serum miRNA marker related to auxiliary diagnosis of ischemic stroke and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| KUMAR V等: "Differential Expression of microRNAs Correlates With the Severity of Experimental Autoimmune Cystitis", 《FRONT IMMUNOL》 * |
| 刘燕芳: "circOGDH结合miR-5112调控缺血半暗带神经元损伤的机制研究", 《中国优秀博硕士学位论文全文数据库(硕士)》 * |
| 康喜龙: "MicroRNA-5112靶向IKKγ调控NF-κB炎性通路的功能研究", 《中国优秀博硕士学位论文全文数据库(博士)农业科技辑》 * |
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