Biomarker and application thereof in cerebral infarction diagnosis

Abstract

The invention provides a biomarker and application thereof in cerebral infarction diagnosis. The expression level of the hsa-miR-450a-5p in blood of a cerebral infarction patient is remarkably higher than that of a healthy population, the area under an AUC curve is calculated and analyzed to be 0.945, the diagnosis sensitivity of the hsa-miR-450a-5p can reach 85.5%, and the specificity reaches up to 90.8%. The hsa-miR-450a-5p gene has a good application prospect in the diagnosis of the cerebral infarction patient, and provides reference for the early diagnosis of the cerebral infarction patient.

Description

Technical Field

[0001] The present invention relates to the technical field of auxiliary diagnosis of cerebral infarction, and in particular to a biomarker and its application in diagnosis of cerebral infarction. Background Art

[0002] Cerebral infarction, also known as ischemic stroke, is a disease of brain tissue necrosis or softening caused by ischemia and hypoxia of local brain tissue due to obstruction of blood supply to the brain. Causes include atherosclerosis of intracranial and cervical arteries, cardiogenic embolism, and high-risk factors such as hypertension, heart disease, diabetes and dyslipidemia. Middle-aged and elderly people are particularly susceptible. Common symptoms of cerebral infarction include hemiplegia, sensory impairment, aphasia, etc. The specific symptoms depend on the site and area of ​​infarction. The disease has an acute onset and severe symptoms. Once related symptoms appear, medical treatment should be sought immediately. Cerebral infarction mainly formulates treatment plans for acute causes, including drug therapy, surgical treatment, interventional treatment, etc., as well as active treatment of the primary disease, such as controlling blood pressure and blood sugar. Cerebral infarction cannot heal itself, and the underlying causes such as chronic diseases such as atherosclerosis cannot be cured, and cerebral infarction has a risk of recurrence. Therefore, early diagnosis of cerebral infarction is very necessary for the prevention and treatment of cerebral infarction.

[0003] MiRNAs are a class of non-coding RNA molecules with a length of about 18-22 nucleotides encoded by endogenous genes. MiRNAs regulate gene expression at the post-transcriptional level by targeting the untranslated region of 3'-mRNA transcripts, and play an important role in a variety of physiological and pathological processes. Many studies have shown that miRNAs can be detected in a variety of body fluids, such as plasma, serum, urine, and cerebrospinal fluid, and it is generally believed that miRNAs released by damaged cells or circulating cells cause increased expression of serum miRNAs. Based on the stability of miRNAs in body fluids, the tissue specificity of miRNAs, and the relatively convenient quantitative detection of miRNAs by methods such as real-time PCR and microarrays, people have shown great interest in using circulating miRNAs as clinical biomarkers.

[0004] Currently, there are no reliable miRNAs that can be used as diagnostic markers for cerebral infarction. Therefore, there is an urgent need to develop new, reliable, non-invasive early diagnostic markers to promote early intervention and treatment of cerebral infarction and prolong the survival of patients with cerebral infarction. Summary of the invention

[0005] In view of the technical problems existing in the prior art, the present invention provides a biomarker and its application in the diagnosis of cerebral infarction. The present invention uses hsa-miR-450a-5p as an auxiliary diagnostic marker for cerebral infarction, and can quickly realize auxiliary detection of cerebral infarction by analyzing its expression level, providing a reference for early diagnosis and early intervention treatment of patients with cerebral infarction.

[0006] One of the objects of the present invention is to provide the use of hsa-miR-450a-5p as a diagnostic marker in the preparation of reagents for auxiliary diagnosis and / or evaluation of cerebral infarction, and the nucleotide sequence of the hsa-miR-450a-5p is shown in SEQ ID No. 1.

[0007] Preferably, the expression level of hsa-miR-450a-5p in patients with cerebral infarction is detected to assist in the diagnosis and / or assessment of cerebral infarction.

[0008] One of the purposes of the present invention is to provide a kit for assisting in the diagnosis and / or assessment of cerebral infarction, wherein the kit contains primers for specifically amplifying hsa-miR-450a-5p.

[0009] Preferably, the primers for specifically amplifying hsa-miR-450a-5p are: the reverse transcription primer sequence is SEQ ID No. 2, the upstream primer sequence is SEQ ID No. 3, and the downstream primer sequence is SEQ ID No. 4.

[0010] Further preferably, the test sample in the kit is blood.

[0011] One of the purposes of the present invention is to use hsa-miR-450a-5p in preparing a kit for assisting in the diagnosis and / or assessment of cerebral infarction.

[0012] One of the purposes of the present invention is to use hsa-miR-450a-5p as a therapeutic target in the preparation of a drug for treating cerebral infarction.

[0013] Preferably, an inhibitor of hsa-miR-450a-5p is used in the preparation of a drug for treating cerebral infarction, wherein the drug uses hsa-miR-450a-5p as a therapeutic target and is used to knock out or reduce the expression level of the gene.

[0014] Preferably, the inhibitor comprises any one or more of the hexapeptides of SEQ ID No. 5-6.

[0015] One of the purposes of the present invention is to provide a therapeutic drug for cerebral infarction, wherein the drug takes hsa-miR-450a-5p as a therapeutic target and is used to knock out or reduce the expression level of the gene.

[0016] Preferably, the drug comprises any one or more of the hexapeptides of SEQ ID No. 5-6.

[0017] Preferably, the drug comprises a pharmaceutically acceptable carrier or excipient.

[0018] The advantages of the present invention are as follows: The present invention found that the expression level of hsa-miR-450a-5p in the blood of patients with cerebral infarction was significantly higher than that of healthy people, and the area under the AUC curve was calculated to be 0.945, the diagnostic sensitivity of hsa-miR-450a-5p could reach 85.5%, and the specificity was as high as 90.8%. It is confirmed that the hsa-miR-450a-5p gene has a good application prospect in the diagnosis of patients with cerebral infarction, providing a reference for the early diagnosis of patients with cerebral infarction. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 . Expression of hsa-miR-450a-5p in patients with cerebral infarction; Figure 2 . Analysis of the diagnostic value of hsa-miR-450a-5p; Figure 3 .hsa-miR-450a-5p inhibitor screening results. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0021] The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the protection scope of the present invention.

[0022] In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0023] Example 1 Analysis of the expression of hsa-miR-450a-5p in patients with cerebral infarction 1. Preparation of experimental materials: All test subjects were patients diagnosed with cerebral infarction by clinicians from January to December 2023. Blood samples from 20 patients were collected for backup; blood samples from 20 healthy people were collected as controls during the same period.

[0024] 2. Test methods 1) Blood total RNA extraction: The total RNA of blood samples from patients and healthy subjects was extracted using the MolPure® Blood RNA kit and stored at -20°C for future use; 2) Reverse transcription cDNA synthesis: The total RNA in step 1) was reverse transcribed using a reverse transcription kit PrimeScript RT reagent Kit to obtain cDNA, which was stored at -20°C for future use; wherein the reverse transcription primer sequence is shown in SEQ ID NO.2.

[0025] 3) Real-time quantitative PCR: Specific primers were designed based on the nucleic acid sequence of hsa-miR-450a-5p (SEQ ID NO.1), wherein the upstream primer and downstream primer of hsa-miR-450a-5p were SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0026] The 50μL PCR reaction system included: 27μL 10×PCR Buffer, 4μL SYBR Green I fluorescent dye, 8μL dNTP, 2μL each of the upstream and downstream primers of hsa-miR-450a-5p, 2μL Taq DNA polymerase and 5μL cDNA; the PCR reaction program was 94℃ pre-denaturation for 5min; 94℃ 60s, 55℃ 45s, 72℃ extension for 60s, for a total of 35 cycles; 72℃ extension for 10min; and storage at 4℃.

[0027] The specificity of the reaction was determined based on the melting curve, and the formula 2 -ΔΔCt The relative expression of hsa-miR-450a-5p was calculated and the results are shown in Figure 1 .Depend on Figure 1 It can be seen that compared with healthy people, the expression level of hsa-miR-450a-5p detected in 20 pairs of blood samples of patients with cerebral infarction was significantly upregulated.

[0028] Example 2 Analysis of the diagnostic value of hsa-miR-450a-5p in patients with cerebral infarction 1. Preparation of experimental materials: All the test subjects were patients diagnosed with cerebral infarction by clinicians from January to May 2024. Blood samples from 10 patients were collected for backup; blood samples from 10 healthy people were collected as controls during the same period.

[0029] 2. Test methods The method and steps were the same as in Example 1, and the sensitivity and specificity were calculated by AUC curve area. Figure 2 As shown in the figure, the area under the AUC curve of hsa-miR-450a-5p was 0.945, and its diagnostic sensitivity could reach 85.5%, and its specificity was as high as 90.8%. The experimental data analysis further confirmed that hsa-miR-450a-5p has good application prospects in the diagnosis of cerebral infarction patients.

[0030] Example 3 Screening of hsa-miR-450a-5p inhibitors C57BL / 6J male mice weighing about 30g and aged 18-20 weeks were selected, and the mouse cerebral infarction model was established by the mouse middle cerebral artery occlusion reperfusion (MCAO / R, occlusion 1.5h, reperfusion 24h). The mouse cerebral vascular endothelial cells in this model were isolated as the research object, and the artificially synthesized random hexapeptide library was used as a potential screening inhibitor. 10ng / mL hexapeptide was added to the mouse cerebral vascular endothelial cells. After incubation for 12h, the expression level of hsa-miR-450a-5p in the mouse cerebral vascular endothelial cells was detected by real-time quantitative PCR. When the expression level of hsa-miR-450a-5p decreased compared with the control group, the added hexapeptide was an hsa-miR-450a-5p inhibitor. Two hexapeptides that inhibited the expression of hsa-miR-450a-5p were identified by this method, and their amino acid sequences are shown in SEQ ID NO.5-6. The hexapeptide can effectively inhibit the expression of hsa-miR-450a-5p in cells (e.g. Figure 3 It has potential value in treating cerebral infarction.

[0031] The above embodiments are intended to illustrate the essential content of the present invention, but are not intended to limit the protection scope of the present invention. Those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and protection scope of the technical solution of the present invention.

Claims

1. Use of hsa-miR-450a-5p as a diagnostic marker in the preparation of a reagent for auxiliary diagnosis and / or evaluation of cerebral infarction, characterized in that: The nucleotide sequence of the hsa-miR-450a-5p is shown in SEQ ID No.

1.

2. The use according to claim 1, characterized in that The method is to detect the expression level of hsa-miR-450a-5p in patients with cerebral infarction to assist in the diagnosis and / or evaluation of cerebral infarction.

3. A kit for assisting diagnosis and / or evaluation of cerebral infarction, characterized in that: Contains primers that specifically amplify hsa-miR-450a-5p.

4. The kit according to claim 3, characterized in that The primers for specifically amplifying hsa-miR-450a-5p are: the upstream primer sequence is SEQ ID No. 2, and the downstream primer sequence is SEQ ID No.

3.

5. The kit according to claim 3, characterized in that The test sample in the kit is blood.

6. Use of hsa-miR-450a-5p in the preparation of a kit for assisting in the diagnosis and / or assessment of cerebral infarction.

7. Application of hsa-miR-450a-5p as a therapeutic target in the preparation of drugs for the treatment of cerebral infarction.

8. Use of an inhibitor of hsa-miR-450a-5p in the preparation of a drug for treating cerebral infarction, wherein the drug uses hsa-miR-450a-5p as a therapeutic target and is used to knock out or reduce the expression level of the gene.

9. A drug for treating cerebral infarction, characterized in that: The drug takes hsa-miR-450a-5p as a therapeutic target and is used to knock out or reduce the expression level of the gene.

10. The therapeutic drug according to claim 9, characterized in that The medicine includes a pharmaceutically acceptable carrier or excipient.

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