CN118531116B - Use of diagnostic markers for high altitude headache - Google Patents

Abstract

The invention belongs to the technical field of molecular biological detection, and discloses application of a diagnosis marker for altitude headache. In particular discloses a miRNA marker hsa-miR-23b-3p related to altitude headache, and also discloses application of hsa-miR-23b-3p in preparation of a kit for diagnosing or assisting in diagnosing altitude headache, and research of a discovery team and a verification team finds that the expression level of hsa-miR-23b-3p is increased and can be used as an assisting diagnosis basis for altitude headache. The invention provides a novel detection marker for the high altitude headache disease, and provides a brand new molecular information and biological basis for diagnosis and treatment of the disease.

Description

Use of diagnostic markers for high altitude headache

Technical Field

The invention belongs to the technical field of molecular biological detection, and in particular relates to the application of a diagnostic marker for plateau headache and a diagnostic kit.

Background Art

High-altitude headache (HAH), also known as plateau headache or high-altitude headache, is defined by the International Headache Society (IHS) as a mild to moderate dull pain or tenderness in the bilateral frontal and temporal regions within 24 hours after people who have lived in low-altitude areas for a long time and have not adapted to the new environment quickly enter a plateau above 2,500 meters above sea level. In geography, a vast area with an altitude of more than 500 meters and relatively flat or undulating terrain is called a plateau. In medicine, a place with an altitude of more than 2,500 meters is called a plateau. The incidence of HAH is high, ranging from 25% to 90% depending on the speed of ascent and the specific altitude (Luks, A.M. and P.H.Hackett, Medical Conditions and High-Altitude Travel. The New England journal of medicine, 2022. 386(4): p. 364-373). The disease is very harmful. It not only seriously affects the daily quality of life of people who suddenly go to high altitudes and reduces their ability to work, but more seriously, if HAH is not effectively controlled and treated, it may even progress to high-altitude pulmonary edema (HAPE) and high-altitude cerebral edema (HACE), which have a high mortality rate (Pham, K., K. Parikhand E.C. Heinrich, Hypoxia and Inflammation: Insights From High-Altitude Physiology. "Frontiers in physiology", 2021. 12: p. 676782).

Although a lot of research has been done on the diagnosis of HAH in recent years, the current diagnosis of HAH at home and abroad is mainly based on the intensity and characteristics of the relevant clinical symptoms exhibited by the patient. According to the second edition of the International Classification of Headache Disorders developed by IHS, the diagnosis of HAH must meet the following four conditions: (1) entering a plateau at an altitude of more than 2,500 meters; (2) headache occurs within 24 hours after arriving at the plateau; (3) headache gradually subsides within 8 hours after descending to a lower altitude; (4) the nature of the headache meets at least 2 of the following 5 items: a. bilateral headache, b. headache location is frontal or frontotemporal, c. pain is dull or tender, d. pain intensity is mild to moderate, and e. headache intensity is aggravated by exertion and movement of the head or body (Headache Classification Subcommittee of the International Headache Society. The international classification of headache disorders: 2nd edition. "Cephalalgia", 2004. 24 Suppl. 1:9-160). In recent years, with the update of the International Classification of Headache Disorders, the diagnosis of HAH has also been updated. According to the third edition of the International Classification of Headache Disorders developed by IHS, the diagnosis of HAH must meet the following conditions: (1) The headache meets criterion 3; (2) The person ascends to an altitude of more than 2500 m above sea level and meets at least 2 of the following 3 items: (a) The onset of headache is temporally related to the altitude increase; (b) One or both of the following 2 items are met: a) The headache significantly worsens with increasing altitude; b) The headache is relieved within 24 hours of leaving the high-altitude environment; (3) The headache meets at least 2 of the following 3 items: (a) Bilateral headache; (b) Mild to moderate headache; (c) Headache is aggravated by exertion, movement, stretching, coughing, and/or bending (Headache Classification Committee of the International Headache Society (IHS). The International Classification of Headache Disorders. 3rd edition, "Cephalalgia", 2018: 1-211). Since the intensity and characteristics of symptoms are highly subjective, finding more reliable objective diagnostic indicators has become another research hotspot in HAH diagnosis.

Summary of the invention

In view of this, the purpose of the present invention is to provide a miRNA marker hsa-miR-23b-3p associated with high altitude headache, as well as related applications and kits, especially to diagnose the onset of high altitude headache by the expression level of hsa-miR-23b-3p in the saliva of the subject.

In order to achieve the above object, the present invention provides the following technical solutions:

1. Use of a reagent for detecting the expression level of hsa-miR-23b-3p in preparing a diagnostic kit for high altitude headache.

Furthermore, the sequence of the hsa-miR-23b-3p is shown in SEQ ID No.3.

In the further described application, the reagent is a reagent for detecting the expression level of hsa-miR-23b-3p in a sample using a high-throughput sequencing method and/or a quantitative PCR method and/or a probe hybridization method.

In the further described application, the primers include a reverse transcription primer for detecting hsa-miR-23b-3p, a specific upstream primer for fluorescence quantitative reaction, and a specific downstream primer.

In the further described application, the sequence of the reverse transcription primer is shown as SEQ ID No.7.

In the further described application, the sample to be detected is plasma or saliva.

2. A diagnostic kit for high altitude headache, comprising a reverse transcription primer that can specifically bind to hsa-miR-23b-3p for reverse transcription.

In the further diagnostic kit, the reverse transcription primer sequence is shown as SEQ ID No.7.

The diagnostic kit further includes primers that specifically bind to the reverse transcription product and perform PCR amplification.

In the further diagnostic kit, the primers include a forward primer and a reverse primer, the forward primer sequence is shown as SEQ ID No.13, and the reverse primer sequence is shown as SEQ ID No.14.

Furthermore, the microRNA reverse transcription reaction system of the diagnostic kit includes, per 10 μl, 1 μl of reverse transcription primer, 2 μl of 5x reverse transcription Buffer, 2 μl of RTase Mix, 3 μl of RNase-free _{H 2} O, and the remaining 2 μl is sample RNA.

Furthermore, the RT reaction temperature parameters of the diagnostic kit are: 42°C for 60 min, 70°C for 10 min.

Furthermore, the diagnostic kit also includes a microRNA qPCR reaction system, and the microRNA qPCR reaction system includes, per 20 μl, 10 μl of SYBR Green Mix, 0.8 μl of miRNA Forward primer, 0.8 μl of miRNA Reverse primer, 6.4 μl of ddH ₂ O, and the rest is 2 μl of RT reaction product.

Furthermore, the reaction conditions of the microRNA qPCR of the diagnostic kit are pre-denaturation at 95°C for 10 min, denaturation at 95°C for 2 s, annealing at 60°C for 20 s, and extension at 70°C for 10 s.

The technical problem to be solved by the present invention is to find one or several microRNA markers that can effectively diagnose whether a patient is HAH, guide the precise treatment and prevention of the disease, and avoid more risks caused by the delay of HAH disease.

The technical solution of the present invention to solve the above problem is: detecting the expression level of hsa-miR-23b-3p in the plasma or saliva of plain people after acutely entering the plateau to determine whether they are diagnosed as HAH patients.

The beneficial effect of the present invention is that the present invention discovered and confirmed the correlation between the expression levels of circulating microRNAs such as hsa-miR-223-5p, hsa-miR-215-5p, hsa-miR-23b-3p or hsa-miR-222-3p in plasma or saliva and the onset of HAH by using the expression levels of circulating microRNAs in saliva of another independent population after acute exposure to plateau, and their expression levels were all upregulated, and P <0.01, that is, there was a very significant statistical difference. The expression of the above target microRNA can be used as a reagent or kit for preparing a diagnosis of high altitude headache. The target microRNAs of the present invention have good diagnostic efficacy for diagnosing HAH, among which the diagnostic efficacy of hsa-miR-223-5p is 0.802, the diagnostic efficacy of hsa-miR-215-5p is 0.780, the diagnostic efficacy of hsa-miR-23-3p is 0.853, and the diagnostic efficacy of hsa-miR-222-3p is 0.868. The diagnostic efficacy of further combining hsa-miR-222-3p and hsa-miR-23-3p is 0.914; the diagnostic efficacy of combining hsa-miR-223-5p, hsa-miR-215-5p and hsa-miR-23-3p is as high as 0.940. When each target microRNA provided by the present invention is used as a diagnostic marker, its detection sensitivity is high and its specificity is strong, which provides a new effective and reliable supplement to the existing laboratory evidence of microRNA as a potential diagnostic biomarker for HAH, and provides a reliable basis for new body fluid detection, thereby guiding disease prevention and treatment and reducing the health risks of plain people after entering the plateau.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

Figure 1 is a volcano plot of the differences in microRNA expression in plasma between HAH patients and healthy people.

Figure 2 is the melting curve of each target microRNA qPCR reaction, where: A: hsa-miR-223-5p; B: hsa-miR-215-5p; C: hsa-miR-23b-3p; D: hsa-miR-222-3p.

Figure 3 is a comparison of the expression levels of each target microRNA in the saliva supernatant of HAH patients and healthy controls, including A: hsa-miR-223-5p; B: hsa-miR-215-5p; C: hsa-miR-23-3p; D: hsa-miR-222-3p; in the figure, HAH is a patient with high altitude headache and Healthy is a healthy control, P <0.05, that is, there is a significant statistical difference, P <0.01, that is, there is a very significant statistical difference.

Figure 4 shows the receiver operating characteristic curves of each target microRNA in the saliva supernatant of HAH patients, including A: hsa-miR-223-5p; B: hsa-miR-215-5p; C: hsa-miR-23-3p; D: hsa-miR-222-3p. The figure shows the ROC curve, area under the curve (AUC), sensitivity, and specificity of HAH patients very well, where AUC reflects the diagnostic efficacy (AUC=0.5, no diagnostic value; 0.5＜AUC＜0.7, very small diagnostic value; 0.7＜AUC＜0.9, quite accurate diagnostic value; 0.9＜AUC＜1, very accurate diagnostic value.

FIG5 is a receiver operating characteristic curve for the combination of hsa-miR-222-3p and hsa-miR-23-3p in salivary supernatants of HAH patients.

FIG6 is a receiver operating characteristic curve for the combination of hsa-miR-223-5p, hsa-miR-215-5p and hsa-miR-23-3p in the salivary supernatant of HAH patients.

DETAILED DESCRIPTION

The preferred embodiment technical scheme of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention. The experimental methods without specifying specific conditions in the embodiments are usually based on conventional conditions or according to the conditions recommended by the manufacturer. The RNA sequences disclosed in the present invention can all be obtained by artificial synthesis methods or other biological methods.

Example 1 Study on the correlation between the expression of MicroRNA in plasma samples and the onset of HAH

First, we compared and analyzed the plasma microRNA expression profiles of 11 HAH patients and 8 healthy controls after acute exposure to high altitude. Then, we detected and compared the expression levels of each microRNA in saliva samples of 37 HAH patients and 35 healthy controls after acute exposure to high altitude. We studied the correlation between each microRNA and the onset of HAH and found sensitive and reliable biological markers for the diagnosis of HAH.

1. Materials

Peripheral blood samples were collected from 11 HAH patients and 8 healthy subjects after they quickly entered the plateau from the plain. HAH patients are people who enter the plateau. According to the international universal diagnostic criteria for HAH, the third edition of the International Classification of Headache Disorders (ICHD-3), HAH and healthy people are distinguished (Wang K. et al., Physiological, hematological and biochemical factors associated with high-altitude headache in young Chinese males following acute exposure at 3700 m. The journal of headache and pain, 2018.19(1): p. 59).

2. Methods:

The plasma collection procedure is to use EDTA-Na anticoagulant tubes to collect 5 mL of fasting peripheral blood in the morning from the above-mentioned people after they quickly enter the plateau from the plains, centrifuge at 3000 rpm, 25°C for 10 minutes, use RNase-free and bacteria-free pipette tips to take the plasma, place it in a sterile enzyme-free EP tube, and store it at -80°C for later use.

Experimental steps in the screening phase:

(1) Extraction and purification of plasma total RNA

500 μl plasma total RNA was extracted using TRIzol LS Reagent (Invitrogen, USA, Catalog No.: 10296010), and then RNA was purified and concentration was measured using RNeasy mini kit (QIAGEN, Germany, Catalog No.: 74106).

(2) Preparation of microRNA fluorescently labeled probes and biochip hybridization reaction

The preparation of fluorescently labeled probes for microRNA was achieved by using miRCURYTM Array Power Labelingkit (Exiqon, Denmark, catalog number: 208032-a). The specific operation steps were carried out according to the instructions. The biochip hybridization experiment used miRCURYTM LNA Array (Exiqon, Denmark, model: v.18.0) and was operated according to the instructions.

(3) Chip image acquisition and analysis

A microarray gene chip scanner (Axon, USA, model: GENEPIX 4000B) was used to scan the biochip signal and collect images. Afterwards, the biochip scanning analysis software GenePix Pro (Axon, USA, model: v.6.0) was used to extract the chip scanning data and perform median normalization on the chip signal. The above operations were performed according to the instrument and software instructions.

(4) Statistical analysis of plasma microRNA expression profile

The R language software package was used to perform statistical analysis on the acquired data, and microRNAs with significant differences between the HAH and healthy controls were screened out (screening criteria: Fold Change ≥ 2, P-value < 0.05). A total of 2091 microRNAs were detected in the screening experiment. After statistical analysis, it was found that some microRNAs including hsa-miR-223-5p in plasma were significantly differently expressed between HAH patients and healthy people. Figure 1 shows the volcano map of the screening results of the differential microRNAs screened between HAH patients and healthy controls. Among them: square points: microRNAs that are significantly upregulated in HAH patients compared with the healthy group; triangle points: microRNAs that are significantly downregulated in HAH patients compared with the healthy group; circular points: microRNAs that are not differently expressed between the two groups; FC: Fold Change, that is, fold difference.

Example 2 Study on the correlation between the expression of microRNA in saliva samples and the onset of HAH

The expression distribution of some microRNAs including hsa-miR-223-5p in HAH patients (37 cases) and healthy controls (35 cases) was detected in another independent population using qPCR technology. During the whole process, RNA from saliva samples was extracted using the microRNA column extraction kit (miRNeasy Serum/Plasma Kit, Catalog No.: 217184) of Qiagen Technology Co., Ltd., Germany, and then the real-time fluorescence quantitative PCR (Bulge-Loop™ miRNA qRT-PCR Starter Kit) method was used to amplify each microRNA and the external reference cel-miR-39; the normalized expression level of each microRNA for each specimen against the external reference cel-miR-39 was calculated, and the results were tested by SPSS 19.0 and MedCalc 19.20. With P < 0.05 as the significance test standard, it was found that there were significant differences in the expression levels of saliva hsa-miR-223-5p, hsa-miR-215-5p, hsa-miR-23-3p and hsa-miR-222-3p between the HAH patient group and the healthy population.

1. Materials and specimen collection description

Saliva specimens of HAH patients were collected from HAH patients who acutely entered the plateau from the plain population, totaling 37 cases. Saliva specimens of normal people were collected from normal healthy people who acutely entered the plateau from the plain population, totaling 35 cases. The diagnosis of HAH was confirmed by the international common diagnostic criteria-the Third Edition of the International Classification of Headache Disorders (ICHD-3). All people did not take any preventive medication before collecting saliva. A total of 5 ml of saliva was collected for each sample using a sterile, enzyme-free 50 ml centrifuge tube. The clinical case characteristics of the patients are shown in Table 1. This study has been reviewed and approved by the Medical Ethics Committee, and the collection of all specimens has obtained the informed consent of the patients.

Table 1 Clinical data of saliva specimens

project Healthy group (n=35) HAH group (n=37) P- value Age (years) 18.0 (1.0) 18.0 (1.0) 0.281 Gender (male/female, number of people) 35/0 37/0 Height (cm) 170.0 (7.0) 172.0 (9.0) 0.982 Weight (kg) 61.8±9.6 60.0 (1.0) 0.968 Body mass index (kg/m ² ) 20.9±2.5 20.2 (2.8) 0.757 Plateau systolic blood pressure (mmHg) 137.0 (11.0) 141.0 (6.0) 0.130 Plateau diastolic blood pressure (mmHg) 79.0±9.4 79.8±10.6 0.716 Plateau heart rate (beats/min) 95.1±12.3 106.4±16.1 <0.001 Plateau blood oxygen saturation (%) 89.9±1.6 85.3±3.9 <0.001

Note: HAH: patients with high altitude headache; Healthy: healthy people; Normal distribution data are expressed as mean ± standard deviation, and non-normal distribution data are expressed as median (interquartile range).

2. Sample Processing and RNA Extraction

2 hours before saliva collection, fasting, water and gargling are prohibited. Use a 50 ml centrifuge tube to collect 5 ml of saliva. After centrifugation at 3000×g and 4°C for 15 minutes, use an RNase-free and bacteria-free pipette to take the supernatant and place it in an RNase-free and bacteria-free EP tube. After that, centrifuge again at 12000×g and 4°C for 10 minutes, use a sterile enzyme-free pipette to take the supernatant, place it in a sterile enzyme-free EP tube and store it at -80°C for standby use, which is the saliva supernatant. Whole saliva is saliva that has not undergone any centrifugation. The RNA in all samples was extracted and purified by the microRNA column extraction kit (miRNeasy Serum/PlasmaKit, catalog number: 217184) of Qiagen Technology Co., Ltd., Germany, according to the operating procedures in the instruction manual.

1. The concentration of salivary microRNA is low, about 25-40ng/μl;

2. cDNA synthesis was performed using the microRNA fluorescent quantitative PCR reverse transcription kit (Bulge-Loop™ miRNAqRT-PCR Starter Kit), 10 μl reverse transcription reaction system: 2 μl RNA stock solution, 1 μl reverse transcription primer (5 μM), 2 μl 5x reverse transcription buffer, 2 μl RTase Mix, 3 μl RNase-free H ₂ O; the reverse transcription primer sequence is shown in Table 2;

3. Reverse transcription (RT) reaction temperature parameters: 42℃ for 60 min, 70℃ for 10 min.

Table 2 Reverse transcription primer sequences

microRNA sequence Serial Number hsa-miR-223-5p 5'--gtcgtatccagtgcagggtccgaggtattcgcactggatacgacaactca --3' SEQ ID No.5 hsa-miR-215-5p 5'-- gtcgtatccagtgcagggtccgaggtattcgcactggatacgacgtctgt --3' SEQ ID No.6 hsa-miR-23b-3p 5'-- gtcgtatccagtgcagggtccgaggtattcgcactggatacgacgtggta --3' SEQ ID No.7 hsa-miR-222-3p 5'--gtcgtatccagtgcagggtccgaggtattcgcactggatacgacacccag --3' SEQ ID No.8

Example 3 Real-time fluorescence quantitative PCR (SYBR dye method)

The microRNA real-time fluorescence quantitative PCR kit (Bulge-Loop™ miRNA qRT-PCR Starter Kit) of Guangzhou Ruibo Biotechnology Co., Ltd., China, was used to amplify hsa-miR-223-5p, hsa-miR-215-5p, hsa-miR-23-3p and hsa-miR-222-3p (target microRNA) and the external reference cel-miR-39 with the cDNA of the sample to be tested as the template; the Ct value (cycle threshold) was obtained respectively, and the expression level of the microRNA of each specimen was calculated by the formula: expression amount = 2 ^{Ct(cel-miR-39)-Ct(target microRNA)} . The specific operation process is shown in the instructions of the kit. The experiment was repeated three times for each sample. The basic information of hsa-miR-223-5p, hsa-miR-215-5p, hsa-miR-23-3p and hsa-miR-222-3p is shown in Table 3.

1. Amplification reaction system: (20 μl system): RT reaction product 2 μl, SYBR Green Mix 10 μl, miRNA Forward primer (5 μM) 0.8 μl, miRNA Reverse primer (5 μM) 0.8 μl, ddH ₂ O 6.4 μl; primer sequences are shown in Table 4.

2. Reaction conditions: pre-denaturation at 95°C for 10 min, denaturation at 95°C for 2 s, annealing at 60°C for 20 s, extension at 70°C for 10 s;

3. The Realtime PCR amplification curves of the detected miRNA and the external reference cel-miR-39 were smooth and "S" shaped, indicating that the designed primers had high amplification efficiency.

4. The melting curves of PCR products are all single peaks, indicating that the amplified target gene has good specificity and the results are reliable. Figure 2 shows the melting curves of each target microRNA qPCR reaction, including: A: hsa-miR-223-5p; B: hsa-miR-215-5p; C: hsa-miR-23b-3p; D: hsa-miR-222-3p.

Table 3 Basic information of microRNA

MicroRNA microRNA sequences Search Number Serial Number hsa-miR-223-5p cguguauuugacaagcugaguu MIMAT0004570 SEQ ID No.1 hsa-miR-215-5p augaccuaugaauugacagac MIMAT0000272 SEQ ID No.2 hsa-miR-23b-3p aucacauugccagggauuaccac MIMAT0000418 SEQ ID No.3 hsa-miR-222-3p agcuacaucuggcuacugggu MIMAT0000279 SEQ ID No.4

Table 4 microRNA qPCR primer sequences

MicroRNA Primer Name qPCR primer sequences Serial Number hsa-miR-223-5p Forward primer 5'-- cgcgcgtgtatttgacaagc --3' SEQ ID No.9 hsa-miR-223-5p Reverse primer 5'-- agtgcagggtccgaggtatt --3' SEQ ID No.10 hsa-miR-215-5p Forward primer 5'-- gcgcgatgacctatgaattg --3' SEQ ID No.11 hsa-miR-215-5p Reverse primer 5'-- agtgcagggtccgaggtatt --3' SEQ ID No.12 hsa-miR-23b-3p Forward primer 5'-- cgatcacattgccagggat --3' SEQ ID No.13 hsa-miR-23b-3p Reverse primer 5'-- agtgcagggtccgaggtatt --3' SEQ ID No.14 hsa-miR-222-3p Forward primer 5'-- gcgcgagctacatctggcta --3' SEQ ID No.15 hsa-miR-222-3p Reverse primer 5'-- agtgcagggtccgaggtatt --3' SEQ ID No.16

Note: qPCR: real-time quantitative polymerase chain reaction.

Example 5 Results Statistics and Analysis Methods

SPSS 19.0 and MedCalc 19.20 were used for statistical analysis. The Shapiro-Wilk method was used for normality test, the independent sample t-test was used for significant differences in normal distribution data, and the Mann-Whitney Test was used for significant differences in non-normal distribution data. The receiver operating characteristic curve (ROC curve) and the area under the curve (AUC) were used to evaluate the diagnostic efficacy of each microRNA (hsa-miR-223-5p, hsa-miR-215-5p, hsa-miR-23-3p, and hsa-miR-222-3p). When P < 0.05, it was considered statistically significant. Among them, AUC reflects the predictive effectiveness (AUC=0.5, no predictive effectiveness; 0.5＜AUC＜0.7, very small predictive value; 0.7＜AUC＜0.9, quite accurate predictive value; 0.9＜AUC＜1, very accurate predictive value).

5. Results Analysis

1. Figure 3 is a comparison of the expression levels of hsa-miR-223-5p, hsa-miR-215-5p, hsa-miR-23-3p and hsa-miR-222-3p in the saliva supernatant of HAH patients and healthy controls; As shown in Figure 3, the expression levels of hsa-miR-223-5p, hsa-miR-215-5p, hsa-miR-23-3p and hsa-miR-222-3p in the saliva supernatant of HAH patients were significantly different from those of healthy controls ( P < 0.01). HAH refers to HAH patients and Healthy refers to healthy controls.

2. Figure 4 shows the receiver operating characteristic curves of hsa-miR-223-5p, hsa-miR-215-5p, hsa-miR-23-3p and hsa-miR-222-3p in the saliva supernatant of HAH patients. As shown in Figure 4, the diagnostic efficacy of each microRNA in the saliva supernatant for HAH patients and healthy controls can be seen from the ROC curve. The diagnostic efficacy of hsa-miR-223-5p in saliva for HAH patients and healthy controls is very good, among which the AUC of hsa-miR-223-5p is 0.802, the AUC of hsa-miR-215-5p is 0.780, the AUC of hsa-miR-23-3p is 0.853, and the AUC of hsa-miR-222-3p is 0.868. The figure shows the ROC curve, area under the curve (AUC), sensitivity, and specificity of HAH patients, where AUC reflects the diagnostic efficacy. It is generally believed that the larger the AUC of the test, the better its diagnostic value. This fully demonstrates that hsa-miR-223-5p, hsa-miR-215-5p, hsa-miR-23-3p, and hsa-miR-222-3p in saliva supernatant are all accurate and feasible as biomarkers for diagnosing HAH patients. The AUC of further combined hsa-miR-222-3p and hsa-miR-23-3p was 0.914, as shown in Figure 5; the AUC of combined hsa-miR-223-5p, hsa-miR-215-5p and hsa-miR-23-3p was 0.940, as shown in Figure 6. The diagnostic efficiency is more accurate, and it can objectively diagnose whether people who suddenly enter the plateau are HAH patients, provide reference value for the clinical diagnosis and targeted treatment of high-altitude headache, and avoid the error of subjective human judgment to a certain extent.

3. Table 5 shows the relative expression data of microRNA in HAH patients and healthy controls, and Table 6 shows the receiver operating characteristic curve data of each microRNA in the saliva supernatant of healthy people and HAH patients.

Table 5 Relative expression data of microRNA in HAH patients and healthy controls

microRNA Healthy group (n=35) HAH group (n=37) P- value hsa-miR-223-5p 0.469±0.183 0.958 (1.00) <0.01 hsa-miR-215-5p 0.412±0.198 0.608 (0.47) <0.01 hsa-miR-23b-3p 0.003±0.002 0.012±0.014 <0.01 hsa-miR-222-3p 0.011±0.012 0.031±0.013 <0.01

Note: HAH: patients with high altitude headache; Healthy: healthy controls; Normal distribution data are expressed as mean ± standard deviation, and non-normal distribution data are expressed as median (interquartile range).

Table 6 Operating characteristic curve data

Note: HAH: high altitude headache; microRNA combination 1: combination of miR-222-3p and miR-23b-3p; microRNA combination 2: combination of miR-223-5p, miR-215-5p and miR-23b-3p.

In summary, all the experiments show that the use of the microRNA biomarkers or the combination of microRNA biomarkers provided in the examples for diagnosing patients with high altitude headache is fully effective.

Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims (6)

1. A method for preparing a diagnostic kit for high altitude headache using a microRNA as a diagnostic marker, wherein the microRNA is hsa-miR-23b-3p. 2. The use according to claim 1, characterized in that the sequence of hsa-miR-23b-3p is shown as SEQ ID No.3. 3. The use according to claim 1, characterized in that the reagent is a reagent for detecting the expression amount of hsa-miR-23b-3p in a sample using a high-throughput sequencing method and/or a quantitative PCR method and/or a probe hybridization method. 4. The use according to claim 3, characterized in that the primers in the quantitative PCR include a reverse transcription primer for detecting hsa-miR-23b-3p, a specific upstream primer for fluorescent quantitative reaction, and a specific downstream primer. 5. The use according to claim 4, characterized in that the sequence of the reverse transcription primer is shown as SEQ ID No.7. 6. The use according to claim 1, characterized in that the sample to be detected is plasma or saliva.