MicroRNA molecular marker composition for screening patients with chronic mountain sickness

Abstract

This invention relates to a micronucleic acid biomarker for early warning of chronic mountain sickness (CMS) risk among permanent residents of high-altitude areas, and the application of said micronucleic acid molecule in the preparation of reagents or kits for screening and diagnosing CMS patients. Furthermore, it provides a kit for detecting hsa-miR-7854-3p and hsa-miR-6513-3p molecules in the plasma of test subjects using real-time quantitative PCR, and the expression levels of these molecules can specifically identify CMS patients from healthy individuals in high-altitude areas. Its use in CMS screening and diagnosis is characterized by high acceptability, non-invasiveness, and excellent sensitivity and specificity. This biomarker is suitable for large-scale CMS screening and early diagnosis in populations long-term residents of high-altitude areas, providing a scientific basis for early warning and precise treatment of CMS, thereby reducing the health damage and life risks caused by CMS among permanent residents of high-altitude areas, improving the quality of life of residents in high-altitude areas, and benefiting the economic development and construction of high-altitude areas.

Description

Technical Field

[0001] This invention belongs to the field of molecular biotechnology and its applications, specifically relating to a plasma microRNA molecular marker for screening and diagnosing the risk of chronic mountain sickness, as well as its applications and related reagent kits. Background Technology

[0002] Chronic mountain sickness (CMS), also known as Monge's disease, is an idiopathic disease commonly seen in people who live at high altitudes (≥2500 meters) for extended periods. Its main pathological features include excessive red blood cell count, severe hypoxemia, and a range of nonspecific clinical symptoms such as headache, fatigue, dizziness, palpitations, shortness of breath, nausea, vomiting, general weakness, insomnia, blurred vision, dyspnea, numbness in the hands and feet, cyanosis of the lips and fingers, tachycardia, and sleep disturbances. CMS has a high incidence rate, ranging from 1.21% to 31.5% in long-term high-altitude residents (Jiang C, Chen J, Liu F, Luo Y, Xu G, Shen HY, Gao Y, Gao W. Chronic mountain sickness in Chinese Han males who migrated to the Qinghai-Tibetan plateau: application and evaluation of diagnostic criteria for chronic mountain sickness. BMC PUBLICHEALTH 2014, 14: 701.). Chronic mountain sickness (CMS) is extremely dangerous. Excessive erythrocyte proliferation leads to increased blood viscosity and microcirculatory dysfunction, resulting in hypoxic damage to multiple organs and severely impairing the work capacity and quality of life of people living at high altitudes (Villafuerte FC, Corante N. Chronic Mountain Sickness: Clinical Aspects, Etiology, Management, and Treatment. HIGH ALT MED BIOL 2016, 17(2): 61-69; Gao Yuqi, Huang Jian. Inflammatory Response and Altitude Sickness. Journal of the Third Military Medical University 2016, 38(3): 215-219.). In severe cases, CMS can even lead to thromboembolism in vital organs such as the heart, brain, kidneys, spleen, and lungs, resulting in sudden death.(Leon-Velarde F, Maggiorini M, Reeves JT, Aldashev A, Asmus I, Bernardi L, Ge RL, Hackett P, Kobayashi T, Moore LG, Penaloza D, Richalet JP, Roach R, Wu T, Vargas E, Zubieta-Castillo G, Zubieta-Calleja G. Consensus statement on chronic and subacute high altitude diseases. HIGH ALT MED BIOL 2005, 6(2): 147-157.).

[0003] CMS has an insidious onset, often going unnoticed by patients. Therefore, early screening and diagnosis are crucial for controlling disease progression and implementing targeted treatment. Currently, the diagnosis of CMS mainly relies on clinical symptoms and laboratory tests, using the "Qinghai Chronic Altitude Sickness Scoring System" for assessment (Chinese Medical Association Plateau Medicine Branch. "Decision on Unifying the Use of the 'Qinghai Standard' for Chronic Altitude (Mountain) Disease." Journal of Plateau Medicine 17.1(2007):2.). This diagnostic standard requires plateau medicine specialists, and the diagnostic process involves a large number of labor-intensive operations and invasive examinations, which is not conducive to large-scale population screening and continuous disease monitoring, severely limiting the effectiveness of CMS prevention and control. Although the Qinghai standard scoring system includes symptoms and hematological (hemoglobin) indicators, in actual clinical practice, it has been found that some people have very high hemoglobin levels but no clinical symptoms, and their quality of life is not affected. These people do not belong to CMS.

[0004] Therefore, there is an urgent need to find a non-invasive, widely accepted, convenient, quick, and effective screening method for chronic mountain sickness, especially a precise detection method using biomarkers, to meet the significant practical needs of disease prevention and control. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a plasma microRNA molecular biomarker for screening and diagnosing the risk of chronic mountain sickness (CMS) in long-term high-altitude populations. This biomarker detects the expression levels of hsa-miR-7854-3p and hsa-miR-6513-3p in the plasma of test subjects, providing early warning and diagnosis of CMS risk. The invention also provides applications and kits for this microRNA molecular biomarker. The combination of circulating hsa-miR-7854-3p and hsa-miR-6513-3p has the potential to serve as a biomarker for early diagnosis of CMS risk in long-term high-altitude individuals, suitable for rapid screening and long-term disease monitoring of CMS in large-scale long-term high-altitude populations, and has high clinical application value.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] 1. A micronucleic acid molecular biomarker for rapid diagnosis of CMS risk in long-term high-altitude populations, wherein the micronucleic acid molecular biomarker is hsa-miR-7854-3p or / and hsa-miR-6513-3p.

[0008] Furthermore, the sequence of hsa-miR-7854-3p is shown in SEQ ID No. 1, and the sequence of hsa-miR-6513-3p is shown in SEQ ID No. 2.

[0009] 2. The use of a reagent for detecting the expression level of the micronucleic acid molecular biomarker as described in claim 1 or 2 in the preparation of reagents or kits for the auxiliary diagnosis of chronic mountain sickness.

[0010] Furthermore, the micronucleic acid molecular biomarkers are derived from plasma samples.

[0011] Furthermore, the plasma is plasma obtained by centrifuging fasting peripheral venous blood in the morning.

[0012] Furthermore, the plasma is peripheral venous blood collected in the morning after fasting from EDTA-Na anticoagulation blood collection tubes. After centrifugation at 3000 rpm and 4°C for 10 minutes, the supernatant (plasma) is collected using an RNase-free and sterile pipette tip and stored in a sterile, enzyme-free EP tube at -80°C for later use.

[0013] In this application, the expression levels of hsa-miR-7854-3p and hsa-miR-6513-3p in the plasma of subjects were detected. The expression of these nucleic acid molecules in the plasma of patients with chronic mountain sickness (CMS) was upregulated compared to that in the plasma of healthy individuals at high altitudes (NC). The difference was statistically significant (P < 0.01).

[0014] 3. A kit for diagnosing chronic mountain sickness, the kit comprising reagents for detecting the expression levels of hsa-miR-7854-3p or hsa-miR-6513-3p.

[0015] Furthermore, the reagent includes primers for reverse transcription of hsa-miR-7854-3p or hsa-miR-6513-3p;

[0016] Furthermore, the CMS screening kit also includes dNTPs, MMLV Reverse Transcriptase, 5 × MMLV RT buffer, and RNase-free H2O.

[0017] Furthermore, the kit also includes a microRNA reverse transcription (RT) reaction system, which, per 10 uL, comprises: 0.6 uL of Stem-loop RT primer, 0.375 uL of dNTP, 0.1 uL of MMLV Reverse Transcriptase, 2 uL of 5 × MMLV RT buffer, 0.925 uL of RNase-free H2O, and the remaining 6 uL of sample RNA.

[0018] Furthermore, the RT reaction temperature parameters of the kit are: 25℃ for 30 min, 42℃ for 30 min, and 85℃ for 5 min.

[0019] Furthermore, the reagent also includes specific amplification primer pairs for the fluorescence quantitative PCR detection of the reverse transcription products.

[0020] Furthermore, the kit also includes reagents for detecting an internal reference gene, which is at least one selected from miR-16-5p, miR-39, or U6 snRNA.

[0021] Furthermore, the primers for reverse transcription of hsa-miR-7854-3p are shown in SEQ ID No. 4; and the primers for reverse transcription of hsa-miR-6513-3p are shown in SEQ ID No. 5.

[0022] Furthermore, the forward primer for quantitative PCR of hsa-miR-7854-3p reverse transcription products is shown in SEQ ID No. 7, the forward primer for quantitative PCR of hsa-miR-6513-3p reverse transcription products is shown in SEQ ID No. 8, and the universal reverse primer is shown in SEQ ID No. 10.

[0023] Furthermore, the kit also includes a microRNA qPCR reaction system, which comprises, per 20 μL: 10 μL of 2 × Real-time Master Mix, 0.2 μL of microRNA qPCR Forward Primer, 0.2 μL of microRNA qPCR Reverse Primer, 0.4 μL of 50 × ROX Reference Dye, 0.2 μL of Tag DNA Polymerase, 6 μL of ddH2O, and the remainder being 3 μL of RT reaction product.

[0024] Furthermore, the microRNA qPCR reaction conditions of the kit are: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 12 s, and annealing extension at 62°C for 40 s.

[0025] The beneficial effects of this invention are as follows: By screening circulating microRNAs in the plasma of long-term high-altitude populations through RNA sequencing, and combining this with the clinical prevalence of chronic mountain sickness (CMS), this invention further discovered significant differences in the levels of circulating hsa-miR-7854-3p and hsa-miR-6513-3p in plasma between CMS patients and healthy high-altitude individuals (NC). Furthermore, the expression levels of circulating hsa-miR-7854-3p and hsa-miR-6513-3p in the plasma of another independent population were detected using real-time quantitative PCR with chimeric fluorescent dyes. This confirmed a close correlation between the expression levels of circulating hsa-miR-7854-3p and hsa-miR-6513-3p in plasma and the prevalence of CMS, demonstrating good early warning and diagnostic efficacy for CMS. This tiny nucleic acid molecule can be used independently or in combination as a biomarker for screening the risk of CMS, providing a scientific basis for the early diagnosis and precise treatment of CMS, thereby reducing the health damage and life risks caused by CMS to people living in high-altitude areas, improving the quality of life of residents in high-altitude areas, and benefiting the economic development and national defense of my country's plateau regions.

[0026] The microRNA biomarker proposed in this invention can be detected through plasma, and the sample acquisition has advantages such as safety, convenience and high acceptance; it is suitable for early large-scale population screening of CMS, improving diagnostic accuracy and reducing misdiagnosis and missed diagnosis. Attached Figure Description

[0027] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

[0028] Figure 1Supervised orthogonal partial least squares principal component analysis (OPLSDA) plot of plasma microRNA sequencing results from patients with chronic mountain sickness (CMS) and healthy individuals at high altitudes (NC);

[0029] Figure 2 Volcano plot showing the difference in microRNA expression in plasma between patients with chronic mountain sickness (CMS) and healthy individuals at high altitudes (NC);

[0030] Figure 3 Cluster heatmap showing the difference in microRNA expression in plasma between patients with chronic mountain sickness (CMS) and healthy individuals at high altitudes (NC);

[0031] Figure 4 The melting curves of the qPCR reaction for hsa-miR-7854-3p (A) and hsa-miR-6513-3p (B) are shown.

[0032] Figure 5 The melting curve of the hsa-miR-6806-3p qPCR reaction;

[0033] Figure 6 To compare the relative expression levels of hsa-miR-7854-3p (A) and hsa-miR-6513-3p (B) in plasma of patients with chronic mountain sickness (CMS) and healthy individuals at high altitudes (NC);

[0034] Figure 7 To compare the relative expression levels of hsa-miR-6806-3p in plasma between patients with chronic mountain sickness (CMS) and healthy individuals at high altitudes (NC);

[0035] Figure 8 The efficacy of hsa-miR-7854-3p (A) and hsa-miR-6513-3p (B) in screening and diagnosing CMS disease when used alone in plasma;

[0036] Figure 9 The efficacy of combined application of hsa-miR-7854-3p and hsa-miR-6513-3p in plasma for screening and diagnosis of CMS pathogenesis is shown in the figure.

[0037] Figure 10 This is a power graph showing the screening and diagnostic efficacy of plasma hsa-miR-6806-3p for CMS pathogenesis. Detailed Implementation

[0038] The preferred embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Experimental methods not specified with specific conditions in the embodiments are generally performed under conventional conditions or as recommended by the manufacturer.

[0039] Example 1: Screening of differentially expressed microRNAs in plasma between patients with chronic mountain sickness and healthy individuals at high altitudes via sequencing

[0040] First, the plasma microRNA expression profile sequencing results of patients with chronic mountain sickness (CMS) and healthy individuals at high altitudes were compared and analyzed. The samples for this expression profile screening came from 7 patients with chronic mountain sickness (CMS) and 7 healthy individuals at high altitudes (NC). The correlation between each microRNA and the risk of CMS was studied, and potential microRNA biomarkers related to CMS were screened out. The clinical data of the microRNA expression profile screening population are shown in Table 1.

[0041] Table 1 Clinical data of the microRNA screening population

[0042]

[0043] Note: BMI: Body Mass Index; HGB: Hemoglobin Level; CMS: Chronic Mountain Sickness; NC: Healthy Individuals at High Altitudes; Normally distributed data are expressed as mean ± standard deviation, and non-normally distributed data are expressed as median (interquartile range).

[0044] I. Description of Materials and Specimen Collection:

[0045] The above-mentioned participants were all volunteers who had moved from the plains to work in a plateau area at an altitude of 4300 meters for more than one year. Plasma samples were collected and physical examinations were conducted after one year of work in the plateau region. All sample collection followed ethical guidelines and informed consent was obtained. Peripheral blood was collected using EDTA anticoagulant tubes, centrifuged at 3000×g for 10 minutes at 4°C, and the supernatant plasma was carefully aspirated and stored at -80°C for later use. The treatment was administered by a plateau specialist according to the "Qinghai Standard for Chronic Altitude Sickness" established at the Sixth World Congress of Plateau Medicine (Chinese Medical Association Plateau Medicine Branch. "Decision on the Unified Use of the 'Qinghai Standard' for Chronic Altitude (Mountain) Sickness." Journal of Plateau Medicine 17.1(2007):2.).

[0046] The specific diagnostic criteria for chronic mountain sickness (CMS) are as follows:

[0047] 1. Moving from plains to high-altitude areas (altitude: > 2500 meters) and living there continuously for more than 6 months;

[0048] 2. Normal lung function, no other chronic lung diseases that aggravate hypoxemia, and exclusion of polycythemia vera and other secondary polycythemia diseases;

[0049] 3. Symptom scoring criteria for the "Qinghai Standard for Chronic Altitude Sickness":

[0050] (1) Dyspnea and / or palpitations: 0 points for none, 1 point for mild, 2 points for moderate, and 3 points for severe;

[0051] (2) Sleep disorders: 0 points for normal sleep, 1 point for inability to fall asleep normally, 2 points for insufficient sleep or waking up frequently, and 3 points for inability to fall asleep.

[0052] (3) Cyanosis: 0 points for no cyanosis, 1 point for mild cyanosis, 2 points for moderate cyanosis, and 3 points for severe cyanosis;

[0053] (4) Vascular dilation: 0 points for no vascular dilation, 1 point for mild vascular dilation, 2 points for moderate vascular dilation, and 3 points for severe vascular dilation.

[0054] (5) Sensory abnormalities: 0 points for no sensory abnormalities, 1 point for mild sensory abnormalities, 2 points for moderate sensory abnormalities, and 3 points for severe sensory abnormalities.

[0055] (6) Headache: 0 points for no headache, 1 point for mild headache, 2 points for moderate headache, and 3 points for severe headache.

[0056] (7) Tinnitus: 0 points for no tinnitus, 1 point for mild tinnitus, 2 points for moderate tinnitus, and 3 points for severe tinnitus;

[0057] (8) Hemoglobin: Males with hemoglobin content (HGB) <210 g / L are scored 0 points, and those with HGB ≥210 g / L are scored 3 points. Females with HGB <190 g / L are scored 0 points, and those with HGB ≥190 g / L are scored 3 points. (9) CMS diagnosis and severity determination: Add the above scores together. No CMS = 0-5 points, mild CMS = 6-10 points, moderate CMS = 11-14 points, and severe CMS ≥15 points.

[0058] II. Experimental steps in the screening phase:

[0059] 1. Extraction and purification of total RNA from plasma

[0060] Total RNA was extracted from 2 mL of plasma using TRIzol LS Reagent (Invitrogen, USA, catalog number: 10296010CN). RNA was then purified and its concentration determined using the RNeasy mini kit (QIAGEN, Germany, catalog number: 74106).

[0061] 2. Quality control of RNA extraction solution, library construction and RNA-seq detection

[0062] (1) RNA fragments of 18-30 nt in length were specifically recovered by gel electrophoresis; (2) 3' and 5' adapters were ligated to the recovered RNA fragments; (3) the above fragments were amplified by RT-PCR to obtain cDNA products; (4) cDNA fragments of 140-160 bp in length were specifically recovered by gel electrophoresis; (5) a cDNA library was constructed using the above fragments; (6) the cDNA library was quality controlled using Agilent 2100 Bioanalyzer and its matching kit Agilent RNA 6000 Nano Kit (detection volume: 1 μL); (7) the quality-controlled cDNA library was then tested using Illumina HiSeq. TM RNA-seq was performed at 2500.

[0063] 3. Bioinformatics analysis of RNA-seq results

[0064] (1) RNA-seq data quality analysis

[0065] The RNA-seq data were filtered according to the following conditions: ① Data with a quality value less than 20 were removed; ② Data with a base number greater than 1 were removed; ③ Data containing N-terminus were removed; ④ Data without 3'-adaptor were removed; ⑤ Data without 5'-adaptor were removed; ⑥ Data without insert fragments were removed; ⑦ Data with short insert fragments (<18 nt) and data containing polyA were removed.

[0066] (2) Standard data analysis of RNA-seq detection results

[0067] Non-coding RNA information (including rRNA, scRNA, snoRNA, snRNA, and tRNA) from GenBank was selected to annotate the tag sequences obtained from RNA-seq. Then, blast 2.2.25 (blastn) software was used with an identity parameter > 97% to identify and remove rRNA, scRNA, snoRNA, snRNA, and tRNA sequences from the data. The Rfam (v.11.0) database was selected to annotate the tag sequences obtained from RNA-seq. Then, blast 2.2.25 (blastn) software was used with an identity parameter > 97% to further remove rRNA, scRNA, snoRNA, snRNA, and tRNA sequences from the data. bowtie (v.1.12) was used to align the tag sequences to the human genome (Homo sapiens hg19) for RNA-seq data annotation. Based on the alignment results, tag sequences from mRNA degradation fragments were removed. Based on the genome alignment results, repetitive tag sequences were removed. Finally, by comparing the human microRNA sequences in the miRbase (v.21) database (http: / / www.mirbase.org / ), the microRNAs in the RNA-seq test results were identified.

[0068] 4. Statistical analysis of plasma microRNA expression profiles

[0069] (1) Summarize the microRNAs identified in each sample and calculate the TPM (tags per million) expression level of each microRNA using the formula TPM = T * 10. 6 / N (T represents microRNA tags, N represents total microRNA tags), and the expression profiles of all samples were obtained.

[0070] (2) Principal component analysis of sample data

[0071] In this study, microRNAs with TPM < 1 were filtered out, and then supervised orthogonal partial least squares principal component analysis (OPLSDA) was performed using OECloud tools (https: / / cloud.oebiotech.com). Figure 1 The OPLSDA analysis graphs for the CMS and NC groups are shown. The results show that the microRNA sequencing data of the two groups can be clearly separated.

[0072] (3) Hierarchical cluster analysis of microRNA

[0073] This study used heat maps to visually represent the expression levels of microRNAs in different datasets (sample size: 14, including 7 CMS sequencing datasets and 7 NC sequencing datasets). Figure 3 Clustering heatmaps show the expression differences between the CMS and NC groups; the redder the color on the heatmap, the higher the expression level, and the bluer the color, the lower the expression level.

[0074] (4) Differential expression microRNA analysis between CMS and NC groups

[0075] The acquired data were statistically analyzed using the R language package to screen for microRNAs with significant differences in expression between patients with chronic mountain sickness (CMS) and healthy individuals at high altitudes (NC) (screening criteria: Fold Change ≥ 2, P-value < 0.05). A total of 2751 microRNAs were detected in the screening experiment, and many microRNAs showed significant differences in expression between patients with chronic mountain sickness (CMS) and healthy individuals at high altitudes (NC). Figure 2 A volcano plot shows the differential microRNA screening results between the selected chronic mountain sickness (CMS) patients and healthy high-altitude individuals (NC). Specifically: red boxes represent microRNAs that were significantly upregulated in the CMS group compared to the NC group; blue triangles represent microRNAs that were significantly downregulated in the CMS group compared to the NC group; gray dots represent microRNAs with no difference in expression between the two groups; FC stands for Fold Change; and pVal represents the statistical p-value.

[0076] To further verify the accuracy of differentially expressed microRNAs in plasma samples in diagnosing patients with chronic mountain sickness, the validation team used real-time quantitative qRT-PCR to detect the expression levels of multiple microRNAs. The results showed that the expression trends of the microRNAs screened in this invention were consistent with the microarray detection results. Only the experimental data relevant to this invention are presented below.

[0077] Example 2: Study on the predictive efficacy of plasma microRNA expression levels on high-altitude adaptation

[0078] I. Description of Materials and Specimen Collection

[0079] The selection criteria for subjects were the same as in Example 1. Forty individuals (CMS group) developed CMS one year after relocating from the plains to work in a plateau region at an altitude of 4300 meters. Forty healthy individuals (NC group) who did not develop CMS one year after relocating from the plains to work in a plateau region at an altitude of 4300 meters were also included in the plasma collection. Detailed clinical data are shown in Table 2. 2.5 mL of peripheral venous blood (fasting in the morning) was collected from these individuals after physical examination. The blood was centrifuged at 3000 rpm for 10 minutes at 4°C. Plasma was then collected using sterile, enzyme-free pipette tips and stored in sterile, enzyme-free EP tubes at -80°C for later use. This study has been approved by the Medical Ethics Committee, and informed consent was obtained from all patients for the collection of all specimens.

[0080] Table 2 Clinical data of subjects with plasma samples

[0081]

[0082] Note: BMI: Body Mass Index; HGB: Hemoglobin Level; CMS: Chronic Mountain Sickness; NC: Healthy Individuals at High Altitudes; Normally distributed data are expressed as mean ± standard deviation, and non-normally distributed data are expressed as median (interquartile range).

[0083] II. Specimen Collection, RNA Extraction, and cDNA Synthesis

[0084] RNA from all plasma samples was extracted and purified using the microRNA column extraction kit (miRNeasy Serum / Plasma Kit, catalog number: 217184) from Qiager GmbH, Germany, following the manufacturer's instructions.

[0085] 1. Plasma RNA concentration: 25-50 ng / uL.

[0086] 2. cDNA synthesis was performed using the microRNA real-time PCR reverse transcription kit (Hairpin-it miRNAs RT-PCR Quantitation Kit).

[0087] 3. microRNA cDNA synthesis system: Basic information of each microRNA is shown in Table 3; the reverse transcription reaction system is prepared as shown in Table 4, and the stem-loop reverse transcription primer sequences of each microRNA are shown in Table 5.

[0088] 4. Reverse transcription (RT) reaction temperature parameters: 25℃ for 30 min; 42℃ for 30 min; 85℃ for 5 min.

[0089] Table 3 Basic Information on microRNA

[0090]

[0091] Table 4. Preparation of microRNA reverse transcription system

[0092]

[0093] Table 5. Stem-loop reverse transcription primer sequences

[0094]

[0095] III. Real-time quantitative PCR (qPCR, chimeric fluorescent dye method)

[0096] Target microRNAs, including hsa-miR-7854-3p, hsa-miR-6513-3p, and hsa-miR-6806-3p, as well as the external reference cel-miR-39, were amplified using real-time quantitative PCR (Hairpin-it miRNAs RT-PCR Quantitation Kit, catalog number: E01008). The cycle threshold (Ct) values ​​were obtained, and the expression level was calculated using the formula: expression level = 2. Ct(cel-miR-39)-Ct(目标microRNA) The expression level of microRNA in each sample was calculated separately. Each sample was tested three times.

[0097] 1. Amplification reaction system: Prepare the qPCR reaction system according to Table 6; Table 7 shows the primer sequences used for microRNA qPCR.

[0098] 2. Reaction conditions: Pre-denaturation at 95℃ for 3 min, denaturation at 95℃ for 12 s, annealing extension at 62℃ for 40 s;

[0099] 3. The Real-time PCR amplification curves of the microRNA to be detected and the external reference cel-miR-39 both showed an "S" shape;

[0100] 4. Figure 4 Melting curves of hsa-miR-7854-3p(A) and hsa-miR-6513-3p(B) microRNA qPCR reactions. Figure 5 The figure shows the melting curves of the hsa-miR-6806-3p qPCR reaction. As shown, the melting curves of the PCR products all exhibit a single peak, indicating that the target gene amplified using the primers of this invention has good specificity and the results are reliable.

[0101] Table 6. Preparation of microRNA qPCR reaction system

[0102]

[0103] Table 7. MicroRNA qPCR Primer Sequences

[0104]

[0105] IV. Statistical Analysis Methods

[0106] Statistical analysis was performed using SPSS 27.0 and MedCalc 23.1.7 software. Normality was tested using the Shapiro-Wilk method. Significant differences in normally distributed data were assessed using the independent samples t-test, while significant differences in non-normally distributed data were assessed using the Mann-Whitney test. The receiver operating characteristic curve (ROC curve) and area under the curve (AUC) were used to evaluate the screening efficacy of each microRNA and its combinations. A p-value < 0.05 was considered statistically significant. AUC reflects predictive efficacy (AUC = 0.5, no diagnostic efficacy; 0.5 < AUC < 0.7, very small diagnostic value; 0.7 < AUC < 0.9, fairly accurate diagnostic value; 0.9 < AUC < 1, very accurate diagnostic value).

[0107] V. Results Analysis

[0108] 1. Figure 6 The expression levels of hsa-miR-7854-3p (A) and hsa-miR-6513-3p (B) in plasma were compared between patients with chronic mountain sickness (CMS) and healthy individuals at high altitudes (NC). Figure 7 This study compared the expression levels of hsa-miR-6806-3p in patients with chronic mountain sickness (CMS) and healthy individuals at high altitudes (NC). As shown in the figure, compared with the NC group, the expression levels of hsa-miR-7854-3p, hsa-miR-6513-3p, and hsa-miR-6806-3p in plasma were significantly different in the CMS group (P < 0.01). A p < 0.05 was considered statistically significant, and a p < 0.01 was considered extremely statistically significant.

[0109] 2. Figure 8 The working characteristic curves of hsa-miR-7854-3p and hsa-miR-6513-3p in the plasma of patients with chronic mountain sickness (CMS) are shown. Figure 9 The combined operating characteristic curves of hsa-miR-7854-3p and hsa-miR-6513-3p; Figure 10The figure shows the operating characteristic curves of hsa-miR-6806-3p in plasma from patients with chronic mountain sickness (CMS). As indicated by the ROC curves, plasma hsa-miR-7854-3p, hsa-miR-6513-3p, or hsa-miR-6806-3p exhibit good screening efficacy between CMS and NC populations, with AUCs of 0.865, 0.826, and 0.903, respectively. The figure clearly displays the ROC curves, area under the curve (AUC), sensitivity, and specificity for CMS patients, with AUC reflecting the screening diagnostic efficacy. Generally, a higher AUC indicates better diagnostic value. This demonstrates that plasma hsa-miR-7854-3p, hsa-miR-6513-3p, or hsa-miR-6806-3p can be used alone as biomarkers for screening and diagnosing CMS patients with good accuracy and feasibility. Further combining the two, such as Figure 9 As shown, the diagnostic efficacy of the combined use of hsa-miR-7854-3p and hsa-miR-6513-3p was improved to 0.924.

[0110] 3. Table 8 shows the receiver operating characteristic curve data of various microRNAs in the plasma of patients with chronic mountain sickness (CMS) and healthy individuals at high altitudes (NC).

[0111] Table 8 Operating characteristic curve data

[0112]

[0113] Note: The combination of the two refers to the combination of hsa-miR-7854-3p and hsa-miR-6513-3p.

[0114] This invention screens microRNAs that are strongly correlated with the risk of chronic mountain sickness (CMS) by analyzing and comparing plasma microRNA expression profiles. Then, it analyzes the expression levels of each microRNA in a large number of independent plasma samples. Finally, it verifies that hsa-miR-7854-3p, hsa-miR-6513-3p, or hsa-miR-6806-3p in plasma have good screening efficacy between CMS patients and healthy individuals at high altitudes. This confirms that these three microRNAs can accurately screen or diagnose CMS patients at an early stage, and have potential clinical value as plasma biomarkers for early screening and early warning evaluation of CMS patients.

[0115] Based on the early diagnostic value of these three microRNAs for chronic mountain sickness, a screening kit for chronic mountain sickness can be developed. This kit includes reverse transcription primers for the microRNAs, and may also include standard reverse transcription reagents such as reverse transcription buffer, reverse transcriptase mixture, and RNase-free water. It may also include real-time quantitative qPCR reagents, such as the forward and reverse primers required for qPCR, and standard reagents for qPCR amplification detection, such as reverse transcriptase, buffer, dNTPs, MgCl2, ddH2O, fluorescent dyes, Taq enzyme, etc., or commercially available standard matching reagents.

[0116] In the screening experiment of Example 1, a total of 2751 microRNAs were detected. Compared with the microRNA biomarkers for detecting acute mountain sickness, the expression levels of hsa-miR-449b-3p and hsa-miR-4791 were not detected. The detection results of the remaining biomarkers in the chronic mountain sickness part are shown in Table 9. It shows that the expression levels of these microRNAs in patients with chronic mountain sickness are not significantly different from those in normal controls, and therefore cannot be used as biomarkers for detecting chronic mountain sickness.

[0117] Table 9. Results of some other microRNAs

[0118]

[0119] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended 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 to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A micronucleic acid molecular biomarker for diagnosing chronic mountain sickness, characterized in that, The micronucleic acid molecular biomarkers are hsa-miR-7854-3p and / or hsa-miR-6513-3p.

2. The micronucleic acid molecular biomarker according to claim 1, characterized in that, The sequence of hsa-miR-7854-3p is shown in SEQ ID No. 1, and the sequence of hsa-miR-6513-3p is shown in SEQ ID No.

2.

3. The use of a reagent for detecting the expression level of the micronucleic acid molecular biomarker as described in claim 1 or 2 in the preparation of reagents or kits for the auxiliary diagnosis of chronic mountain sickness.

4. The application according to claim 3, characterized in that, The micronucleic acid molecular biomarkers are derived from plasma samples.

5. A reagent kit for diagnosing chronic mountain sickness, characterized in that, The kit contains reagents for detecting the expression levels of hsa-miR-7854-3p or hsa-miR-6513-3p as described in claim 1.

6. The reagent kit according to claim 5, characterized in that, The reagents include primers for reverse transcription of hsa-miR-7854-3p or hsa-miR-6513-3p.

7. The reagent kit according to claim 6, characterized in that, The reagents also include specific amplification primer pairs for the quantitative real-time PCR detection of reverse transcription products.

8. The reagent kit according to any one of claims 5-7, characterized in that, The kit also includes reagents for detecting an internal reference gene, which is at least one selected from miR-16-5p, miR-39, or U6 snRNA.

9. The reagent kit according to claim 8, characterized in that, The primers for reverse transcription of hsa-miR-7854-3p are shown in SEQ ID No. 4; the primers for reverse transcription of hsa-miR-6513-3p are shown in SEQ ID No.

5.

10. The reagent kit according to claim 8, characterized in that, The forward primers for quantitative PCR of hsa-miR-7854-3p reverse transcription products are shown in SEQ ID No. 7, the forward primers for quantitative PCR of hsa-miR-6513-3p reverse transcription products are shown in SEQ ID No. 8, and the universal reverse primers are shown in SEQ ID No. 10.

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