A pd diagnosis and staging kit based on serum exosomal mirna

Abstract

The application discloses a PD diagnosis and staging kit based on serum exosome miRNA. By detecting the expression level of miRNA of a specific sequence of a PD patient, different onset stages of PD, such as an early stage like stage II, are recognized, so that treatment means can be taken as early as possible to prevent the disease from further deterioration. The PD diagnosis and staging marker adopted by the application is derived from exosome miRNA, and compared with peripheral circulation miRNA commonly used for detection, has the advantages of high stability, strong sensitivity and good specificity.

Description

Technical Field

[0001] This invention relates to disease diagnostic screening reagents, specifically to the use of serum exosomal miRNA for PD diagnosis and staging. Background Technology

[0002] Parkinson's disease (PD) is an insidious and severe neurodegenerative disease (NDD). By the time obvious symptoms appear, approximately 70% of dopamine neurons have been irreversibly lost, making early diagnosis of PD crucial. Real-time quantitative reverse transcription PCR (qRT-PCR) is a method primarily used to detect the relative expression levels of specific nucleic acids in various samples. It features a wide detection range, high sensitivity and accuracy, and avoids cross-contamination, making it particularly suitable for testing with small sample volumes. It has been widely used in the diagnosis of various NDDs.

[0003] miRNAs are a class of non-coding single-stranded small RNA molecules composed of 18–25 nucleotides. Multiple studies have shown that changes in peripheral blood miRNA levels in different NDDs have become an important indicator for revealing their developmental stages. However, circulating miRNAs are easily degraded by abundant RNases in the peripheral circulation system, and cannot accurately reflect the state of the central nervous system (CNS), making it difficult to screen for biomarkers for clinical diagnosis from circulating miRNAs.

[0004] Exosomes are extracellular vesicles (EVs) with a diameter of 30–150 nm that can be secreted by almost all cells in the body and can be found in body fluids such as cerebrospinal fluid and blood. miRNAs can be transported out of cells and encapsulated in exosomes (i.e., forming exosomal miRNAs) and carriers such as Argonaute 2 protein, serving as messengers for intercellular communication. Exosomal miRNAs (ex-miRNAs) play an important role in the diagnosis of various NDDs. Compared with circulating miRNAs, ex-miRNAs have the following advantages: (1) The average content of miRNAs in exosomes (42%–48%) is higher than that in plasma (30%); (2) Exosomes can enrich and stabilize miRNAs, preventing miRNAs from being degraded by RNases present in the peripheral circulation system; (3) Exosomes can freely cross the blood-brain barrier, directly reflecting changes in the state of the central nervous system; (4) The extraction, sequencing, and quantification techniques for exosomal RNAs are mature.

[0005] Chinese patent CN110872628A discloses the use of extracellular vesicle biomarkers, such as hsa-miR-199a-3p, in cancer diagnosis. It also points out that the expression levels of extracellular vesicle biomarkers, such as cytokines and exoDNA, can be used to determine whether degenerative diseases such as PD are in an aging state.

[0006] Currently, there is no experimental evidence to predict the biological function of miRNAs based on sequence similarity, and there are no reports of using miRNAs derived from serum exosomes for early diagnosis of PD. Summary of the Invention

[0007] The purpose of this invention is to provide a PD diagnosis and staging kit based on serum exosomal miRNA, which solves the problem in the prior art that miRNA cannot be used to stage PD for early diagnosis.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A PD diagnostic kit includes real-time quantitative PCR primers (e.g., SEQ.ID.NO.1, SEQ.ID.NO.2) for detecting the expression level of biomarkers, wherein the biomarkers are hsa-miR-374a-5p and / or hsa-miR-374b-5p.

[0010] A stage II PD patient staging kit includes real-time quantitative PCR primers (e.g., SEQ.ID.NO.4, SEQ.ID.NO.5) for detecting the expression level of biomarkers, said biomarkers being hsa-miR-199a-3p and / or hsa-miR-195-5p.

[0011] A staged (stage III) kit for PD patients includes real-time quantitative PCR primers (e.g., SEQ.ID.NO.6) for detecting the expression level of a biomarker, said biomarker being hsa-miR-28-5p.

[0012] A staged (stage IV) kit for PD patients includes real-time quantitative PCR primers (e.g., SEQ.ID.NO.3, SEQ.ID.NO.7) for detecting the expression level of biomarkers, said biomarkers being hsa-miR-22-5p and / or hsa-miR-151a-5p.

[0013] A PD diagnostic and staging kit includes real-time quantitative PCR primers for detecting the expression levels of any one or two biomarkers of hsa-miR-374a-5p and hsa-miR-374b-5p, and real-time quantitative PCR primers for detecting the expression levels of any one or more biomarkers of hsa-miR-199a-3p, hsa-miR-195-5p, hsa-miR-28-5p, hsa-miR-22-5p, and hsa-miR-151a-5p.

[0014] Preferably, all of the above markers are extracted from peripheral blood.

[0015] Preferably, the peripheral blood sampling volume is 5–15 mL / individual (e.g., 9–11 mL / individual).

[0016] Preferably, the above markers are extracted from serum exosomes in peripheral blood.

[0017] The above miRNAs (hsa-miR-374a-5p, hsa-miR-374b-5p, hsa-miR-199a-3p, hsa-miR-195-5p, hsa-miR-28-5p, hsa-miR-22-5p, hsa-miR-151a-5p) and their reverse transcripts as biomarkers are used in the preparation of early diagnostic kits and reagents for PD.

[0018] The above miRNAs (hsa-miR-374a-5p, hsa-miR-374b-5p, hsa-miR-199a-3p, hsa-miR-195-5p, hsa-miR-28-5p, hsa-miR-22-5p, hsa-miR-151a-5p) and their reverse transcripts as biomarkers are used in the preparation of PD patient staging kits and reagents.

[0019] The above real-time quantitative PCR primers (sequences shown in SEQ.ID.NO.1~SEQ.ID.NO.7) are used in the preparation of PD early diagnostic kits and reagents.

[0020] Preferably, the kit further includes universal primers for real-time quantitative PCR amplification reaction and internal control sequence amplification primers (e.g., SEQ.ID.NO.8 and SEQ.ID.NO.9).

[0021] The above real-time quantitative PCR primers (sequences shown in SEQ.ID.NO.1~SEQ.ID.NO.7) are used in the preparation of PD patient staging kits and reagents.

[0022] Preferably, the kit further includes universal primers for real-time quantitative PCR amplification reaction and internal control sequence amplification primers (e.g., SEQ.ID.NO.8 and SEQ.ID.NO.9).

[0023] The beneficial effects of this invention are reflected in:

[0024] This invention can identify different stages of PD (e.g., early stages such as stage II) by detecting the expression level of miRNAs of specific sequences in PD patients, thereby enabling early treatment to prevent further deterioration of the disease.

[0025] Furthermore, the PD diagnostic and staging biomarkers used in this invention are derived from exosomal miRNAs, which have the advantages of high stability, high sensitivity, and good specificity compared with peripheral circulating miRNAs commonly used for detection. Attached Figure Description

[0026] Figure 1 The expression of exosomal miRNAs in different individuals: A. hsa-miR-374a-5p; B. hsa-miR-374b-5p; * indicates p<0.05, **** indicates p<0.0001; stage2\3\4 correspond to phases II, III, and IV.

[0027] Figure 2 The expression of exosomal miRNAs in different individuals: A. hsa-miR-199a-3p; B. hsa-miR-195-5p; * indicates p<0.05, ** indicates p<0.001, **** indicates p<0.0001; stage2, 3, and 4 correspond to phases II, III, and IV.

[0028] Figure 3 Expression of exosomal miRNA (hsa-miR-28-5p) in different individuals: **** indicates p<0.0001; stage2\3\4 correspond to phases II, III, and IV.

[0029] Figure 4 The expression of exosomal miRNAs in different individuals: A. hsa-miR-22-5p; B. hsa-miR-151a-5p; **** indicates p<0.0001; stage2\3\4 correspond to phases II, III, and IV.

[0030] Figure 5A To analyze the expression level of hsa-miR-374a-5p using ROC curves.

[0031] Figure 5BTo analyze the expression level of hsa-miR-374b-5p using ROC curves.

[0032] Figure 6A To analyze the expression level of hsa-miR-199a-3p using ROC curves.

[0033] Figure 6B To analyze the expression level of hsa-miR-195-5p using ROC curves.

[0034] Figure 7 To analyze the expression level of hsa-miR-28-5p using ROC curves.

[0035] Figure 8A To analyze the expression level of hsa-miR-22-5p using ROC curves.

[0036] Figure 8B To analyze the expression level of hsa-miR-151a-5p using ROC curves.

[0037] Figure 9 Gaussian kernel density distribution of peripheral blood exosomal miRNA sequencing data from PD patients at different stages. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The embodiments described are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0039] Part 1: PD Diagnosis and Staging Procedure Trial

[0040] (a) Collect peripheral blood from individuals and extract serum exosomes. The inclusion and exclusion criteria for patients are as follows:

[0041] 1.1 Inclusion Criteria

[0042] According to the Chinese Medical Association's "Diagnostic Criteria for Parkinson's Disease in China (2016 Edition)":

[0043] 1) Patients with primary Parkinson's disease.

[0044] 2) Patients with H&Y stage II, III, or IV, the specific staging criteria are shown in Table 1-0.

[0045] Table 1-0. H&Y Installment Standards

[0046]

[0047] 3) No age or gender restrictions.

[0048] 1.2 Exclusion Criteria

[0049] 1) Patients who do not meet the inclusion criteria.

[0050] 2) Patients with Alzheimer's disease, Huntington's disease, hydrocephalus, neurocysticercosis, acute cerebral hemorrhage, and brain tumors, etc.

[0051] 3) Individuals with serious primary diseases of the liver, kidneys, hematopoietic system, etc.

[0052] 4) Suspected or confirmed drug users, alcoholics (more than 14 units per week, 1 unit = 8g of pure alcohol) or smokers (more than 5 cigarettes per day).

[0053] 5) Those with poor compliance who are not expected to complete blood collection and follow-up according to the study protocol.

[0054] 6) Other situations where selection is not appropriate.

[0055] 2. Collect peripheral blood from PD patients and normal controls.

[0056] 1) Collect approximately 10 mL of blood from an individual using a serum tube that does not contain anticoagulants.

[0057] 2) Let stand at room temperature for 30 minutes, then let stand at 4°C for 3-5 hours or overnight, until blood clots are visible.

[0058] 3) Centrifuge at 3000g for 15 minutes at 4℃, and carefully transfer the serum into a new 15mL centrifuge tube to ensure the serum quality to the greatest extent possible.

[0059] 4) After centrifugation, extract exosomes from the serum obtained in a timely manner or store it in a -80℃ refrigerator for later use.

[0060] 3. Extraction of exosomes

[0061] 1) All accessories for the ultracentrifuge are sterilized in a high-pressure steam sterilizer in advance, and the ultracentrifuge tubes are sterilized by irradiating them with ultraviolet light overnight.

[0062] 2) Remove the serum from the refrigerator, thaw it in a water bath, and dilute it to 11 mL with clean PBS, ensuring that each tube of serum has the same volume.

[0063] 3) Centrifuge at 3000g and 4℃ for 10 minutes, and carefully transfer all the supernatant to a new 15mL centrifuge tube.

[0064] 4) Centrifuge at 10000g and 4℃ for 30min; after centrifugation, transfer the supernatant to an ultracentrifuge tube and balance with PBS.

[0065] 5) Centrifuge at 100000g and 4℃ for 70 minutes, then turn off the centrifuge brake; after centrifugation, leave about 1.5mL of supernatant, add PBS to resuspend and balance.

[0066] 6) Centrifuge at 100,000g, 4℃ for 70 min, then turn off the centrifuge brake; discard the supernatant until the bottom layer has 50-100 μL remaining; transfer the remaining bottom layer (mainly containing serum exosomes) to an RNase-free EP tube for the next experiment or store at -80℃ for later use.

[0067] (II) Acquisition and Preprocessing of High-Throughput Sequencing Data

[0068] High-throughput sequencing of small RNAs was performed on peripheral blood exosomes from patients with different stages of PD, yielding 103 miRNA sequencing data points. After normalization using the TMM method, Gaussian kernel density distribution maps were plotted. Figure 9 The results showed that the expression levels of each sample were generally consistent, with the Log2 counts per million (Logcpm) mostly ranging from -5 to 15, indicating potential for further analysis. Based on the PD stages of the samples, the miRNA sequencing data were divided into normal control, stage 2, stage 3, and stage 4. Differential expression analysis was performed between any two groups using both edgeR and t-test methods (i.e., control vs. stage 2, control vs. stage 3, control vs. stage 4, stage 2 vs. stage 3, stage 2 vs. stage 4, stage 3 vs. stage 4), yielding a total of 185 differentially expressed miRNAs. Of these, 71 were identified using the edgeR method, 158 using the t-test, and 44 showed differences using both methods.

[0069] (III) Screening for miRNAs expressed at different stages of PD using WGCNA

[0070] Clustering trees were constructed at each phase of Phase PD using all 185 differentially expressed miRNAs. The trees were built based on their dissimilarity, and the resulting clustered modules were then fused. All generated modules were exported as point-edge files, imported into Cytoscape, and their colors were set to obtain a visualized co-expression network diagram. Ultimately, 9 effective modules were obtained in the normal control group, 7 in Phase II, 7 in Phase III, and 11 in Phase IV.

[0071] In the co-expression networks constructed for different stages, to further determine the connections between different stages in the progression of Parkinson's disease (PD), miRNAs that play a role throughout the entire PD pathogenesis were screened. The node relationships of miRNAs within different stages established in the previous step were transformed into module relationships to identify miRNAs with characteristics indicative of the entire PD pathogenesis stage. By constructing connections between modules of different stages, modules containing miRNAs appearing in different stages of PD were linked together. Modules containing these miRNAs are associated; the more identical miRNAs present in a module, the stronger the association between the two modules, and the more critical the role of that module in the disease. Based on this, a total of 21 modules and 114 miRNAs were obtained. These modules and miRNAs had a weak correlation with PD. To further screen them, the obtained miRNAs were compared with PD-related miRNAs in the Human miRNA Disease Database (HMDD). Modules containing miRNAs that could not be matched were deleted, ultimately resulting in 11 PD-related modules and 30 PD-related miRNAs. Two modules were obtained in phase II, three modules in phase III, and six modules in phase IV. Four miRNAs (hsa-miR-374a-5p, hsa-miR-374b-5p, hsa-miR-19b-3p, and hsa-miR-9-5p) were associated with phases II, III, and IV, which may reflect the common biological characteristics of different PD stages.

[0072] After obtaining miRNAs reflecting the evolution of PD using WGCNA, we analyzed phase-specific miRNAs reflecting the characteristics of different PD stages. From 185 miRNAs, we selected a subset and again constructed cluster trees for each PD stage using WGCNA. Unlike before, when constructing the cluster trees for this stage, we only used the differentially expressed miRNAs between this stage and other stages; a total of 145 such miRNAs were used. Similarly, after determining the soft threshold β (β = 0.43 for stage II, β = 0.05 for the rest), we obtained cluster trees for different stages and fused the modules. These were then imported into Cytoscape to obtain a visualized co-expression network diagram. Ultimately, we obtained 9 effective modules in the normal control group, 11 effective modules in stage II, 8 effective modules in stage III, and 9 effective modules in stage IV.

[0073] After obtaining the co-expression networks for each stage, in order to obtain miRNAs that reflect the specific biological characteristics of different stages of PD and reveal the staged disease characteristics of PD, we performed miRNA functional enrichment analysis within each stage of PD to identify modules and miRNAs closely related to different stages of PD disease progression from a biological perspective. The miRNAs in each phase module were functionally enriched using the TAM database (Version 2.0, http: / / www.lirmed.com / tam2 / ). Entries with a false discovery rate (FDR) < 0.05 were selected, and 15 biological functions closely related to the occurrence and development of PD were chosen: neurotoxicity, aging, cell death, cell differentiation, regulation of the Nf-κb signaling pathway, hormone-mediated signaling pathway, inflammation, neural stem cell differentiation, apoptosis, regulation of the Akt signaling pathway, DNA damage response, immune response, regulation of stem cell, neuron development, and cell reprogramming. The modules containing miRNAs with these functional entries were then retained, resulting in 18 modules and 88 miRNAs. To strengthen the correlation between these modules and miRNAs and PD, these miRNAs were compared with PD-related miRNAs in HMDD, and the modules containing the miRNAs that could be matched were retained, ultimately yielding 16 PD-related modules and 25 PD-related miRNAs. Among them, 5 modules were obtained in phase II, 4 modules in phase III, and 7 modules in phase IV; 3 miRNAs were specifically related to phase II only, namely hsa-miR-199a-3p, hsa-miR-195-5p, and hsa-miR-28-3p.One miRNA, hsa-miR-28-5p, is specifically associated with phase III only, while nine miRNAs are specifically associated with phase IV only: hsa-miR-151a-3p, hsa-miR-183-3p, hsa-miR-29a-3p, hsa-miR-151a-5p, hsa-miR-205-5p, hsa-miR-29b-3p, hsa-miR-29c-3p, hsa-miR-30b-5p, and hsa-miR-22-5p. These 13 miRNAs are likely phase-specific miRNAs of PD, reflecting the biological characteristics of different stages in the development and progression of PD.

[0074] (iv) Screening of miRNAs expressed at different stages of PD using receiver operating characteristic (ROC) curve analysis and qRT-PCR experiments.

[0075] Using methods such as constructing a co-expression network with WGCNA, ROC curve analysis, and qRT-PCR experiments, six miRNAs that are expressed only in serum exosomes and have diagnostic value for different stages of PD were finally screened. These are: hsa-miR-374a-5p, hsa-miR-374b-5p, hsa-miR-199a-3p, hsa-miR-28-5p, hsa-miR-22-5p, and hsa-miR-151a-5p.

[0076] Part Two: Acquisition of PD cases at different stages and analysis of miRNAs with diagnostic value for different stages of PD

[0077] (I) Peripheral blood samples were collected again from the Department of Neurology and the Physical Examination Center of Xijing Hospital, with the consultation period from June 2020 to October 2020. Among them, there were 10 normal controls, 7 cases of stage II, 12 cases of stage III, and 11 cases of stage IV. The inclusion and exclusion criteria were the same as before.

[0078] (II) qRT-PCR detection

[0079] 1. Primers

[0080] qRT-PCR primers were synthesized by Shanghai Sangon Biotech Co., Ltd., and the primer sequences are shown in Tables 1-1 and 1-2:

[0081] Table 1-1. Specific primers for miRNA real-time quantitative PCR amplification reaction

[0082]

[0083]

[0084] The universal primers for real-time quantitative PCR amplification of the above miRNAs are the primers in the miScript II RT kit (cat#218161, QIAGEN).

[0085] Table 1-2. Primers for internal reference sequences in real-time quantitative PCR

[0086]

[0087] 2. Reverse transcription to synthesize cDNA

[0088] 1) Prepare the reaction mixture as shown in Table 2 in an RNase-free centrifuge tube, and gently pipette to mix, avoiding shaking; the template RNA is total RNA obtained by extracting RNA contained in serum exosomes (i.e., serum exosomal RNA) using the Trizol method.

[0089] Table 2. Reverse transcription reaction system

[0090]

[0091] 2) Set the PCR instrument program according to the steps shown in Table 3. After the reaction is complete, cDNA can be obtained, which can be used for subsequent experiments or stored in a -20℃ refrigerator for later use.

[0092] Table 3. Reverse Transcription Process

[0093]

[0094] 3. Real-time quantitative PCR reaction

[0095] 1) Dilute the cDNA sample (referring to the quantitative peripheral blood serum exosomal RNA reverse transcription product of each individual) 5 times.

[0096] 2) Prepare the reaction system mixture as shown in Table 4, gently mix by blowing, centrifuge at 3000 rpm at room temperature, and keep away from light.

[0097] Table 4. Real-time quantitative PCR reaction system

[0098]

[0099] 3) Amplify using a real-time quantitative PCR instrument, following the steps shown in Table 5.

[0100] Table 5. Real-time quantitative PCR reaction program

[0101]

[0102] After obtaining the Ct values ​​for each group of samples (PD patients at different stages and normal controls), they were calculated according to Formula 2. -ΔΔCt The final quantitative results were calculated. All experiments were performed in triplicate, and the average of the three results was taken. Statistical analysis was performed using GraphPad Prism8, with one-way ANOVA used to analyze the results. Differences between groups were analyzed using Dunnett's multiple comparisons test. Receiver operating characteristic (ROC) curve analysis was then conducted.

[0103] Part Three: PD Diagnosis and Staging Result Verification

[0104] 3.1 Verification of miRNA expression related to each phase

[0105] In the preliminary results (Part 1), two miRNAs were found to be associated with phases II, III, and IV: hsa-miR-374a-5p and hsa-miR-374b-5p. After validation in 40 patients, it was found that hsa-miR-374a-5p (…) was associated with phases II, III, and IV, respectively. Figure 1 A) and hsa-miR-374b-5p Figure 1 The expression level of B) showed differences in stages II, III, and IV (significantly higher than the normal control group; hsa-miR-374a-5p: control 1.032, stage II 3.431, stage III 7.503, stage IV 6.393; hsa-miR-374b-5p: control 1.081, stage II 3.176, stage III 6.640, stage IV 7.502).

[0106] ROC curve analysis revealed that hsa-miR-374a-5p ( Figure 5A ) and hsa-miR-374b-5p ( Figure 5BThis assay can distinguish between stages II, III, and IV and the normal control group. Specifically, the area under the curve (AUC) and corresponding 95% confidence intervals (95% CI) for hsa-miR-374a-5p were 0.758 (95% CI: 57.21–99.40, p = 0.026), 0.780 (95% CI: 67.33–88.58, p < 0.0001), and 0.763 (95% CI: 62.13–90.36, p < 0.026), respectively. =0.0012); the AUC values ​​and corresponding 95% CIs of hsa-miR-374b-5p were 0.798 (95% CI: 60.84-98.84, p = 0.0101), 0.742 (95% CI: 62.40-85.98, p = 0.0004), and 0.761 (95% CI: 61.82-90.38, p = 0.0013), respectively.

[0107] 3.2 Verification of miRNA expression specifically associated with phases II, III, and IV.

[0108] In the preliminary results (Part 1), two miRNAs, hsa-miR-199a-3p and hsa-miR-195-5p, were found to be specifically associated only with phase II; one miRNA, hsa-miR-28-5p, was specifically associated only with phase III; and two miRNAs, hsa-miR-151a-5p and hsa-miR-22-5p, were specifically associated only with phase IV.

[0109] Similarly, after validation, among the two miRNAs specifically associated with phase II, hsa-miR-199a-3p showed the highest concentration compared to control, phase III, and phase IV miRNAs. Figure 2 A), hsa-miR-195-5p Figure 2 The expression level of hsa-miR-199a-3p showed a significant difference only in stage II (significantly lower than in control, stage III, and stage IV; hsa-miR-199a-3p: control 1.018, stage II 0.2857, stage III 1.027, stage IV 1.096; hsa-miR-195-5p: control 1.004, stage II 0.5966, stage III 1.069, stage IV 1.522); ROC curve results showed that hsa-miR-199a-3p could distinguish stage II from stage III, stage IV, and the normal control group. Figure 6AThe AUC values ​​and corresponding 95% CIs were 0.738 (95% CI: 55.97-91.61, p = 0.0402), 0.729 (95% CI: 54.93-90.91, p = 0.0416), and 0.756 (95% CI: 55.97-95.17, p = 0.0348), respectively. However, hsa-miR-195-5p could not differentiate between stage II, stage III, stage IV, and the normal control group. Figure 6B ).

[0110] Similarly, after validation, the expression level of only one miRNA specifically associated with phase III (hsa-miR-28-5p) showed a significant difference only in phase III compared to control, phase II, and phase IV (significantly higher than control, phase II, and phase IV). Figure 3 The control group (P<0.036) was 1.036, stage II (P<0.016) was 1.244, stage III (P<0.016) was 5.626, and stage IV (P<0.016) was 1.414. The ROC curve results showed that hsa-miR-28-5p could differentiate stage III from stage II, stage IV, and the normal control group. Figure 7 The AUC values ​​and corresponding 95% CIs were 0.746 (95% CI: 63.47-85.68, p = 0.0004), 0.789 (95% CI: 64.07-93.67, p = 0.0103), and 0.738 (95% CI: 61.69-85.93, p = 0.0019), respectively.

[0111] Similarly, after validation, among the two miRNAs specifically associated with phase IV, hsa-miR-22-5p showed a higher concentration compared to control, phase II, and phase III miRNAs. Figure 4 A), hsa-miR-151a-5p Figure 4 The expression level of hsa-miR-22-5p showed significant differences only in stage IV (the former was significantly higher than the latter and significantly lower than the control, stage II, and stage III; hsa-miR-22-5p: control 1.063, stage II 1.115, stage III 1.454, stage IV 4.945; hsa-miR-151a-5p: control 1.045, stage II 1.120, stage III 1.035, stage IV 0.6633). The ROC curve results showed that hsa-miR-22-5p could distinguish stage IV from the normal control group, stage II, and stage III. Figure 8AThe AUC values ​​and corresponding 95% CIs were 0.817 (95% CI: 70.23-93.12, p<0.0001), 0.773 (95% CI: 54.95-99.59, p=0.0244), and 0.700 (95% CI: 57.13-82.91, p=0.0089), respectively; hsa-miR-151a-5p can differentiate stage IV from the normal control group, stage II, and stage III. Figure 8B The AUC values ​​and corresponding 95% CIs were 0.741 (95% CI: 60.65-87.44, p = 0.0031), 0.796 (95% CI: 60.0-99.09, p = 0.0147), and 0.708 (95% CI: 58.12-83.44, p = 0.0066), respectively.

[0112] 3.3 Reagent Kit Interpretation

[0113] Based on the validation results, the biomarkers used in the kit for PD diagnosis were determined to be hsa-miR-374a-5p and hsa-miR-374b-5p. The relative expression level of the biomarkers was determined to be 3. That is, if the expression level is higher than this threshold (since the relative expression level of the normal control is about 1, it can also be determined as "expression"), the individual is identified as a PD patient.

[0114] Based on the validation results, the biomarker used in the kit for staging PD patients (Stage II) was determined to be hsa-miR-199a-3p, and the relative expression level threshold for the biomarker was set at 0.3; that is, PD patients were classified as Stage II if their expression level was below this threshold. Based on the validation results, the biomarker used in the kit for staging PD patients (Stage III) was determined to be hsa-miR-28-5p, and the relative expression level threshold for the biomarker was set at 5; that is, PD patients were classified as Stage III if their expression level was above this threshold.

[0115] Based on the validation results, the biomarker used in the kit for PD patient staging (Stage IV) can be hsa-miR-22-5p, with a relative expression level threshold of 5; PD patients are classified as Stage IV if their relative expression level is above this threshold. Alternatively, the biomarker used in the kit for PD patient staging (Stage IV) can also be hsa-miR-151a-5p, with a relative expression level threshold of 0.7; PD patients are classified as Stage IV if their relative expression level is below this threshold.

[0116] Combined with clinical symptom verification, the results show that using the above biomarkers for kit detection can accurately locate the stage of PD, thereby effectively helping clinicians to carry out different treatment methods according to the different stages of PD patients' disease development, and providing reliable clinical test evidence for the early diagnosis and treatment of PD. <110> Air Force Medical University of the Chinese People's Liberation Army <120> A diagnostic and staging kit for PD based on serum exosomal miRNA <160> 9 <210> 1 <211> twenty two <212> DNA <213> Artificial synthesis <400> 1 ttataataca acctgataag tg 22 <210> 2 <211> twenty two <212> DNA <213> Artificial synthesis <400> 2 atataataca acctgctaag tg 22 <210> 3 <211> twenty three <212> DNA <213> Artificial synthesis <400> 3 tgtaaacatc ctacactctc agc 23 <210> 4 <211> twenty two <212> DNA <213> Artificial synthesis <400> 4 acagtagtct gcacattggt ta 22 <210> 5 <211> twenty one <212> DNA <213> Artificial synthesis <400> 5 tagcagcaca gaaatattgg c 21 <210> 6 <211> twenty two <212> DNA <213> Artificial synthesis <400> 6 aaggagctca cagtctattg ag 22 <210> 7 <211> twenty one <212> DNA <213> Artificial synthesis <400> 7 tcgaggagct cacagtctag t 21 <210> 8 <211> 16 <212> DNA <213> Artificial synthesis <400> 8 gctcgcttcg gcagca 16 <210> 9 <211> 20 <212> DNA <213> Artificial synthesis <400> 9 aacgcttcac gaatttgcgt 20

Claims

1. A PD diagnosis and staging kit, characterized in that: This includes real-time quantitative PCR primers for detecting the expression level of the biomarker hsa-miR-374a-5p, and real-time quantitative PCR primers for detecting the expression level of the biomarker hsa-miR-22-5p.

2. The application of any one or more of hsa-miR-374a-5p and hsa-miR-199a-3p, hsa-miR-195-5p, hsa-miR-28-5p, hsa-miR-22-5p, and hsa-miR-151a-5p in the preparation of PD diagnostic and PD patient staging kits and reagents.

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