Molecular marker for diagnosing severity of Parkinson's disease and application of molecular marker

Abstract

The invention relates to a marker for predicting the severity or staging of Parkinson's disease and application of the marker. Specifically, exosome miRNA (micro Ribonucleic Acid) is two or more of hsa-miR-106b-5p, hsa-miR-142-5p and hsa-miR-421. The molecular marker provided by the invention is applied to prediction of the severity or staging of the Parkinson's disease and has better diagnosis efficiency, and a new way is provided for diagnosis or auxiliary diagnosis of the severity or staging of the Parkinson's disease.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, more specifically, to the field of RNA technology, and in particular to a molecular marker for diagnosing the severity of Parkinson's disease and an application thereof. Background Art

[0002] Parkinson's disease (PD) is a chronic progressive neurological disease that usually occurs in middle-aged and elderly people. The disease is mainly characterized by the loss of dopaminergic neurons and motor dysfunction. According to the severity of the disease and the manifestation of symptoms, Parkinson's disease can be divided into five stages, and the severity of symptoms increases step by step. The diagnosis of Parkinson's disease is usually confirmed through a doctor's clinical evaluation and related examinations, such as neurological examinations and brain imaging examinations.

[0003] Since PD has atypical symptoms in the early stages of the disease and is easily confused with diseases such as multiple system atrophy and progressive supranuclear palsy, early diagnosis of PD is still a difficult problem. PD patients often fall into the dilemma of entering the middle and late stages once diagnosed, causing many PD patients to miss the opportunity for early diagnosis and early treatment. Parkinson's disease has a serious impact on the patient's quality of life and daily life functions, so timely diagnosis and treatment are crucial.

[0004] Biomarkers are beneficial for early diagnosis and prediction of disease progression, which will facilitate treatment and more accurate disease monitoring, ultimately improving the prognosis of PD patients. Although some progress has been made in the study of PD blood markers, there are still some challenges: (1) low specificity and sensitivity make it difficult to distinguish different disease stages; (2) some potential biomarkers, such as α-synuclein oligomers, are difficult to detect and require more sensitive detection technology; (3) PD is a heterogeneous disease, and the pathophysiological mechanisms of different patients may be different, which poses a challenge to finding universal blood markers.

[0005] With the development of molecular biology technology, miRNA, as a type of small non-coding RNA, has attracted attention due to its regulatory role in a variety of diseases. Studies have shown that the expression level of specific miRNA changes in PD patients, which may be closely related to the occurrence and development of the disease. However, there is currently no widely recognized miRNA marker for PD staging prediction. Summary of the invention

[0006] The purpose of the present invention is to provide a group of molecular markers, kits and applications thereof for diagnosing or assisting in diagnosing the severity of Parkinson's disease with high sensitivity and good specificity.

[0007] In order to solve the above problems, the present invention adopts the following technical solutions:

[0008] On the one hand, the present invention provides the use of a reagent for detecting molecular markers in the preparation of a product for diagnosing or assisting in the diagnosis of Parkinson's disease severity or staging, wherein the molecular markers are two or more of hsa-miR-106b-5p, hsa-miR-142-5p and hsa-miR-421.

[0009] Preferably, the molecular markers include at least hsa-miR-106b-5p, hsa-miR-142-5p and hsa-miR-421.

[0010] Preferably, the molecular markers also include one or more of hsa-let-7b, hsa-let-7g-5p, hsa-miR-191-5p, hsa-miR-22-5p, hsa-miR-26a-5p, hsa-miR-26b-5p and hsa-miR-27b.

[0011] Preferably, the reagents include miRNA primers and / or probes, and the miRNA primers include reverse transcription primers, RT-PCR upstream primers and RT-PCR downstream primers.

[0012] Preferably, the reverse transcription primer sequence of hsa-miR-106b-5p is shown in SEQ ID NO: 7, the RT-PCR upstream primer sequence is shown in SEQ ID NO: 8, the RT-PCR downstream primer sequence is shown in SEQ ID NO: 34, and the probe sequence is shown in SEQ ID NO: 9;

[0013] The reverse transcription primer sequence of the hsa-miR-142-5p is shown in SEQ ID NO: 10, the RT-PCR upstream primer sequence is shown in SEQ ID NO: 11, the RT-PCR downstream primer sequence is shown in SEQ ID NO: 34, and the probe sequence is shown in SEQ ID NO: 12;

[0014] The reverse transcription primer sequence of the hsa-miR-421 is shown in SEQ ID NO: 28, the RT-PCR upstream primer sequence is shown in SEQ ID NO: 29, the RT-PCR downstream primer sequence is shown in SEQ ID NO: 34, and the probe sequence is shown in SEQ ID NO: 30.

[0015] Further preferably, the reagent also includes a reagent for detecting the internal reference hsa-miR-423-5p.

[0016] More preferably, the reverse transcription primer sequence of the hsa-miR-423-5p is shown in SEQ ID NO: 31, the RT-PCR upstream primer sequence is shown in SEQ ID NO: 32, the RT-PCR downstream primer sequence is shown in SEQ ID NO: 34, and the probe sequence is shown in SEQ ID NO: 33.

[0017] Preferably, the molecular marker is derived from exosomes, and the exosomes are derived from one or more of blood, saliva, urine and sputum.

[0018] On the other hand, the present invention provides a kit for diagnosing or assisting in the diagnosis of the severity or staging of Parkinson's disease, the kit comprising a reagent for detecting the expression levels of two or more of the molecular markers hsa-miR-106b-5p, hsa-miR-142-5p and hsa-miR-421.

[0019] Preferably, the kit further comprises a reagent for detecting the expression level of one or more of the molecular markers hsa-let-7b, hsa-let-7g-5p, hsa-miR-191-5p, hsa-miR-22-5p, hsa-miR-26a-5p, hsa-miR-26b-5p and hsa-miR-27b.

[0020] Preferably, the reagents include miRNA primers and probes, and the miRNA primers include reverse transcription primers, RT-PCR upstream primers and RT-PCR downstream primers.

[0021] Preferably, the kit further comprises RT-PCR detection reagents and / or standards.

[0022] In another aspect, the present invention provides a device for diagnosing or assisting in the diagnosis of Parkinson's disease severity or stage, the device comprising an information acquisition module, a calculation module and a diagnosis module, wherein:

[0023] The information acquisition module is used to perform an operation of acquiring test information of the subject, wherein the test information includes Ct values ​​of two or more of hsa-miR-106b-5p, hsa-miR-142-5p and hsa-miR-421, and the Ct values ​​are normalized by an internal reference miRNA;

[0024] The calculation module is used to perform the operation of substituting the Ct value of the molecular marker, the age and gender of the subject into the diagnostic model to obtain the model value;

[0025] The diagnosis module is used to execute an operation of determining the severity or stage of Parkinson's disease suffered by the subject according to the model value.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention provides the use of a reagent for detecting molecular markers in diagnosing or assisting in the diagnosis of Parkinson's severity or staging, which has high sensitivity and high specificity, and provides a more accurate prediction for the staging of PD. At the same time, the present invention predicts the PD staging through blood exosome specimens, which has the characteristics of non-invasiveness and high accuracy. The specificity of the combined detection of multiple miRNAs for the interpretation of PD staging is 76.3%, the sensitivity is 81.8%, and the compliance rate reaches 77.4%. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the identification result of the exosomes extracted in Example 1 of the present invention, wherein Part A is an electron micrograph of brain-derived exosomes, in which a typical saucer-like vesicle structure can be seen; Part B is a particle size distribution diagram of brain-derived exosomes, 90% of which are between 30-150nm, meeting the exosome standard; Part C is the quality inspection result of brain-derived exosome small RNA, with a peak in the range of 15-40nt.

[0029] Figure 2 This is a heat map of the sequencing results of some differentially expressed miRNAs in Example 2 of the present invention.

[0030] Figure 3 This is the ROC curve of the multivariate model constructed based on the random forest algorithm combining gender and age factors.

[0031] Figure 4 The figure is a scatter plot of the relative expression of targets in samples from different cohorts.

[0032] Figure 5 This is a ROC curve diagram of the auxiliary staging diagnosis model provided by the present invention. DETAILED DESCRIPTION

[0033] In order to make the technical solutions and beneficial effects of the present invention more clearly understandable, the following is a detailed description by listing specific embodiments. The drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0034] After extensive and in-depth research, the inventors unexpectedly screened out molecular markers closely related to Parkinson's stage through high-throughput sequencing and bioinformatics analysis, and the expression patterns of these markers in PD patients were significantly different from those in healthy controls. The markers are brain-derived exosomal miRNAs derived from plasma, including 10 exosomal miRNAs and corresponding combinations, wherein the 10 exosomal miRNAs are: hsa-let-7b, hsa-let-7g-5p, hsa-miR-106b-5p, hsa-miR-142-5p, hsa-miR-191-5p, hsa-miR-22-5p, hsa-miR-26a-5p, hsa-miR-26b-5p, hsa-miR-27b and hsa-miR-421. In particular, when a combination of hsa-miR-106b-5p, hsa-miR-142-5p and hsa-miR-421 is selected, it has high sensitivity and high specificity, and the diagnostic model constructed by combining age and gender improves the stability and accuracy of the detection. The experiment further shows that the marker of the present invention can be used to predict the severity or stage of Parkinson's disease. On this basis, the present invention was completed.

[0035] As used in the present invention, "plurality" means any integer. Preferably, the "plurality" in "one or more" can be any integer within a range, including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12.

[0036] As used herein, the term "about" encompasses a range of ±25% of a given value. In other embodiments, the term "about" encompasses a range of ±20%, ±15%, ±10%, or ±5% of a given value. For example, in one embodiment, "about 3 grams" means a value of 2.7-3.3 grams (i.e., 3 grams ±10%), etc.

[0037] As used in the present invention, the term "exosome" refers to a small vesicle with a complete membrane structure, with a diameter between 30-150nm, secreted by a variety of cells and widely present and distributed in body fluids including blood, saliva, urine, pleural effusion, etc., and is an important medium for intercellular information transmission. Exosomes contain a large number of inclusions such as miRNA, mRNA and protein secreted by their source cells, so exosomes can become liquid biopsy components.

[0038] As used in the present invention, microRNA (miRNA) is a type of endogenous short-chain RNA with a length of about 20-24 nucleotides. It is a non-coding RNA that participates in the expression regulation of various target genes in the post-transcriptional stage. Almost all types of cells release miRNAs into the circulatory system passively (e.g., through apoptotic bodies) or actively (e.g., in exosomes or microvesicles). These molecules can affect the homeostasis of other tissues through mechanisms similar to paracrine signals, or trigger some pathogenic mechanisms, including the transformation of normal cells into tumor cells and the promotion of tumor cell proliferation. A large number of miRNAs only work after being packaged in exosomes for transport.

[0039] As used in the present invention, "miRNA", "microRNA", "miRNA marker of the present invention" and "exosomal miRNA" are used interchangeably to refer to the exosomal miRNA of the present invention for diagnosing or assisting in the diagnosis of the severity of Parkinson's disease.

[0040] It should be noted that the “hsa” before the miRNA represents the corresponding species, hsa stands for human, and the “5p” or “3p” after the miR is used to distinguish which arm the miRNA is formed by.

[0041] The exosomal miRNA of the present invention includes one or more of: hsa-let-7b, hsa-let-7g-5p, hsa-miR-106b-5p, hsa-miR-142-5p, hsa-miR-191-5p, hsa-miR-22-5p, hsa-miR-26a-5p, hsa-miR-26b-5p, hsa-miR-27b and hsa-miR-421. The expression patterns of these markers in PD patients are significantly different from those in healthy controls, and can be used to determine or predict the stage of Parkinson's disease in patients.

[0042] The sequences of the 10 markers of the present invention are shown in Table 1:

[0043] Table 1

[0044]

[0045]

[0046] On the one hand, the present invention provides the use of a reagent for detecting molecular markers in the preparation of a product for diagnosing or assisting in the diagnosis of Parkinson's disease severity or staging, wherein the molecular markers are two or more of hsa-miR-106b-5p, hsa-miR-142-5p and hsa-miR-421.

[0047] In certain embodiments, the molecular markers include at least hsa-miR-106b-5p, hsa-miR-142-5p and hsa-miR-421.

[0048] In certain embodiments, the molecular markers further include one or more of hsa-let-7b, hsa-let-7g-5p, hsa-miR-191-5p, hsa-miR-22-5p, hsa-miR-26a-5p, hsa-miR-26b-5p and hsa-miR-27b.

[0049] In certain embodiments, the reagents include miRNA primers and / or probes, and the miRNA primers include reverse transcription primers, RT-PCR upstream primers, and RT-PCR downstream primers.

[0050] As used in the present invention, "primer" refers to a nucleic acid molecule with a specific nucleotide sequence that guides synthesis at the start of nucleotide polymerization. Primers are usually two artificially synthesized oligonucleotide sequences, one primer is complementary to a DNA template strand at one end of the target region, and the other primer is complementary to another DNA template strand at the other end of the target region, and its function is to serve as the starting point of nucleotide polymerization. Primers artificially designed in vitro are widely used in polymerase chain reaction (PCR), qPCR, sequencing and probe synthesis, etc. Typically, primers are designed to amplify products with a length of 50 to 150 bp, 60 to 140 bp, 70 to 130 bp, 80 to 120 bp. The design method of PCR primers for templates is known in the art. Preferably, the product length is 50 to 100 bp.

[0051] As used in the present invention, "probe" mainly refers to a DNA or RNA sequence that hybridizes with a target sequence under stringent conditions. Usually, the 5' end of the probe sequence is labeled with a fluorescent reporter group, and the 3' end is labeled with a quencher group. When discussing primers or probes, "recognition" or "hybridization" as described herein refers to hybridization of a primer or probe with a template sequence under stringent or highly stringent conditions, which are well known in the art. For example, the highly stringent conditions may be hybridization at 65°C and washing the membrane in a solution of 0.1×SSPE (or 0.1×SSC), 0.1% SDS.

[0052] In certain embodiments, the reverse transcription primer sequence of hsa-miR-106b-5p is shown in SEQ ID NO: 7, the RT-PCR upstream primer sequence is shown in SEQ ID NO: 8, the RT-PCR downstream primer sequence is shown in SEQ ID NO: 34, and the probe sequence is shown in SEQ ID NO: 9;

[0053] The reverse transcription primer sequence of the hsa-miR-142-5p is shown in SEQ ID NO: 10, the RT-PCR upstream primer sequence is shown in SEQ ID NO: 11, the RT-PCR downstream primer sequence is shown in SEQ ID NO: 34, and the probe sequence is shown in SEQ ID NO: 12;

[0054] The reverse transcription primer sequence of the hsa-miR-421 is shown in SEQ ID NO: 28, the RT-PCR upstream primer sequence is shown in SEQ ID NO: 29, the RT-PCR downstream primer sequence is shown in SEQ ID NO: 34, and the probe sequence is shown in SEQ ID NO: 30.

[0055] Further preferably, the reagent also includes a reagent for detecting the internal reference hsa-miR-423-5p.

[0056] More preferably, the reverse transcription primer sequence of the hsa-miR-423-5p is shown in SEQ ID NO: 31, the RT-PCR upstream primer sequence is shown in SEQ ID NO: 32, the RT-PCR downstream primer sequence is shown in SEQ ID NO: 34, and the probe sequence is shown in SEQ ID NO: 33.

[0057] The reverse transcription primers, RT-PCR upstream primers and RT-PCR downstream primers involved in this application are shown in Table 2:

[0058] Table 2

[0059]

[0060]

[0061] In the present application, the reverse universal primer for the markers hsa-let-7g-5p and hsa-miR-26b-5p is universal primer-R2, as shown in SEQ ID NO:35; the reverse universal primer for other markers is universal primer-R1, as shown in SEQ ID NO:34.

[0062] In the present application, the specific probe of the present invention is a fluorescent probe, and the fluorescent group is a conventional fluorescent group in the art. The present application does not impose any limitation on the fluorescent group, as long as it can realize RT-PCR real-time fluorescence quantitative detection.

[0063] In certain embodiments, the molecular marker is derived from exosomes, and the exosomes are derived from one or more of blood, saliva, urine and sputum.

[0064] On the other hand, the present invention provides a kit for diagnosing or assisting in the diagnosis of the severity or staging of Parkinson's disease, the kit comprising a reagent for detecting the expression levels of two or more of the molecular markers hsa-miR-106b-5p, hsa-miR-142-5p and hsa-miR-421.

[0065] In certain embodiments, the kit further includes a reagent for detecting the expression levels of one or more of the molecular markers hsa-let-7b, hsa-let-7g-5p, hsa-miR-191-5p, hsa-miR-22-5p, hsa-miR-26a-5p, hsa-miR-26b-5p and hsa-miR-27b.

[0066] In certain embodiments, the reagents include miRNA primers and probes, wherein the miRNA primers include reverse transcription primers, RT-PCR upstream primers, and RT-PCR downstream primers.

[0067] In certain embodiments, the kit further comprises RT-PCR detection reagents and / or standards.

[0068] The present invention also provides a method for detecting exosome-derived miRNA markers for diagnosis or auxiliary diagnosis of Parkinson's severity or staging. Specifically, the method includes reverse transcription and real-time fluorescence quantitative polymerase chain reaction (RT-PCR).

[0069] In addition, the detection method of the above miRNA is not limited to the RT-PCR method provided by the present invention. Other methods such as sequencing, microarray, Northern blot, bioluminescence and probe method can also be used to detect the molecular markers.

[0070] In another aspect, the present invention provides a device for diagnosing or assisting in the diagnosis of Parkinson's disease severity or stage, the device comprising an information acquisition module, a calculation module and a diagnosis module, wherein:

[0071] The information acquisition module is used to perform an operation of acquiring test information of the subject, wherein the test information includes Ct values ​​of two or more of hsa-miR-106b-5p, hsa-miR-142-5p and hsa-miR-421, and the Ct values ​​are normalized by an internal reference miRNA;

[0072] The calculation module is used to perform the operation of substituting the Ct value of the molecular marker, the age and gender of the subject into the diagnostic model to obtain the model value;

[0073] The diagnosis module is used to execute an operation of determining the severity or stage of Parkinson's disease suffered by the subject according to the model value.

[0074] In the present invention, for the test sample, when the score is less than the threshold, the result is judged to be negative, which means that the patient is an early PD patient, as shown by the H+Y stage of 1 / 2; otherwise, it is positive, which means that the patient is a mid-to-late PD patient, as shown by the H+Y stage of 3 / 4 / 5.

[0075] When the exosomal miRNA is selected from the above three miRNAs, the detection model is Y = 0.163 ╳ Age - 0.867 ╳ Gender +1.785 ╳ hsa-miR-106b-5p-1.596 ╳ hsa-miR-142-5p-1.462 ╳ hsa-miR-421-6.007, the threshold value is 0.353.

[0076] In the present invention, the level or concentration of the exosomal miRNA in the sample is expressed as a Ct value, and the patient's gender is converted into a numerical value, where a male is assigned a value of 1 and a female is assigned a value of -1.

[0077] As used in the present invention, "Ct value" or "Cycle Threshold" refers to the number of amplification cycles corresponding to when the fluorescence signal of the amplified product in quantitative PCR reaches the set fluorescence threshold. Simply put, the Ct value represents the number of cycles when the starting template amplification reaches a certain amount of product. The higher the concentration of the starting template, the smaller the Ct value; the lower the concentration of the starting template, the larger the Ct value.

[0078] The specific operation of the present invention for screening the key differential miRNA for preventing PD stages is as follows:

[0079] (1) Brain-derived exosome extraction: Magnetic beads coated with ATP1A3 antibodies are used to specifically capture brain-derived exosomes in plasma. Enrichment of brain-derived exosomes can better detect neuronal lesions in the brain, reduce background signal interference from exosomes from other sources, and improve the accuracy of PD diagnosis. (2) Exosome nucleic acid extraction: Small nucleic acids from brain-derived exosomes are extracted using a miRNA extraction kit.

[0080] (3) Library construction and sequencing: The small RNA library was constructed using the QiaSeq miRNA library construction kit, and the library was sequenced using the Ion Torrent540 chip. The data was mapped to the HG19 and miRBase v21 databases, and each read was normalized to RPM for data analysis to screen for differentially expressed miRNA targets in the cohort samples.

[0081] (4) Reverse transcription and real-time fluorescence quantitative PCR: The extracted miRNA is reverse transcribed into cDNA, and then the content of the target miRNA is detected by qPCR to verify the diagnostic performance of the target screened by sequencing.

[0082] (5) Diagnostic model establishment: Based on the validation set data, a diagnostic model is created in combination with factors such as age and gender, and the algorithm is transformed. Finally, the evaluation score is calculated and the PD stage is determined based on the score.

[0083] As used herein, the term "sample" or "specimen" refers to material specifically associated with a subject from which specific information about the subject can be determined, calculated or inferred. A sample may consist entirely or in part of biological material from a subject.

[0084] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sam brook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0085] Example 1: Extraction of exosome miRNA

[0086] Clinical cohort sample collection:

[0087] The discovery cohort consisted of 274 patients from the First Affiliated Hospital of Zhongshan Hospital in Guangzhou (89 healthy controls, 26 H&Y stage 1, 59 H&Y stage 2, 63 H&Y stage 3, and 37 H&Y stage 4);

[0088] The validation set cohort consisted of 74 cases from the First Affiliated Hospital of Zhongshan University in Guangzhou (including 22 healthy controls, 11 H&Y stage 1, 22 H&Y stage 2, 6 H&Y stage 3, and 13 H&Y stage 4); and 140 cases from the Second Affiliated Hospital of Soochow University in Suzhou (including 74 healthy controls, 20 H&Y stage 1 / 1.5, 33 H&Y stage 2 / 2.5, 12 H&Y stage 3, and 1 H&Y stage 5).

[0089] Inclusion criteria:

[0090] 1. Age > 50 years old;

[0091] 2. Able to speak Mandarin (or able to communicate effectively with doctors in dialect);

[0092] 3. The applicant and his / her family sign the informed consent form;

[0093] 4.MMSE≥25;

[0094] 5. Diagnosed with Parkinson's disease according to the Chinese Parkinson's disease diagnostic criteria;

[0095] 6. According to HOEHN&YAHR classification, it belongs to level 0-4.

[0096] Exclusion criteria:

[0097] (II) Exclusion criteria for Parkinson's disease group

[0098] 1. Hearing impairment, visual impairment, poor cooperation;

[0099] 2. Have a lifetime history of schizophrenia, schizoaffective disorder or bipolar disorder;

[0100] 3. Subjects currently taking antidepressants: Subjects currently suffering from depression or with a history of severe depression in the past two years;

[0101] 4. Have received electroconvulsive therapy in the past;

[0102] 5. A history of alcohol abuse or alcohol dependence after two years of screening;

[0103] 6. Long-term heavy drinking history (alcohol content greater than 42% liquor, greater than 3 liang / day, drinking for more than 12 months);

[0104] 7. History of malignant tumors that have not been stably controlled within one year (including tumors of the nervous system and tumors outside the nervous system);

[0105] 8. Any serious systemic disease or unstable condition that makes it difficult to comply with the protocol, including: history of severe lung disease (COPD, pulmonary encephalopathy); history of severe cardiovascular disease (heart failure, severe hypertension);

[0106] 9. Subjects with the following clinically significant symptoms or medical history: history of severe nervous system infection of any cause, history of multiple sclerosis, history of immune encephalitis, history of Hashimoto's encephalopathy, etc.; epileptic seizures currently not stably controlled due to various causes in the past; history of genetic diseases affecting cognitive function (such as Huntington's disease, Down syndrome, CADASIL syndrome, adrenoleukodystrophy, mitochondrial encephalopathy, etc.); infection and immune-related diseases affecting the central nervous system (systemic lupus erythematosus, inadequately treated HIV infection or history of CNS spirochetal infection, etc.); metabolic diseases and endocrine disorders affecting the central nervous system (thyroid dysfunction requiring new treatment or adjustment of current treatment, folic acid or vitamin B12 deficiency); epileptic seizures currently not stably controlled due to various causes in the past;

[0107] 10. Subjects with the following diseases or clinical symptoms: including atypical features of Parkinson's syndrome (progressive supranuclear palsy (PSP), multiple system atrophy (MSA), corticobasal degeneration (CBD)), including cerebellar symptoms, supranuclear gaze palsy, apraxia and other cortical symptoms, or prominent autonomic failure;

[0108] 11. A history of clinically significant and unstable gastrointestinal diseases in the past two years, including ulcers, severe or occult gastrointestinal bleeding;

[0109] 12. Systemic diseases affecting the nervous system function, and exclusion criteria related to abnormal liver and kidney function (abdominal dialysis or hemodialysis during the screening period; AST or ALT ≥ 3 × upper limit of normal (ULN) or total bilirubin ≥ 2 × ULN during the screening period)

[0110] 13. Uncontrolled diabetes;

[0111] 14. History of unstable malignant tumors (including tumors of the nervous system and tumors outside the nervous system) within 1 year;

[0112] 15. Drugs that need to be excluded from the study include:

[0113] 1) Use of narcotic drugs, methyldopa, and clonidine within 4 weeks before screening;

[0114] 2) Use of short-term antianxiety drugs or sedative hypnotic drugs more than twice a week within 4 weeks before the screening (Note: sedative drugs should not be used within 72 hours after the screening);

[0115] 3) use of systemic corticosteroids within 3 months before screening;

[0116] 4) Use of anticonvulsant drugs (such as phenytoin, phenobarbital, carbamazepine) within 2 months before screening;

[0117] 5) Use of warfarin (Coumadin) within 4 weeks before screening;

[0118] 6) Use of phosphodiesterase inhibitors within 4 weeks prior to screening.

[0119] Brain-derived exosome miRNA extraction

[0120] The method for preparing immune capture microspheres in this embodiment is as follows: using Dynabeads TM ATP1A3 antibody (Thermo Fisher, MA3-915) was coupled to Dynabeads M-270 epoxy resin microspheres using an antibody coupling kit (Thermo Fisher, 14311D) according to the manufacturer's instructions, with a coupling amount of 5 μg antibody / mg microspheres.

[0121] 1 mL of clinical plasma samples were collected from the cohort and centrifuged at 3000 g for 10 min and 10000 g for 20 min to remove impurities. The supernatant was used to extract total exosomes using the Exoquick kit (SBI, EXOQ5TM-1) according to the manufacturer's instructions, and a total of 500 uL of total exosomes was obtained.

[0122] Specific enrichment of exosomes from brain neurons in total exosomes using immunocapture microspheres

[0123] Add 1 mg of microspheres to the above 500 μL total exosomes and rotate and mix overnight at 4°C. Use a magnetic stand for solid-liquid separation, remove the supernatant, wash the microspheres three times with 500 μL PBST (0.01M phosphate buffer containing 0.05% Tween 20), remove the washing solution by magnetic separation, resuspend the microspheres with 200 μL PBS, and enrich the brain exosomes on the microspheres. Use the miRcute serum plasma miRNA extraction and separation kit (Tiangen Biochemical, DP503) to extract brain exosome miRNA according to the instructions. Electron microscopy and particle size detection of the enriched brain-derived exosomes are performed as follows:

[0124] Electron microscopy: 1. Take the exosome sample out of the -80℃ refrigerator and place it in an ice box. Centrifuge it slightly after dissolving. Use a pipette to draw 15μL of the exosome sample onto a copper grid and let it stand for 1 minute.

[0125] 2. Use filter paper to absorb the exosome sample on the copper mesh, and then use a pipette to draw 15 μL of 2% uranyl acetate staining solution and stain at room temperature for 1 minute.

[0126] 3. Use filter paper to absorb the exosome sample on the copper mesh, place the stained sample under the light for 10 minutes, observe and take pictures, and save the pictures.

[0127] Electron microscopy results are shown in Figure 1 In part A, saucer-like vesicle structures can be seen under the microscope, and the size is within the standard range of exosomes.

[0128] The particle size and particle concentration were measured using a particle size analyzer (Spectradyne, model nCS1) using the microfluidic resistive pulse sensing (MRPS) method as follows:

[0129] Turn on the machine, load the cleaning cartridge, and run the instrument prime program.

[0130] Take an appropriate amount of EVs, dilute with PBST, and pipette 4 μL into the well of the cartridge (chip);

[0131] The cartridge is loaded into the instrument and the particle size detection program is run;

[0132] After completion, replace the cleaning cartridge and clean the instrument pipeline with purified water.

[0133] Particle size distribution diagram Figure 1 In part B, 90% of the particles (D90) were below 135 nm in size, and the particle size distribution was within the standard range of exosomes.

[0134] The concentration of extracted miRNA was determined using the Qubit microRNA detection kit (Thermo Fisher Scientific, catalog number Q32881) in combination with a Qubit4 fluorescence spectrophotometer, and the extracted miRNA was quality controlled using a small RNA detection chip in combination with a 2100 bioanalyzer (Agilent). Figure 1 In the middle C part, the peak shape conforms to the conventional morphology of small RNA.

[0135] Example 2 Discovery cohort sample miRNA NGS sequencing and differential miRNA target screening

[0136] A small RNA library construction kit (Qiagen, Qiagen miRNA library kit) was used to construct the library, and 540Chips were used for sequencing on the Ion Torrent (Thermo Fisher) platform. The data were mapped to the HG19 and miRBase v21 databases, and each read was normalized to RPM for data analysis. The miRNAs with high abundance expression in the cohort samples and significant differences between normal subjects and PD patients were screened.

[0137] The markers screened in this example are hsa-let-7b, hsa-let-7g-5p, hsa-miR-106b-5p, hsa-miR-142-5p, hsa-miR-191-5p, hsa-miR-22-5p, hsa-miR-26a-5p, hsa-miR-26b-5p, hsa-miR-27b and hsa-miR-421. The heat map of some miRNAs with higher expression abundance is shown in FIG. Figure 2 , showing that it is differentially expressed in different PD stages.

[0138] Using the random forest algorithm, a multivariate model was constructed based on age, gender, and normalized numerical RPM of sequencing to view the diagnostic efficacy of candidate targets and provide support for further screening of targets for subsequent qPCR verification. The ROC curve is shown in Figure 3 , the area under the curve AUC = 0.873, the sensitivity was 93.51%, the specificity was 54.99%, and the accuracy was 81.02%.

[0139] Example 3 Validation of molecular markers

[0140] The validation set cohort samples were collected and brain-derived exosomal miRNA was extracted according to the method in Example 2.

[0141] Reverse transcription kit TaqMan TM The miRNA was reverse transcribed into cDNA using the MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, 4366597). A 15 μL reverse transcription system was used. The amounts of the components were shown in Table 3. The reverse transcription primers (see Table 2) were included in the TaqMan TM Assays (Thermo Fisher, 4440886) reagent, the reaction conditions are: 16°C for 30 min, 42°C for 30 min, 85°C for 5 min, and maintain at 4°C.

[0142] Table 3

[0143] Reverse transcription system Volume (μL) dNTPs and dTTP (100 mM) 0.3 Reverse transcriptase 3 10× Reverse Transcription Buffer 1.5 RNase inhibitor (20U / μL) 0.19 RNase-free water 1.01 Reverse transcription primer library 6 Sample miRNA 3 Total volume 15

[0144] Using preamplification reagents TaqMan TM The reverse transcription product obtained above was preamplified using PreAmp premix (Thermo Fisher Scientific, 4488593). The reaction system was 25 μL. The amount of components used was shown in Table 4. The primers (see Table 2) were included in Assay (Thermo Fisher, 4440886) reagent, the reaction conditions are: 95°C 10min, 55°C 2min, 72°C 2min, (95°C 15s, 60°C 4min) 12 cycles, 99.9°C 10min, 4°C hold. After the reaction, 175μL of 0.1×TE, pH 8.0 was added to each reaction well for dilution, and the final volume was 200μL.

[0145] Table 4

[0146]

[0147] use Fast Advanced premix (2×) (Thermo Fisher, 4444557) was used for RT-qPCR detection of the Ct value of exosomal miRNA markers. A 10 μL system was used. The amounts of each component were shown in Table 5. The primers and probes (see Table 2) were included in Assay (Thermo Fisher, 4440886) reagent, the reaction conditions were: 50°C for 2 min, 95°C for 20 s, (95°C for 1 s, 60°C for 20 s) for 40 cycles.

[0148] Table 5

[0149]

[0150] The hsa-miR-423-5p, which was stably expressed in all samples as shown by the NGS sequencing results, was used as the internal reference. The relative expression levels of hsa-let-7b, hsa-let-7g-5p, hsa-miR-106b-5p, hsa-miR-142-5p, hsa-miR-191-5p, hsa-miR-22-5p, hsa-miR-26a-5p, hsa-miR-26b-5p, hsa-miR-27b, and hsa-miR-421 were obtained according to the normalized Ct values. The relative expression levels were grouped into normal group, early PD (H&Y stage 1, 2), and mid-to-late PD (H&Y stage 3 and above) according to different cohorts. The relative expression level dot plots are shown in the figure. Figure 4 ,Depend on Figure 4 It can be seen that in the validation of a multicenter cohort, hsa-miR-106b-5p, hsa-miR-142-5p and hsa-miR-421 had obvious expression differences in the early and late stages of PD, and the targets showed the ability to distinguish different stages.

[0151] Example 4 Establishment of PD staging diagnosis model

[0152] The staging model was established based on the ΔCt values ​​of the target and the internal reference measured by RT-PCR. Binary logistic regression was used to train one or more of hsa-let-7b, hsa-let-7g-5p, hsa-miR-106b-5p, hsa-miR-142-5p, hsa-miR-191-5p, hsa-miR-22-5p, hsa-miR-26a-5p, hsa-miR-26b-5p, hsa-miR-27b, and hsa-miR-421 in combination with age and gender to obtain the logistic regression equation (judgment formula). The diagnostic performance of each target is shown in Table 6.

[0153] Table 6

[0154]

[0155]

[0156] Combining several targets with higher AUC, the fitting function is: Y = 0.163 ╳ Age - 0.867 ╳ Gender +1.785 ╳ hsa-miR-106b-5p-1.596 ╳ hsa-miR-142-5p-1.462 ╳ hsa-miR-421-6.007, ROC curve is shown in Figure 5, AUC = 0.852, where the patient's gender is converted into a numerical value, where males are assigned a value of 1 and females are assigned a value of -1. Each marker in the fitting function is represented by the normalized ΔCt value of each marker. When the score is less than the threshold, the judgment result is negative, which means that the patient is an early PD patient, as shown by the H+Y stage of 1 / 2. Otherwise, it is positive, which means that the patient is a mid-to-late PD patient, as shown by the H+Y stage of 3 / 4 / 5. When the cutoff value is 0.353, the sensitivity is 81.8% and the specificity is 76.3%.

[0157] According to the cohort inclusion criteria in Example 1, 216 samples were collected as a test set, and the model validation test was performed according to the above detection method. The results are shown in Table 7.

[0158] Table 7

[0159]

[0160] It should be understood that the above embodiments are exemplary and are not intended to include all possible implementations included in the claims. Various modifications and changes may be made on the basis of the above embodiments without departing from the scope of the present disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only express several implementations of the present invention and do not limit the scope of protection of the patent of the present invention.

Claims

1. Use of a reagent for detecting molecular markers in the preparation of a product for diagnosing or assisting in the diagnosis of Parkinson's disease severity or staging, characterized in that: The molecular markers are two or more of hsa-miR-106b-5p, hsa-miR-142-5p and hsa-miR-421.

2. The use according to claim 1, characterized in that: The molecular markers include at least hsa-miR-106b-5p, hsa-miR-142-5p and hsa-miR-421.

3. The use according to claim 1, characterized in that: The molecular markers also include one or more of hsa-let-7b, hsa-let-7g-5p, hsa-miR-191-5p, hsa-miR-22-5p, hsa-miR-26a-5p, hsa-miR-26b-5p and hsa-miR-27b.

4. The use according to any one of claims 1 to 3, characterized in that: The reagents include miRNA primers and / or probes, and the miRNA primers include reverse transcription primers, RT-PCR upstream primers and RT-PCR downstream primers.

5. The use according to claim 4, characterized in that: The reverse transcription primer sequence of hsa-miR-106b-5p is shown in SEQ ID NO: 7, the RT-PCR upstream primer sequence is shown in SEQ ID NO: 8, the RT-PCR downstream primer sequence is shown in SEQ ID NO: 34, and the probe sequence is shown in SEQ ID NO: 9; The reverse transcription primer sequence of the hsa-miR-142-5p is shown in SEQ ID NO: 10, the RT-PCR upstream primer sequence is shown in SEQ ID NO: 11, the RT-PCR downstream primer sequence is shown in SEQ ID NO: 34, and the probe sequence is shown in SEQ ID NO: 12; The reverse transcription primer sequence of the hsa-miR-421 is shown in SEQ ID NO: 28, the RT-PCR upstream primer sequence is shown in SEQ ID NO: 29, the RT-PCR downstream primer sequence is shown in SEQ ID NO: 34, and the probe sequence is shown in SEQ ID NO:

30.

6. The use according to claim 1, characterized in that: The molecular markers are derived from exosomes, and the exosomes are derived from one or more of blood, saliva, urine and sputum.

7. A kit for diagnosing or assisting in the diagnosis of Parkinson's disease severity or staging, characterized in that: The kit includes reagents for detecting the expression levels of two or more of the molecular markers hsa-miR-106b-5p, hsa-miR-142-5p and hsa-miR-421.

8. The kit according to claim 7, characterized in that The kit further includes a reagent for detecting the expression level of one or more of the molecular markers hsa-let-7b, hsa-let-7g-5p, hsa-miR-191-5p, hsa-miR-22-5p, hsa-miR-26a-5p, hsa-miR-26b-5p and hsa-miR-27b.

9. The kit according to claim 7 or 8, characterized in that The reagents include miRNA primers and probes, wherein the miRNA primers include reverse transcription primers, RT-PCR upstream primers and RT-PCR downstream primers; and / or, the kit also includes RT-PCR detection reagents and / or standards.

10. A device for diagnosing or assisting in the diagnosis of Parkinson's disease severity or staging, characterized in that: The device includes an information acquisition module, a calculation module and a diagnosis module, wherein: The information acquisition module is used to perform an operation of acquiring test information of the subject, wherein the test information includes Ct values ​​of two or more of hsa-miR-106b-5p, hsa-miR-142-5p and hsa-miR-421, and the Ct values ​​are normalized by an internal reference miRNA; The calculation module is used to perform the operation of substituting the Ct value of the molecular marker, the age and gender of the subject into the diagnostic model to obtain the model value; The diagnosis module is used to execute an operation of determining the severity or stage of Parkinson's disease suffered by the subject according to the model value.

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