A MAPKAPK2 molecule and its application to auxiliary diagnosis of Parkinson's disease

Abstract

The application discloses a MAPKAPK2 molecule and application thereof to auxiliary diagnosis of Parkinson's disease. + The expression level of the MAPKAPK2 gene in peripheral blood, substantia nigra and prefrontal lobe of PD patients and healthy people is significantly different, and the MAPKAPK2 gene can be used as a potential molecular marker or target point of Parkinson's disease, applied to early clinical diagnosis and targeted treatment of Parkinson's disease, and is favorable for further elucidation of the occurrence and development mechanism and signal pathways of Parkinson's disease, and has great application prospect and theoretical value.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and biomedicine, and relates to the application of the MAPKAPK2 gene, a potential biomarker for Parkinson's disease, in the clinical diagnosis, treatment, or scientific research of Parkinson's disease. Background Technology

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease, with a prevalence of 1% in people over 60 years of age in industrialized countries and 3% in those over 80 years of age. In my country, the prevalence of PD in people over 65 years of age is approximately 1.7%. PD is a common and complex neurological disorder. Its motor symptoms include resting tremor, bradykinesia, rigidity, postural abnormalities, and gait disturbances, accompanied by non-motor symptoms such as depression, constipation, and sleep disorders, placing a heavy burden on patients, families, and society. The main pathological feature of PD is the progressive loss of dopaminergic neurons in the substantia nigra pars compacta and the pathological accumulation of α-synuclein in the substantia nigra, the latter being a major component of Lewy bodies (LB). It is known that the accumulation and aggregation of pathological oligomeric α-synuclein forms α-synuclein fibers, which further accumulate to form Lewy bodies. All three are neurotoxic, causing the death of dopaminergic neurons and the development of PD through pathways such as mitochondrial abnormalities, endoplasmic reticulum-Golgi transport abnormalities, and inhibition of the autophagy-proteasome pathway.

[0003] The etiology and pathogenesis of Parkinson's disease remain unclear. Currently, it is generally accepted that genetic factors, environmental factors, aging, and oxidative stress are all risk factors for Parkinson's disease. The development of Parkinson's disease is a dynamic process involving multiple factors and genes. Therefore, exploring the molecular mechanisms of its dynamic development, identifying Parkinson's disease-related molecular markers, further clarifying its pathogenesis, and developing reagents or products applicable to early clinical diagnosis, treatment, and prognostic assessment are of great significance for improving the diagnosis and treatment of Parkinson's disease.

[0004] The MAPKAPK2 gene, located at chromosome 1q32, is approximately 49 kb in length, consists of 10 exons, and encodes the 400-amino acid MAPKAPK2 protein. The protein encoded by the MAPKAPK2 gene is a multifunctional protein kinase and an important effector substrate of MAPK. Activated MAPK kinase 2 participates in various cellular events, such as inflammation, cell cycle, cell migration, and apoptosis. It can regulate cell cycle and cell adhesion functions by modulating the tumor suppressor protein tuberin; it can affect the antioxidant function of lung epithelial cells; it can also activate heat shock proteins HSP27 and HSP25, inhibiting apoptosis and cell motility; and it can influence the function of macrophages and dendritic cells by regulating the secretion of tumor necrosis factor TNF, thus regulating the body's resistance to exogenous infections. Under radiation damage, MAPKAPK2 can modulate the cell cycle checkpoint initiated by DNA repair genes ATM or ATR, affecting the body's cellular repair of DNA damage.

[0005] MAPKAPK2 has been shown to be involved in the occurrence and development of many tumors; however, there are very few reports on its application in Parkinson's disease. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide the application of the MAPKAPK2 molecule in the preparation of biomarkers for the auxiliary diagnosis of Parkinson's disease. Another objective of the present invention is to provide the application of the aforementioned MAPKAPK2 molecule in the pathogenesis of Parkinson's disease and α-synuclein-targeted therapy. The present invention successfully identified high expression of the MAPKAPK2 gene in the peripheral blood, substantia nigra, and prefrontal cortex of Parkinson's disease patients. It can serve as a potential molecular marker or target for Parkinson's disease, applicable to the early clinical diagnosis or targeted therapy of Parkinson's disease, and is beneficial for further elucidating the pathogenesis and signaling pathways of Parkinson's disease, demonstrating significant application prospects and theoretical value.

[0007] The technical solution of this invention is as follows.

[0008] We constructed a cell model of α-synuclein pathological aggregation in Parkinson's disease and identified the MAPKAPK2 gene as the core gene for α-synuclein pathological aggregation.

[0009] Database samples revealed that the MAPKAPK2 gene was highly expressed in the peripheral blood, substantia nigra, and prefrontal cortex of Parkinson's disease patients.

[0010] The screening and identification of the MAPKAPK2 gene as a core gene of DNB involves the following method: using the Dynamic Network Biomarker (DNB) analysis method to analyze key molecules that undergo dramatic changes during the critical period of α-synuclein aggregation from normal state to pathological state.

[0011] The identification of high expression of the MAPKAPK2 gene in the peripheral blood of Parkinson's disease patients was carried out by analyzing the differences in gene expression levels between peripheral blood samples from PD patients and healthy controls using a database.

[0012] The identification of high expression of the MAPKAPK2 gene in the substantia nigra of Parkinson's disease patients was carried out by analyzing the difference in gene expression levels between substantia nigra samples from PD patients and healthy controls using a database.

[0013] The identification of high expression of the MAPKAPK2 gene in the prefrontal cortex of Parkinson's disease patients was carried out by analyzing the difference in gene expression levels between prefrontal cortex samples from PD patients and healthy controls using a database.

[0014] The identification of high expression of the MAPKAPK2 gene in Lewy body dementia patients was carried out by analyzing the differences in gene expression levels between Lewy body dementia patients and healthy controls using a database.

[0015] The MAPKAPK2 gene, and reagents for detecting MAPKAPK2 include specific primer pairs for amplifying the MAPKAPK2 gene;

[0016] The specific primer pair used to amplify the MAPKAPK2 molecule has the following sequence:

[0017] F: 5'-CCCACCCAGAGAATGACCATC-3';

[0018] R: 5'-ACAGAATCCTCTGCTCACAACC-3'.

[0019] The kit for the auxiliary diagnosis of Parkinson's disease using the MAPKAPK2 gene.

[0020] Furthermore, it can be used for the auxiliary diagnosis or prognostic assessment of Parkinson's disease.

[0021] Furthermore, the reagent kit is characterized by comprising the following steps:

[0022] (1) Design and synthesize the MAPKAPK2 gene molecule and internal reference gene primer pair;

[0023] (2) Extract total RNA from the sample;

[0024] (3) Prepare sample cDNA;

[0025] (4) qPCR reaction solution.

[0026] In the above method, step (2) specifically involves: lysing SH-SY5Y cells with cell lysis buffer and rapidly extracting total RNA from the cell sample using the TransZolUp Plus RNA Kit.

[0027] In the above method, step (3) specifically involves: determining the concentration of the RNA from step (2) using an ultra-micro spectrophotometer, and rapidly reverse transcribing the RNA into cDNA using the HiScript II Q Select RT SuperMix for qPCR kit and storing it in a -20°C freezer.

[0028] In the above method, step (4) specifically involves: using the cDNA extracted in (3) as a template and β-actin as an internal control, adding SYBR Green fluorescent dye to the reaction system under light-protected conditions, centrifuging slightly, placing it in a qPCR instrument, setting the program, and preparing a kit for the auxiliary diagnosis of Parkinson's disease.

[0029] Compared with existing technologies, this invention successfully induced a pathological cell model of α-synuclein aggregation. The MAPKAPK2 gene was found to be highly expressed in the peripheral blood, substantia nigra, and prefrontal cortex of this model and Parkinson's disease patients. This can serve as a potential molecular marker for early clinical diagnosis of Parkinson's disease. Alternatively, it can be identified as a therapeutic target for α-synuclein, such as blocking its diffusion, inhibiting its aggregation, or promoting its clearance, which is significant for the clinical treatment of Parkinson's disease. Furthermore, it helps to further elucidate the pathogenesis and signaling pathways of Parkinson's disease, demonstrating great application potential and theoretical value. Attached Figure Description

[0030] Figure 1a A pathological cell model of α-synuclein (α-Syn) aggregation;

[0031] Figure 1b The earliest pathological aggregation of α-Syn occurred 12 hours after induction;

[0032] Figure 2 Sequencing results of the MAPKAPK2 gene in a cell model of pathological aggregation of α-synuclein;

[0033] Figure 3 The results of qPCR for the MAPKAPK2 gene in a pathological cell model of α-synuclein aggregation.

[0034] Figure 4The expression of HSF1 and SERPINE1 genes in blood samples from PD patients and healthy individuals;

[0035] Figure 5 Results of MAPKAPK2 expression in peripheral blood of Parkinson's patients and healthy individuals from the GEO database;

[0036] Figure 6 Results of MAPKAPK2 expression in the substantia nigra of Parkinson's patients and healthy individuals from the GEO database;

[0037] Figure 7 Results of MAPKAPK2 expression in the prefrontal cortex of Parkinson's patients and healthy individuals from the GEO database;

[0038] Figure 8 The expression results of MAPKAPK2 in Lewy body dementia patients and healthy individuals in the GEO database (ns: p≥0.05; *: p<0.05; ****: p<0.0001. HC: healthy controls; PD: Parkinson's disease patients; DLB: Lewy body dementia; MSA: multiple system atrophy). Detailed Implementation

[0039] Example 1: Construction of a cell model of pathological aggregation of α-synuclein

[0040] Remove the cryovials of the SH-SY5Y human neuroblastoma cell line from the liquid nitrogen tank and quickly thaw them in a 37°C water bath. Transfer the cells to 15mL centrifuge tubes under a clean bench, add SH-SY5Y medium (cryopreservative to medium volume ratio 1:6–1:8) to ensure complete removal of DMSO. After centrifugation, discard the supernatant, resuspend the cells in an appropriate amount of SH-SY5Y medium, mix thoroughly, and then seed them into culture dishes.

[0041] Cell culture: Cultured in a 37℃, 5% CO2 cell incubator.

[0042] Drug treatment: Weigh 0.01g MPP using a 0.01g balance. + Iodide (297.13 g / mol), this 0.01 g was dissolved in 5 mL of SH-SY5Y medium, in which MPP + The iodide concentration was 6731.06 μmol / L, which is MPP. + Stock solution. Take MPP. + Add 37.14 μL of the stock solution to 49 mL of SH-SY5Y medium, and then add 962.86 μL of SH-SY5Y medium.

[0043] Using 5μM MPP +Cells were induced and cultured to construct a cell model of pathological aggregation of α-synuclein, with a control group used for comparison. Immunofluorescence staining was performed on cells at 0h, 4h, 8h, 12h, and 24h after induction using two antibodies, 5G4 and p-α-Syn. Results are as follows: Figure 1a and Figure 1b The average immunofluorescence intensity at 0h in the induction group was not significantly different from that at 4h and 8h, but was significantly different from that at 12h and 24h. The pathological aggregation of α-synuclein occurred as early as 12h after induction.

[0044] Total RNA was collected from cells at 0h, 4h, 8h, and 12h after induction, and transcriptome sequencing was performed, with four parallel samples at each time point. Differential gene and DNB (Dynamic Network Biomarker) analyses were performed on the sequencing data to screen for core DNB genes, including MAPKAPK2.

[0045] Example 2: Expression level of MAPKAPK2 gene in a cell model of pathological aggregation of α-synuclein

[0046] Using MPP + SH-SY5Y cells were induced with iodide, and cells were collected at corresponding time points (0h, 4h, 8h, 12h). Total RNA was rapidly extracted from the cell samples using the TransZol Up Plus RNA Kit. A portion of the cell sample RNA was sequenced using the Illumina NovaSeq 6000 sequencing platform, with 6GB of data per sample and four replicates at each time point. Sequencing data are shown below. Figure 2 As shown in the figure. The concentration of the other portion of RNA was determined using an ultra-micro spectrophotometer, and the RNA was rapidly reverse transcribed into cDNA using the HiScript II QSelect RT SuperMix for qPCR kit and stored at -20°C for qPCR experiments. Triple replicates were performed at each time point. The qPCR results are shown in the figure. Figure 3 As shown.

[0047] The mRNA levels of MAPKAPK2, HSF1, SERPINE, and housekeeping gene ACTB in peripheral blood and cell samples were quantified. The designed qPCR primers are shown in the table below. ACTB expresses β-actin and was used as an internal control in the qPCR experiment. The ChamQ SYBR qPCR Master Mix kit was used, with four auxiliary wells. The qPCR system and reaction conditions are shown below. SYBR Green fluorescent dye was added to the reaction system under dark conditions, and after slight centrifugation, the mixture was placed in the qPCR instrument, and the reaction program was set. Relative expression levels were used to measure gene expression. The internal control gene ACTB and a cell sample induced for 0 h / one healthy control sample HC24 were used as control samples. Two... -ΔΔCt Relative expression levels were calculated using standardized formulas. GraphPad Prism 8.0.1 statistical software was used for plotting and statistical analysis. Nonparametric two-tailed t-tests were used to analyze gene expression levels between the PD and HC groups.

[0048]

[0049]

[0050] Generally, a final primer concentration of 0.2 μM in the reaction system is sufficient to obtain good amplification results. When the reaction performance is poor, the primer concentration can be adjusted within the range of 0.1-1.0 μM.

[0051] If the template type is undiluted cDNA stock solution, the volume used should not exceed 1 / 10 of the total qPCR reaction volume. 2×ChamQ SYBR qPCR Master Mix and 50×ROX Reference Dye 1 should be added in the dark. After adding all components, invert the container to mix thoroughly and centrifuge to remove air bubbles.

[0052] Reaction procedure:

[0053]

[0054] Example 3: Expression level of the MAPKAPK2 gene in Parkinson's disease patients and healthy controls

[0055] Forty-four subjects were collected from Guangdong Provincial People's Hospital, including 27 Parkinson's disease (PD) patients as the case group and 17 healthy controls as the control group. The recruited PD patients were those who visited the outpatient or inpatient department of neurology at Guangdong Provincial People's Hospital between March and December 2019. After diagnosis, patients did not receive medication, and peripheral blood samples were collected from them immediately into anticoagulant tubes. Control group samples were obtained from the Guangdong Provincial People's Hospital Health Examination Center. All enrolled PD patients met the clinical diagnostic criteria of the UKPD Brain Bank and were diagnosed according to the "Diagnostic Criteria for Parkinson's Disease (2016 Edition)" formulated by the Parkinson's Disease and Movement Disorders Group of the Chinese Medical Association Neurology Branch and the Parkinson's Disease and Movement Disorders Professional Committee of the Chinese Medical Doctor Association Neurology Branch. The collection and testing of samples complied with the Declaration of Helsinki and the requirements of the International Conference on Unified Good Clinical Practice (World Medical Association, 1997). This study has been approved by the Medical Research Ethics Committee of Guangdong Provincial People's Hospital (Guangdong Academy of Medical Sciences) [Guangdong Medical Science 2019490H(R1)], and written informed consent has been obtained from the study subjects or their families for blood sample analysis.

[0056] qPCR experiment: The mRNA levels of HSF1, SERPINE1, MAPKAPK2, and the housekeeping gene ACTB in peripheral blood and cell samples were quantified. The designed qPCR primers are shown in the table below. The ACTB gene expresses β-actin and was used as an internal control in the qPCR experiment. qPCR results are shown below. Figure 4 As shown.

[0057]

[0058] Data on three diseases associated with pathological aggregation of α-synuclein, including Parkinson's disease (PD), dementia with Lewy body (DLB), and multiple system atrophy (MSA), were obtained using GEO, as shown in the table below. Probe IDs were annotated with gene names using GPL platform files. For the peripheral blood data collected from the four PD patients, the removeBatchEffect algorithm in the R package limma was used for batch removal. Box plots and principal component analysis (PCA) were used to evaluate the batch removal effect. Compared with healthy individuals, the MAPKAPK2 gene was significantly overexpressed in the peripheral blood, substantia nigra, and prefrontal cortex of Parkinson's disease patients, as shown in the table below. Figure 5 , 6 As shown in Figures 7 and 8.

[0059] Dementia with Lewy body (DLB) and multiple system atrophy (MSA) are also synucleinopathies, in which pathological accumulation of α-synuclein occurs. Analysis of relevant clinical datasets in the GEO database revealed differences in MAPKAPK2 gene expression levels between healthy individuals and patients. In both neurodegenerative diseases, MAPKAPK2 was significantly overexpressed in the brain tissue and peripheral blood of patients with Parkinson's disease (PD). Figure 8 As shown.

[0060]

[0061]

Claims

1. Application of reagents for detecting MAPKAPK2 gene expression levels in the preparation of kits for the auxiliary diagnosis of Parkinson's disease.

2. The application according to claim 1, characterized in that, The reagent includes primer pairs that specifically recognize the MAPKAPK2 gene, and the sequences of the primer pairs are shown below: F: 5'-CCCACCCAGAGAATGACCATC-3'; R: 5'-ACAGAATCCTCTGCTCACAACC-3'.

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