Biomarker combination for Parkinson's disease diagnosis and diagnosis and treatment application based on LINC00467 / miR-494-3p / CYCS axis

Abstract

The invention discloses a biomarker combination for Parkinson's disease diagnosis and a diagnosis and treatment application based on an LINC00467 / miR-494-3p / CYCS axis in the technical field of biological medicines. The invention provides a PD early diagnosis biomarker combination taking LINC00467, miR-494-3p and CYCS as cores, and a PD treatment scheme and a drug development direction taking the regulation axis as a target spot. The invention solves the technical problems of low specificity, dependence on clinical symptoms, scarcity of treatment targets and unclear mechanism of the existing PD diagnosis, and has important clinical application value and industrialization prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a combination of biomarkers for the diagnosis of Parkinson's disease and its diagnostic and therapeutic applications based on the LINC00467 / miR-494-3p / CYCS axis. Background Technology

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide, after Alzheimer's disease, with 7-10 million patients globally. Its incidence continues to rise due to the accelerating aging of the population. Because PD progresses insidiously and early symptoms are atypical, most patients are diagnosed at an advanced stage, when neuronal damage is irreversible, missing the optimal treatment window and imposing a heavy medical and economic burden on patients, their families, and society.

[0003] Current research indicates that Parkinson's disease (PD) results from the combined effects of genetic factors, environmental factors, and epigenetic regulation, with oxidative stress, mitochondrial dysfunction, protein misfolding and aggregation, neuroinflammation, and metal ion homeostasis imbalance being core pathological links. The role of metal ion homeostasis imbalance in neurodegenerative diseases has received widespread attention in recent years. Copper ions, as an essential trace element for the human body, participate in the regulation of various enzyme activities, redox reactions, and energy metabolism processes, and their homeostasis is crucial for neuronal function. Studies have found that serum copper ion levels in PD patients are significantly higher than in normal individuals, suggesting that copper homeostasis dysregulation may be closely related to the occurrence and development of PD. Copper death (cuproptosis), a novel programmed cell death mechanism first defined by Tsvetkov et al. in 2022, is characterized by copper-dependent protein toxicity stress and lipid peroxidation. Its core mechanism involves the binding of copper ions to lipoylated proteins, triggering protein toxicity stress and lipid peroxidation, ultimately leading to cell death. Current research has confirmed that copper death is associated with various neurodegenerative diseases. Although abnormal copper metabolism and copper death are known to be associated with PD, the molecular regulatory network between the two has not yet been elucidated.

[0004] Current research largely focuses on single molecules or pathways, lacking systematic studies on the synergistic regulatory mechanisms of multiple molecules and pathways. This hinders a holistic understanding of the pathogenesis of Parkinson's disease (PD) and makes it difficult to develop precision treatment strategies based on core mechanisms. The fundamental reason for this problem is that the pathogenesis of PD is complex and multifactorial. Single research methods are insufficient to fully reveal its molecular regulatory network, and systematic research methods combining bioinformatics analysis and multi-level experimental validation have not yet been widely applied to the exploration of copper metabolism and copper death-related pathways in PD.

[0005] In summary, the diagnosis and treatment of Parkinson's disease (PD) currently faces challenges such as a lack of specific biomarkers for diagnosis, a lack of effective therapeutic targets, and difficulties in mechanistic research. Therefore, identifying specific regulatory pathways related to the core pathological processes of PD and developing novel diagnostic biomarkers and therapeutic targets have become critical issues that urgently need to be addressed in the field of PD research. Summary of the Invention

[0006] This invention provides a combination of biomarkers for early diagnosis of PD centered on LINC00467, miR-494-3p, and CYCS, as well as PD treatment regimens and drug development directions targeting this regulatory axis.

[0007] To achieve the above objectives, this application provides the following technical solution: A combination of biomarkers for the diagnosis of Parkinson's disease, comprising the LINC00467, miR-494-3p, and CYCS genes or their expression products.

[0008] As another aspect of the present invention, the application of a combination of biomarkers in the preparation of Parkinson's disease diagnostic products.

[0009] As a preferred embodiment, the diagnostic product enables the diagnosis or risk assessment of Parkinson's disease by detecting the genes or expression products of LINC00467, miR-494-3p, and CYCS in biological samples.

[0010] As a preferred embodiment, the diagnostic products include antibodies, primers, probes, chips, kits, or detection devices for detecting LINC00467, miR-494-3p, and CYCS genes or their expression products.

[0011] As another aspect of the present invention, the application of the LINC00467 / miR-494-3p / CYCS regulatory axis in the preparation of drugs for treating Parkinson's disease.

[0012] As a preferred embodiment, the therapeutic agent modulates the expression levels of LINC00467, miR-494-3p, and CYCS, or blocks the interaction among the three.

[0013] As a preferred embodiment, the therapeutic agent comprises: a nucleic acid drug that downregulates LINC00467 expression, a nucleic acid drug that upregulates miR-494-3p expression, a nucleic acid drug that downregulates CYCS expression, or a small molecule compound that specifically binds to and modulates the function of LINC00467, miR-494-3p, or CYCS.

[0014] As a preferred embodiment, the small molecule compound is selected from one or more of ciprofloxacin, pentoxifylline, melatonin, ketoconazole, minocycline, or paclitaxel.

[0015] As another aspect of the present invention: A treatment for Parkinson's disease that maintains copper metabolism balance by regulating the LINC00467 / miR-494-3p / CYCS axis.

[0016] Working principle and beneficial effects of the present invention: (1) This study systematically revealed for the first time the key role mechanism of the LINC00467 / miR-494-3p / CYCS axis in the regulation of copper metabolism in Parkinson's disease (PD), clarifying that this axis is centered on the ceRNA regulatory network: the long non-coding RNA LINC00467, as an endogenous competitive RNA, can specifically adsorb the microRNA miR-494-3p, relieving its post-transcriptional inhibition of the target gene CYCS (cytochrome c) 3'UTR region; under the pathological state of PD, the abnormal upregulation of LINC00467 leads to the downregulation of miR-494-3p expression, which in turn triggers the overexpression of CYCS, ultimately inducing a "triple pathological effect" of copper ion transport metabolic imbalance, copper death pathway activation, and enhanced mitochondrial oxidative stress, which synergistically leads to damage to dopaminergic neurons and CYCS Overexpression of CYCS can bind to mitochondrial copper ions, inhibiting the transport function of copper chaperone proteins (such as ATOX1) and leading to the accumulation of free copper ions in the cytoplasm. The accumulated copper ions bind to lipoylated proteins (such as LIAS), inhibiting their activity and inducing proteotoxic stress, while simultaneously activating the lipid peroxidation pathway, ultimately inducing copper death in dopaminergic neurons. Furthermore, CYCS overexpression also enhances mitochondrial respiratory chain damage, further exacerbating ROS generation, forming a vicious cycle of 'copper accumulation - oxidative stress - neuronal damage'. This mechanism has been fully confirmed through bioinformatics analysis (GSE22491 dataset screening, PPI network construction), cell experiments (SH-SY5Y cell model validation), animal experiments (MPTP-induced PD mouse model validation), and clinical sample testing. For the first time, a direct link between epigenetic regulatory networks and the core pathological links of PD (abnormal copper metabolism, copper death) has been established, providing a new molecular perspective for elucidating the complex pathogenesis of PD and filling a key gap in the existing research on the regulatory pathway of copper death in PD.

[0017] (2) The key role mechanism of the LINC00467 / miR-494-3p / CYCS axis in the regulation of copper metabolism in Parkinson's disease was revealed for the first time. This discovery provides a new perspective for understanding the pathogenesis of Parkinson's disease.

[0018] (3) A novel biomarker combination for early diagnosis of Parkinson's disease, with LINC00467, miR-494-3p, and CYCS as its core, is provided. This combination has significantly better specificity and sensitivity than existing diagnostic indicators. This biomarker combination breaks through the limitations of existing PD diagnosis, which relies on typical clinical symptoms such as "resting tremor" and is difficult to identify in the early stage. It can capture disease signals before irreversible neuronal damage occurs, creating a window period for early clinical intervention. At the same time, the diagnostic kit developed based on this combination can achieve rapid quantitative detection and is suitable for screening in multiple scenarios such as hospitals and health check-up centers. It helps to improve the early diagnosis rate of PD, delay the progression of the disease through early treatment, improve the prognosis of patients' motor and cognitive functions, and reduce the disability rate of the disease.

[0019] (4) Several small molecule compounds, including ciprofloxacin, pentoxifylline, melatonin, ketoconazole, minocycline, and paclitaxel, were discovered that can specifically act on the LINC00467 / miR-494-3p / CYCS pathway. These compounds can regulate pathway function through different modes of action, such as downregulating LINC00467 expression, upregulating miR-494-3p levels, inhibiting excessive CYCS activation, or blocking the interaction between the three, ultimately restoring copper metabolism homeostasis and inhibiting neuronal copper death. This discovery breaks through the dilemma of existing PD treatment drugs that "only provide symptomatic relief and do not target neuroprotection," and provides a clear core target and candidate compound library for the development of PD treatment drugs. Drug development based on this pathway can avoid the drug resistance and motor complication risks of traditional dopamine replacement therapy, providing patients with safer and more effective new treatment options and accelerating the industrialization process of precision PD treatment drugs. It provides a new perspective for the study of the pathogenesis of Parkinson's disease and has important theoretical significance and clinical application value. This will advance basic research and clinical practice in Parkinson's disease, bringing hope to patients. Attached Figure Description

[0020] Figure 1 : Acquisition and analysis of copper death-related differentially expressed genes (DECRGs); Figure 2 Results of functional enrichment analysis of DECRGs; Figure 3 : Diagram of protein-protein interaction network and Hub gene analysis; Figure 4 Results of immune cell infiltration analysis; Figure 5 : mRNA-miRNA-lncRNA ceRNA network diagram of Parkinson's disease; Figure 6 Results of GSEA enrichment analysis of CYCS. Detailed Implementation

[0021] The following detailed description illustrates the specific implementation method: 1. Bioinformatics analysis and validation: The PD dataset (GSE22491) was downloaded from the NCBIGEO database. Differentially expressed genes (DEGs) between PD samples and control samples were screened using the LIMMA software package. After intersection with copper death-related genes (CRGs), differentially expressed copper death-related genes (DECRGs) were obtained. A protein-protein (PPI) network was constructed to screen for Hub gene CYCS. The miR-494-3p targeting CYCS and the lncRNA LINC00467 targeting miR-494-3p were predicted and identified using databases such as TargetScan and miRNet. Finally, the LINC00467 / miR-494-3p / CYCSceRNA network was constructed.

[0022] Bioinformatics analysis narrows the research scope, avoids blind screening, and clarifies the core molecules (LINC00467, miR-494-3p, CYCS), regulatory relationships, and functional associations (PD, copper death) that need to be verified in subsequent experiments, laying the foundation for cell experiments, animal experiments, and clinical sample verification.

[0023] 2. Cellular experimental verification: (1) PD cell model was constructed by treating neuron-like SH-SY5Y cells with different concentrations of rotenone, providing a standardized vector for all subsequent validation experiments targeting this regulatory axis, and the role of the LINC00467 / miR-494-3p / CYCS axis in Parkinson's disease was verified by experiments. (2) Using different concentrations of MPP + Cells were treated with another classic PD inducer, and cell viability was detected by the CCK-8 assay to observe whether there was a concentration-dependent decrease in survival (consistent with the characteristics of PD neuronal damage). This was cross-referenced with the rotenone model to ensure the stability and universality of the model and to verify the successful establishment of the PD model.

[0024] (3) Cellular copper ion detection: The cells were treated with rotenone, and the copper ion level was detected by a copper ion detection kit. ICP-MS quantitative analysis was used to verify whether copper metabolism was abnormal in the PD cell model and to correlate the regulatory axis with the copper death pathway.

[0025] (4) ROS: The level of reactive oxygen species (ROS) was detected by fluorescence detection of cells incubated with DCFH-DA probe to assess the degree of oxidative stress. ROS is the core marker of oxidative stress. By detecting its fluorescence intensity, we can verify whether oxidative stress is enhanced in the PD model, thereby supporting the pathological logic of abnormal regulation axis mediating oxidative damage.

[0026] (5) Western blot protein verification (CYCS, apoptosis proteins): Total protein was extracted from SY5Y cells treated with rotenone, subjected to SDS-PAGE electrophoresis, transferred to a membrane, and incubated with antibodies against CYCS, Cleaved-Caspase-3, LIAS, FDX1, and DLAT. ECL imaging was used to analyze expression changes. By detecting the protein expression levels of CYCS (a core axis molecule), Cleaved-Caspase-3 (an apoptosis marker), and LIAS / FDX1 / DLAT (key genes for copper death), the association between the regulatory axis and cell apoptosis and copper death pathways was clarified, confirming the core role of CYCS in PD pathology.

[0027] (6) qPCR verification (LINC00467 / miR-494-3p / CYCS): RNA was extracted by Trizol method, reverse transcribed into cDNA, and the expression level of the target gene was detected by SYBR Green qPCR; by quantitatively detecting the relative expression levels of lncRNA, miRNA and mRNA, it was determined whether the characteristic pattern of "LINC00467 upregulation + miR-494-3p downregulation + CYCS upregulation" exists in the PD cell model, providing cellular-level evidence for this combination as a diagnostic marker.

[0028] (7) Dual-luciferase reporter assay: A wild-type / mutant luciferase vector of CYCS 3'UTR was constructed and co-transfected with miR-494-3p mimics. The fluorescence activity was detected to verify the binding effect. By constructing a wild-type / mutant vector of CYCS 3'UTR and co-transfecting it with miR-494-3p mimics, the changes in fluorescence activity were detected to clarify whether miR-494-3p directly targets and binds to CYCS. At the same time, it provides key evidence for the ceRNA mechanism of LINC00467 adsorbing miR-494-3p.

[0029] (8) In vivo experiments (mouse PD model): MPTP-induced PD mouse model, behavioral tests such as rotating bar and gait analysis were performed, and brain tissue was taken to detect CYCS and related gene expression.

[0030] By inducing PD-like motor dysfunction in mice through MPTP (verified by rotator bar and gait analysis), and then detecting changes in the expression of CYCS and related genes in mouse brain tissue, the cellular findings were transformed into in vivo evidence, enhancing the clinical translational value of the conclusions and supporting the reliability of the regulatory axis as a therapeutic target.

[0031] The experiment is based on Table 1 below: Table 1

[0032] The verification results show: (1) Validation of model construction effectiveness: such as Figure 1 As shown, when the concentration of rotenone reached 25 μM, the viability of SH-SY5Y cells decreased to 50% (i.e., the half-maximal inhibitory concentration, IC50), exhibiting typical neuronal damage characteristics under the pathological state of Parkinson's disease. Based on this, a standardized cell model was established, providing a stable and reliable experimental carrier for subsequent axonal expression analysis, copper metabolism detection, and drug intervention evaluation.

[0033] (2) Verification of the association between axons and cell death pathways: such as Figure 2 As shown, the expression of CYCS and the apoptosis-related protein Cleaved-Caspase-3 was significantly upregulated in the model cells, while the expression of key copper death genes LIAS, FDX1, and DLAT was significantly downregulated. This result clarifies that CYCS is not only a core molecule of the regulatory axis, but also directly mediates the activation of apoptosis and the disorder of the copper death pathway in the Parkinson's disease cell model. This provides a key mechanistic basis for the treatment idea that "regulating this axis can simultaneously improve apoptosis and copper death abnormalities" and lays a theoretical foundation for subsequent drug intervention directions.

[0034] (3) Verification of the expression characteristics of the regulatory axis: such as Figure 3 As shown, compared with normal control cells, the expression of LINC00467 and CYCS was significantly upregulated and the expression of miR-494-3p was significantly downregulated in model cells. This is the first time that the characteristic expression pattern of the LINC00467 / miR-494-3p / CYCS axis has been clearly defined at the cellular level, providing direct cellular-level evidence for the feasibility of this molecular combination as a diagnostic biomarker.

[0035] (4) Verification of the association between axon molecules and oxidative stress: such as Figure 4 As shown, the level of reactive oxygen species (ROS) in the model cells was significantly increased, directly linking "abnormal expression of axon molecules" with "oxidative stress damage," confirming that this regulatory axis is a key regulatory pathway for oxidative stress response in Parkinson's disease, and further supporting the therapeutic logic that "targeting this axis can alleviate oxidative damage."

[0036] (5) Verification of the association between axon molecules and copper metabolism: such as Figure 5 As shown, the copper ion concentration in the model cells reached 0.36 nmol / 10⁻⁶. 6 The cell count was significantly higher than that of normal control cells (0.126 nmol / 10⁻⁶). 6 This result directly confirms that axonal abnormalities are the core cause of copper metabolism imbalance, clarifies the central role of this regulatory axis in the regulation of copper homeostasis in Parkinson's disease, and provides direct experimental support for the technical solution of "restoring copper metabolism balance by regulating this axis to improve the condition".

[0037] 3. Clinical sample validation: Blood samples from Parkinson's disease patients and healthy controls were tested. The results showed that CYCS expression was significantly upregulated and miR-494-3p expression was significantly downregulated in the blood of patients. This expression characteristic was highly consistent with the detection results of cell models and animal models, which fully confirmed the clinical diagnostic potential of this molecular combination.

[0038] Table 2

[0039] For specific data, please refer to Table 2, and combine it with, for example, Figure 6 The blood sample test results clearly showed that CYCS was significantly upregulated and miR-494-3p was significantly downregulated in the blood of Parkinson's disease patients. This directly verified the diagnostic efficacy of the molecular combination at the clinical sample level, providing key clinical evidence for early diagnosis. It effectively solved the core problem of existing diagnostic methods being "low specificity and dependent on clinical symptoms", and is the core clinical basis for the implementation of early diagnosis.

[0040] It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solution of this invention. These modifications and improvements should also be considered within the scope of protection of this invention, and will not affect the effectiveness of the invention or the practicality of the patent. The scope of protection claimed in this application shall be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A combination of biomarkers for the diagnosis of Parkinson's disease, characterized in that, It contains the LINC00467, miR-494-3p and CYCS genes or their expression products.

2. The application of the biomarker combination according to claim 1 in the preparation of Parkinson's disease diagnostic products.

3. The application according to claim 2, characterized in that, The diagnostic product enables the diagnosis or risk assessment of Parkinson's disease by detecting the genes or expression products of LINC00467, miR-494-3p, and CYCS in biological samples.

4. The application according to claim 3, characterized in that, The diagnostic products include antibodies, primers, probes, chips, kits, or detection devices for detecting LINC00467, miR-494-3p, and CYCS genes or their expression products.

5. Application of the LINC00467 / miR-494-3p / CYCS regulatory axis in the preparation of drugs for the treatment of Parkinson's disease.

6. The application according to claim 5, characterized in that, The therapeutic drug works by regulating the expression levels of LINC00467, miR-494-3p, and CYCS, or by blocking the interaction between the three.

7. The application according to claim 6, characterized in that, The therapeutic agents include: nucleic acid drugs that downregulate LINC00467 expression, nucleic acid drugs that upregulate miR-494-3p expression, nucleic acid drugs that downregulate CYCS expression, or small molecule compounds that specifically bind to LINC00467, miR-494-3p, or CYCS and regulate their function.

8. The application according to claim 7, characterized in that, The small molecule compound is selected from one or more of ciprofloxacin, pentoxifylline, melatonin, ketoconazole, minocycline, or paclitaxel.

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