Parkinson's disease marker lncRNA USP28-6 and application thereof

Abstract

The invention relates to a Parkinson's disease marker lncRNA USP28-6 and application thereof, in particular to application of the lncRNA USP28-6 in preparation of a diagnostic reagent and / or a therapeutic drug for Parkinson's disease, and aims to provide a completely novel diagnostic mode and / or targeted therapy mode for diagnosis and / or treatment of Parkinson's disease.

Description

Technical Field

[0001] The present invention belongs to the field of biomarkers, and specifically relates to a Parkinson's disease marker, lncRNA USP28-6, and applications thereof. Background Art

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disease, and its early diagnosis and precise treatment remain major clinical challenges.

[0003] Diagnostic lag: PD patients often have lost 50%-70% of their dopaminergic neurons by the time they are diagnosed. Existing technologies can only detect late-stage pathological changes and cannot provide early warning. Clinical diagnostic techniques rely on subjective symptom assessment. Current PD diagnosis is primarily based on motor symptoms (tremor, rigidity, bradykinesia). However, by the time symptoms appear, irreversible neuronal loss has often already occurred, missing the window for early intervention. Secondly, imaging diagnostic technology is limited.

[0004] Invasive limitations: Cerebrospinal fluid α-synuclein (α-syn) oligomer detection requires lumbar puncture, which has poor patient compliance and the invasive procedure limits its clinical application.

[0005] Limitations of markers: Reported PD-related lncRNAs (such as NEAT1 and MALAT1) mostly rely on brain tissue or cerebrospinal fluid samples and cannot be used for non-invasive detection such as peripheral blood; their expression levels have no clear quantitative correlation with PD stage and inflammation severity.

[0006] Mechanistic fragmentation: Existing studies only describe the single association between a certain lncRNA and inflammation or α-syn aggregation in isolation, without clarifying its upstream regulatory network (such as transcription factor binding) and downstream effector molecules.

[0007] In view of this, the present invention is proposed. Summary of the Invention

[0008] The present invention provides a Parkinson's disease biomarker, lncRNA USP28-6, a detection reagent and / or targeting reagent thereof, and its use in the diagnosis and / or treatment of Parkinson's disease. The present invention aims to provide a completely novel diagnostic method and / or targeted treatment for the diagnosis and / or treatment of Parkinson's disease.

[0009] In a first aspect, the present invention provides a biomarker for Parkinson's disease, wherein the biomarker is lncRNA USP28-6.

[0010] In some specific embodiments, the biomarker is peripheral blood or neuron-derived lncRNA USP28-6.

[0011] In some specific embodiments, the Parkinson's disease is idiopathic Parkinson's disease.

[0012] In a second aspect, the present invention provides use of lncRNA USP28-6 in preparing a diagnostic reagent for Parkinson's disease.

[0013] In some specific embodiments, the lncRNA USP28-6 has a sequence as shown in SEQ ID NO: 1.

[0014] In some specific embodiments, the diagnostic reagent includes a detection reagent for detecting the mRNA expression level of lncRNA USP28-6 in a subject.

[0015] In some specific embodiments, the detection reagent is a PCR primer.

[0016] In some specific embodiments, the PCR primers include a primer pair consisting of SEQ ID NO:4 and SEQ ID NO:5.

[0017] In some specific embodiments, the lncRNA USP28-6 is lncRNA USP28-6 derived from peripheral blood and / or neuronal cells of a subject.

[0018] In some specific embodiments, the Parkinson's disease is idiopathic Parkinson's disease.

[0019] In some specific embodiments, the diagnostic reagent further comprises a reagent for detecting the expression level of ZBTB16 and / or α-syn; preferably, it further comprises a reagent for detecting the mRNA expression level of ZBTB16 and / or α-syn; more preferably, it further comprises:

[0020] (1) a primer pair consisting of SEQ ID NO: 2 and SEQ ID NO: 3; and / or,

[0021] (2) A primer pair consisting of SEQ ID NO: 6 and SEQ ID NO: 7.

[0022] In a third aspect, the present invention also provides use of a detection reagent for lncRNA USP28-6 in the preparation of a diagnostic reagent for Parkinson's disease.

[0023] In some specific embodiments, the lncRNA USP28-6 has a sequence as shown in SEQ ID NO: 1.

[0024] In some specific embodiments, the detection reagent is a detection reagent for detecting the mRNA expression level of lncRNA USP28-6 in a subject.

[0025] In some specific embodiments, the detection reagent is a PCR primer.

[0026] In some specific embodiments, the PCR primers include a primer pair consisting of SEQ ID NO:4 and SEQ ID NO:5.

[0027] In some specific embodiments, the diagnostic reagent further comprises a reagent for detecting the expression level of ZBTB16 and / or α-syn, preferably a reagent for detecting the mRNA expression level of ZBTB16 and / or α-syn; more preferably, the diagnostic reagent further comprises:

[0028] (1) a primer pair consisting of SEQ ID NO: 2 and SEQ ID NO: 3; and / or,

[0029] (2) A primer pair consisting of SEQ ID NO: 6 and SEQ ID NO: 7.

[0030] In some specific embodiments, the lncRNA USP28-6 is lncRNA USP28-6 derived from peripheral blood and / or neuronal cells of a subject.

[0031] In some specific embodiments, the Parkinson's disease is idiopathic Parkinson's disease.

[0032] In a fourth aspect, the present invention further provides a diagnostic kit for Parkinson's disease, comprising a reagent for detecting the expression level of lncRNA USP28-6 in a subject.

[0033] In some specific embodiments, the diagnostic kit further comprises a reagent for detecting the expression level of ZBTB16 and / or α-syn.

[0034] In some specific embodiments, the diagnostic kit includes reagents for detecting the mRNA expression level of lncRNA USP28-6, ZBTB16 and / or α-syn.

[0035] In some specific embodiments, the diagnostic kit comprises:

[0036] (1) a primer pair consisting of SEQ ID NO: 4 and SEQ ID NO: 5;

[0037] (2) a primer pair consisting of SEQ ID NO: 2 and SEQ ID NO: 3; and / or,

[0038] (3) A primer pair consisting of SEQ ID NO: 6 and SEQ ID NO: 7.

[0039] In some specific embodiments, the Parkinson's disease is idiopathic Parkinson's disease.

[0040] In a fifth aspect, the present invention also provides a use of a lncRNA USP28-6 targeting agent in the preparation of a drug for treating Parkinson's disease.

[0041] In some specific embodiments, the Parkinson's disease is idiopathic Parkinson's disease.

[0042] Beneficial effects:

[0043] This study, based on the original discovery that lnc-USP28-6 (preferably peripheral blood lnc-USP28-6) is significantly elevated in Parkinson's disease (especially primary Parkinson's disease) and correlates with motor symptoms in Parkinson's patients, provides an integrated solution for the early diagnosis and targeted treatment of Parkinson's disease. This solution addresses the core challenges of early diagnosis and the lack of therapeutic targets in the field of Parkinson's disease, and possesses both clinical translational and commercial potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Figure 2 is a graph showing differentially expressed genes and their correlation between the normal control group and the PD group; Figure 1 A is a correlation diagram of differentially expressed genes. Green indicates genes with reduced expression levels in the PD group compared with the normal control group, and red indicates genes with increased expression levels in the PD group compared with the normal control group. Figure 1 B is the expression level of lncUSP28-6 in the normal control group and PD disease group.

[0045] Figure 2 Real-time fluorescence quantitative PCR was used to analyze the expression differences of Lnc-USP28-6 in the normal group, Parkinson's disease group and cerebral infarction group.

[0046] Figure 3 Real-time fluorescence quantitative PCR was used to analyze the expression level of Lnc-USP28-6 in control and Parkinson's disease cell models.

[0047] Figure 4 Real-time fluorescence quantitative PCR was used to analyze the expression levels of lncUSP28-6, ZBTB16 and α-syn (α-synuclein) in Parkinson's disease dopaminergic cells SH-SY5Y cells before and after overexpression of lnc-USP28-6.

[0048] Figure 5Real-time fluorescence quantitative PCR was used to analyze the expression levels of lncUSP28-6, ZBTB16, and α-syn (α-synuclein) in SH-SY5Y cells in the lnc-USP28-6 group and the lnc-USP28-6 + neurotoxin rotenone group (Parkinson's disease cell model). DETAILED DESCRIPTION

[0049] To better understand the present invention, specific embodiments will be given to further illustrate the present invention. However, it should be understood that the embodiments described are exemplary embodiments and that the present invention can be implemented in various forms and should not be limited by the embodiments described herein. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0050] Example 1

[0051] RNA sequencing was used to determine the genes differentially expressed in the peripheral blood of healthy controls and PD patients: Total RNA was extracted from peripheral blood mononuclear cells of healthy controls and PD patients, and the purity and quantity of RNA were evaluated using OD260 / OD280, followed by RNA sequencing analysis. Finally, the cDNA library was sequenced on Illumina NovaSeq6000 in 2x 150bp paired-end sequencing mode to obtain FastQ data. DESeq2 was used to analyze the differential expression of genes in the healthy control group and PD group. The identification criteria for significantly differentially expressed genes were p value <0.05 and |log2FC|>1. Based on the known upstream and downstream association relationships of genes, a correlation diagram between differentially expressed genes was constructed. Figure 1 A- Figure 1 As shown in Figure B, the expression levels of lnc-USP28-6 and its associated gene ZBTB16 were increased in the peripheral blood of PD patients compared with healthy controls.

[0052] Example 2

[0053] The expression levels of lnc-USP28-6 in peripheral blood samples of different subjects were detected by PCR:

[0054] 1) Blood samples to be tested were collected into cryovials and stored at -80°C. Peripheral blood samples were collected from 25 healthy individuals, 30 patients with Parkinson's disease, and 20 patients with cerebral infarction. 2) Real-time quantitative PCR analysis: Total RNA was extracted using RNAisoPlus (Takara Bio, Tokyo, Japan), and RNA purity was assessed by measuring the optical density ratio of OD260 / OD280. cDNA was synthesized using the PrimeScript RT Reagent Kit (Perfect Real-Time) (Takara Bio, Tokyo, Japan). mRNA levels were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) using the SYBR Green Expression Detection System (Takara Bio) on a CFX96 Real-Time PCR Detection System (Bio-Rad). PCR conditions included an initial denaturation at 95°C for 30 seconds, followed by 35 cycles of denaturation at 95°C for 5 seconds and annealing at 60°C for 30 seconds. The relative expression levels of the target gene and the reference transcript were calculated using the formula 2^-ΔΔCt.

[0055] Test results such as Figure 2 For the first time, the expression level of lnc-USP28-6 was identified to be correlated with PD (P < 0.0001), but the expression of lnc-USP28-6 in patients with cerebral infarction was not statistically significant compared with the normal group, as shown in Figure 2. Figure 2 Therefore, it has high specificity in diagnosing Parkinson's disease, and for the first time revealed that peripheral blood lnc-USP28-6 is a specific marker for Parkinson's disease.

[0056] Example 3

[0057] Construction of a rotenone ROT-induced Parkinson's disease dopaminergic SH-SY5Y neuronal injury model: 1) Cell culture: SH-SY5Y cells were purchased from Praxair Life Sciences (CL-0208, CL-0493, Wuhan, China). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing high glucose, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The culture conditions were maintained in a humidified incubator at 37°C with an ambient humidity of 5% carbon dioxide. 2) Construction of a Parkinson's disease cell model: Rotenone was first dissolved in DMSO to prepare a 100 mM stock solution, which was then diluted to a working concentration of 2 μM in DMEM. To induce the rotenone effect, SH-SY5Y cells were treated with this rotenone solution and incubated for 36 hours under the standard culture conditions described previously.

[0058] The expression level of lnc-USP28-6 in the model was detected by real-time quantitative PCR (refer to Example 2 for the experimental method). Figure 3As shown in the figure, compared with the control, the expression level of lnc-USP28-6 in SH-SY5Y cells was significantly increased after rotenone ROT induction.

[0059] Example 4

[0060] lncUSP28-6 was overexpressed in SH-SY5Y cells, and then the expression of lncUSP28-6, ZBTB16, and α-syn was detected by real-time PCR (the culture of SH-SY5Y cells refers to Example 3, and the real-time PCR refers to Example 2). Figure 4 As shown, overexpression of lncUSP28-6 upregulated the expression of ZBTB16 and α-syn.

[0061] Example 5

[0062] lncUSP28-6 was overexpressed in SH-SY5Y cells and stimulated with rotenone. Real-time PCR was then used to detect the expression of lncUSP28-6, ZBTB16, and α-syn after overexpression of lncUSP28-6 (refer to Example 3 for SH-SY5Y cell culture and rotenone stimulation, and to Example 2 for real-time PCR). Figure 5 As shown, rotenone enhanced the expression of lncUSP28-6, ZBTB16, and α-syn in the lncUSP28-6 + rotenone group compared with the lncUSP28-6 alone overexpression group.

[0063] The sequence of lnc-USP28-6 of the present invention is as follows:

[0064] Table 1 lnc-USP28-6 sequence information

[0065]

[0066]

[0067] The primers involved in real-time PCR in Examples 2-5 are shown in the following table.

[0068] Table 2 Realtime PCR primer sequences

[0069]

[0070] The above descriptions are only specific embodiments of the present invention. These examples are not intended to limit the present invention to the precise forms disclosed. Various choices and improvements made by those skilled in the art based on the present invention are within the scope of protection of the present invention.

Claims

1. Application of lncRNA USP28-6 in the preparation of diagnostic reagents for Parkinson's disease.

2. The use according to claim 1, characterized in that The lncRNA USP28-6 has the sequence shown in SEQ ID NO:

1.

3. The use according to claim 1, characterized in that The diagnostic reagent includes a detection reagent for detecting the mRNA expression level of lncRNA USP28-6 in a subject.

4. The use according to claim 3, characterized in that The detection reagent is a PCR primer.

5. The use according to claim 4, characterized in that The PCR primers include a primer pair consisting of SEQ ID NO: 4 and SEQ ID NO:

5.

6. The use according to any one of claims 1 to 5, characterized in that The lncRNA USP28-6 is lncRNA USP28-6 derived from the peripheral blood and / or neuronal cells of the subject.

7. A diagnostic kit for Parkinson's disease, characterized in that: The diagnostic kit includes a reagent for detecting the expression level of lncRNA USP28-6 in a subject.

8. The diagnostic kit according to claim 7, characterized in that The diagnostic kit further comprises a reagent for detecting the expression level of ZBTB16 and / or α-syn.

9. The diagnostic kit according to claim 7 or 8, characterized in that The diagnostic kit includes reagents for detecting the mRNA expression levels of lncRNA USP28-6, ZBTB16 and / or α-syn.

10. The diagnostic kit according to any one of claims 7 to 9, characterized in that The diagnostic kit comprises: (1) a primer pair consisting of SEQ ID NO: 4 and SEQ ID NO: 5; (2) a primer pair consisting of SEQ ID NO: 2 and SEQ ID NO: 3; and / or, (3) A primer pair consisting of SEQ ID NO: 6 and SEQ ID NO: 7.

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