CN113308535A - PD diagnosis and staging kit based on serum exosome miRNA - Google Patents
PD diagnosis and staging kit based on serum exosome miRNA Download PDFInfo
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Abstract
The invention discloses a PD diagnosis and staging kit based on serum exosome miRNA. By detecting the expression level of miRNA of specific sequences of PD patients, different pathogenic stages of PD, such as early stage II and other early stages, can be identified, and further disease deterioration can be prevented by adopting treatment measures as early as possible. The PD diagnosis and staging marker adopted by the invention is derived from exosome miRNA, and has the advantages of high stability, strong sensitivity and good specificity compared with the peripheral circulating miRNA commonly used for detection.
Description
Technical Field
The invention relates to a disease diagnosis screening reagent, in particular to a method for diagnosing and staging PD by using serum exosome miRNA.
Background
Parkinson Disease (PD) is a neurodegenerative disease (NDDs) with hidden onset and serious harm, and DA neurons are irreversibly lost by about 70 percent when obvious symptoms appear, so that early diagnosis of PD is very important. The real-time reverse transcription quantitative PCR (qRT-PCR) is a method mainly used for detecting the relative expression quantity of specific nucleic acid in various samples, has the characteristics of wide detection range, high sensitivity and precision and avoidance of cross contamination, is particularly suitable for completing detection under the condition of small sample quantity, and is widely applied to the diagnosis of various NDDs.
miRNA is a non-coding single-stranded small molecule RNA consisting of 18-25 nucleotides. Multiple studies indicate that the level change of peripheral blood miRNA in different NDDs becomes one of important indexes for revealing the development stage of the NDDs, but the circulating miRNA is easily affected by the degradation of rich RNase (RNase) in the peripheral circulation system and cannot accurately reflect the state of the Central Nervous System (CNS), so that certain difficulty exists in screening markers which can be used for clinical diagnosis from the circulating miRNA.
The exosome is an Extracellular Vesicle (EVs) which can be secreted by almost all body cells and has the diameter of 30-150 nm, and can be found in body fluids such as cerebrospinal fluid, blood and the like. mirnas can be transported from cells and encapsulated in vectors such as exosomes (i.e., forming exosome mirnas) and Argonaute 2 proteins as messengers for intercellular information transfer. Exosome mirnas (ex-mirnas) can play an important role in the diagnosis of various NDDs, and compared to circulating mirnas, ex-mirnas mainly have the following advantages: (1) the mean content of mirnas in exosomes (42-48%) was higher than the mean content in plasma (30%); (2) the exosome can enrich and stabilize miRNA, and the miRNA is prevented from being degraded by RNase in a peripheral circulation system; (3) exosomes can freely pass through a blood brain barrier, and the state change of a central nervous system is intuitively reflected; (4) the extraction and sequencing of exosome RNA and the quantitative technology are mature.
Chinese patent CN110872628A discloses the use of biomarkers of extracellular vesicles, such as hsa-miR-199a-3p, in cancer diagnosis, and also indicates that the expression levels of biomarkers of extracellular vesicles, such as cytokines, exoDNA, can be used to determine that degenerative diseases such as PD are in an aging state.
At present, there is no experimental basis for predicting the biological function of miRNA according to sequence similarity, and reports of PD early diagnosis by using miRNA derived from serum exosome are not seen.
Disclosure of Invention
The invention aims to provide a PD diagnosis and staging kit based on a serum exosome miRNA, which solves the problem that the PD cannot be staged by using the miRNA in the prior art so as to realize early diagnosis.
In order to achieve the purpose, the invention adopts the following technical scheme:
a PD diagnostic kit comprising real-time quantitative PCR primers (e.g., seq.id.no.1, seq.id.no.2) for expression level detection of markers that are hsa-miR-374a-5p and/or hsa-miR-374b-5 p.
A PD patient staging (phase ii) kit comprising real-time quantitative PCR primers (e.g., seq.id.no.4, seq.id.no.5) for expression level detection of markers that are hsa-miR-199a-3p and/or hsa-miR-195-5 p.
A PD patient staging (stage iii) kit comprising real-time quantitative PCR primers (e.g., seq. id. No.6) for detection of the expression level of a marker that is hsa-miR-28-5 p.
A PD patient staging (stage iv) kit comprising real-time quantitative PCR primers (e.g., seq.id.no.3, seq.id.no.7) for expression level detection of markers that are hsa-miR-22-5p and/or hsa-miR-151a-5 p.
A PD diagnosis and staging kit comprises real-time quantitative PCR primers for detecting the expression level of any one or two of hsa-miR-374a-5p and hsa-miR-374b-5p, and real-time quantitative PCR primers for detecting the expression level of any one or more of hsa-miR-199a-3p, hsa-miR-195-5p, hsa-miR-28-5p, hsa-miR-22-5p and hsa-miR-151a-5 p.
Preferably, each of the above markers is extracted from peripheral blood.
Preferably, the peripheral blood sample volume is 5-15 mL/subject (e.g., 9-11 mL/subject).
Preferably, each of the above markers is extracted from serum exosomes in peripheral blood.
The miRNA (hsa-miR-374a-5p, hsa-miR-374b-5p, hsa-miR-199a-3p, hsa-miR-195-5p, hsa-miR-28-5p, hsa-miR-22-5p and hsa-miR-151a-5p) serving as the markers and the application of the reverse transcription product thereof in preparing PD early diagnosis kits and reagents.
The miRNA (hsa-miR-374a-5p, hsa-miR-374b-5p, hsa-miR-199a-3p, hsa-miR-195-5p, hsa-miR-28-5p, hsa-miR-22-5p and hsa-miR-151a-5p) serving as the markers and the application of the reverse transcription product thereof in preparing a PD patient stage kit and a reagent.
The real-time quantitative PCR primer (the real-time quantitative PCR amplification reaction specific primer with the sequence shown in SEQ. ID.NO. 1-SEQ. ID.NO.7) is applied to the preparation of PD early diagnosis kits and reagents.
Preferably, the kit further comprises real-time quantitative PCR amplification reaction universal primers and internal reference sequence amplification primers (e.g., seq.id No.8 and seq.id No. 9).
The real-time quantitative PCR primer (the real-time quantitative PCR amplification reaction specific primer with the sequence shown in SEQ. ID.NO. 1-SEQ. ID.NO.7) is applied to the preparation of PD patient staging kits and reagents.
Preferably, the kit further comprises real-time quantitative PCR amplification reaction universal primers and internal reference sequence amplification primers (e.g., seq.id No.8 and seq.id No. 9).
The invention has the beneficial effects that:
the invention can identify different pathogenic stages (for example, early stages such as II) of PD by detecting the expression level of miRNA of a specific sequence of a PD patient, thereby preventing further worsening of the disease by adopting a treatment means as early as possible.
Furthermore, the PD diagnosis and staging marker adopted by the invention is derived from exosome miRNA, and has the advantages of high stability, strong sensitivity and good specificity compared with the peripheral circulating miRNA commonly used for detection.
Drawings
Figure 1 shows the expression of different in vitro exosome mirnas: hsa-miR-374a-5 p; hsa-miR-374b-5 p; p <0.05, p < 0.0001; stage2\3\4 corresponds to II, III and IV periods.
Figure 2 shows the expression of different in vitro exosome mirnas: hsa-miR-199a-3 p; expression of hsa-miR-195-5 p; p <0.05, p <0.001, p < 0.0001; stage2\3\4 corresponds to II, III and IV periods.
FIG. 3 shows the expression of different in vitro exosome miRNAs (hsa-miR-28-5 p): represents p < 0.0001; stage2\3\4 corresponds to II, III and IV periods.
Figure 4 shows the expression of different in vitro exosome mirnas: hsa-miR-22-5 p; hsa-miR-151a-5 p; represents p < 0.0001; stage2\3\4 corresponds to II, III and IV periods.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples. The examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention.
A first part: PD diagnosis and staging procedure test
First, collecting peripheral blood of individual and extracting serum exosome
1. Peripheral blood from PD patients and normal controls (controls) were collected
1) About 10mL of blood was collected from the subject using a serum tube without anticoagulant.
2) Standing for 30min at room temperature, and then standing for 3-5 hours or overnight at 4 ℃ until blood clots are separated out.
3) Centrifuging at 4 deg.C for 15min at 3000g, and carefully transferring the serum into a new 15mL centrifuge tube to ensure the quality of the serum to the maximum extent.
4) The serum obtained after centrifugation is used for timely extracting exosome or stored in a refrigerator at minus 80 ℃ for later use.
2. Extraction of exosomes
1) Sterilizing related accessories of the ultracentrifuge in an autoclave in advance, and irradiating the ultracentrifuge tube under an ultraviolet lamp overnight for sterilization.
2) Serum was taken from the freezer, thawed in a water bath, and diluted to 11mL with clean PBS, keeping the same volume of serum in each tube.
3) Centrifuge at 3000g for 10min at 4 ℃ and carefully transfer all supernatants to a new 15mL centrifuge tube.
4) Centrifuging at 4 deg.C for 30min at 10000 g; after centrifugation, the supernatant was transferred to an ultracentrifuge tube and trimmed with PBS.
5) Centrifuging at 100000g and 4 deg.C for 70min, and closing the centrifuge to brake; after centrifugation, approximately 1.5mL of supernatant was left and resuspended in PBS and then allowed to equilibrate.
6) Centrifuging at 100000g and 4 deg.C for 70min, and closing the centrifuge to brake; discarding the supernatant until 50-100 mu L of the bottom layer is left; transfer the remaining portion of the bottom layer (containing mainly serum exosomes) to rnase-free EP tube for further experiments or storage in a-80 ℃ freezer for future use.
(II) qRT-PCR detection
1. Primer and method for producing the same
The qRT-PCR primers were synthesized by Shanghai Sangon bioengineering, Inc., and the sequences of the primers are shown in tables 1-1 and 1-2:
TABLE 1-1.miRNA real-time quantitative PCR amplification reaction specific primers
The universal primers for real-time quantitative PCR amplification reaction of each miRNA are the primers in the MiScript II RT kit (cat #218161, QIAGEN).
TABLE 1-2 primers for amplification of reference sequence for real-time quantitative PCR
2. Reverse transcription to synthesize cDNA
1) Preparing a reaction system mixed solution shown in the table 2 in an RNase-free centrifuge tube, and lightly blowing, beating and uniformly mixing to avoid oscillation; wherein the template RNA is total RNA obtained by extracting RNA contained in the serum exosome (i.e., serum exosome RNA) by the Trizol method.
TABLE 2 reverse transcription reaction System
2) The procedure of the PCR instrument was set up as shown in Table 3, and after the reaction was completed, cDNA was obtained, which was used for subsequent experiments or stored in a refrigerator at-20 ℃ for future use.
TABLE 3 reverse transcription procedure
3. Real-time quantitative PCR reaction
1) cDNA samples (referring to quantitation of peripheral blood serum exosome RNA reverse transcription products per individual) were diluted 5-fold.
2) A reaction system mixture shown in Table 4 was prepared, gently blown and mixed, centrifuged at 3000rpm at room temperature, and then protected from light.
TABLE 4 real-time quantitative PCR reaction system
3) Amplification was performed using a real-time quantitative PCR instrument, and the reaction was performed according to the procedure shown in Table 5.
TABLE 5 real-time quantitative PCR reaction procedure
After Ct values of each group of samples (different stages of PD patients and normal controls) are obtained, the Ct values are respectively calculated according to the formula 2-ΔΔCtAnd calculating a final quantitative result. All experiments were performed in 3 replicates and the results were averaged over 3. Statistical analysis was performed by GraphPad Prism8, results were analyzed using One-way ANOVA, and differences between groups were tested using Dunnett's multiple complexes.
A second part: acquisition of different staged PD cases
Peripheral blood samples of 40 patients were taken again from the department of neurology and physical examination in the Cijing hospital, and the visit time ranged from 6 months in 2020 to 10 months in 2020. Wherein, the normal comparison is 10 cases, 7 cases in II period, 12 cases in III period and 11 cases in IV period. The case inclusion and exclusion criteria are as follows.
2.1 inclusion criteria:
according to Chinese medical society, "diagnostic criteria for Parkinson's disease in China (2016 edition)":
1) patients with primary Parkinson's disease.
2) The stage H & Y is patients in stage II, III and IV, and the specific staging standard is shown in Table 6.
TABLE 6H & Y staging criteria
3) Age and sex are not limited.
2.2 exclusion criteria:
1) patients who did not meet inclusion criteria.
2) Alzheimer's disease, Huntington's chorea, hydrocephalus, brain cysticercus, acute cerebral hemorrhage, and brain tumor.
3) There are serious primary diseases of liver, kidney, hematopoietic system, etc.
4) Suspected or confirmed drug addicts, alcoholism (14 units per week, 1 unit-8 g pure alcohol) or smoking (5 units per day).
5) Compliance is poor and blood draws and follow-up cannot be expected to be completed following the study protocol.
6) Other cases are not suitable for selection.
Exosome mirnas were extracted and detected by qRT-PCR according to the experimental procedure of the first part.
And a third part: PD diagnosis and staging result verification
3.1 validation of miRNA expression profiles associated with each phase
In the results obtained in the previous period, 2 miRNAs are found to be related to stages II, III and IV, and are hsa-miR-374a-5p and hsa-miR-374b-5 p. After verification, the expression levels of hsa-miR-374a-5p (figure 1A) and hsa-miR-374B-5p (figure 1B) show differences in stages II, III and IV compared with a normal control group (obviously higher than the normal control group; hsa-miR-374a-5 p: control 1.032, 3.431 in stage II, 7.503 in stage III, 6.393 in stage IV; hsa-miR-374B-5 p: control 1.081, 3.176 in stage II, 6.640 in stage III and 7.502 in stage IV).
3.2 verification of miRNA expression conditions specifically related to stages II, III and IV
In the results obtained in the early stage, 2 miRNAs are found, namely hsa-miR-199a-3p and hsa-miR-195-5p are only related to the specificity of the II stage; there are 1 mirnas, hsa-miR-28-5p, that are only associated with stage iii specificity; there are 2 miRNAs specifically associated only with stage IV, hsa-miR-151a-5p and hsa-miR-22-5p, respectively. After the same verification, the expression levels of hsa-miR-199a-3p (figure 2A) and hsa-miR-195-5p (figure 2B) in 2 miRNAs only specifically related to the stage II show significant difference only in the stage II compared with the stages control, III and IV (obviously lower than the stages control, III and IV; hsa-miR-199a-3 p: control 1.018, II 0.2857, III 1.027, IV 1.096; hsa-miR-195-5 p: control 1.004, II 0.5966, III 1.069 and IV 1.522); only 1 miRNA (hsa-miR-28-5p) specifically related to stage III shows significant difference of expression level only in stage III compared with control, stage II and stage IV (obviously higher than control, stage II and stage IV, figure 3; control 1.036, stage II 1.244, stage III 5.626 and stage IV 1.414). Of the 2 miRNAs specifically related to phase IV only, the expression levels of hsa-miR-22-5p (FIG. 4A) and hsa-miR-151a-5p (FIG. 4B) showed significant difference only in phase IV compared with control, phase II and phase III (the former was significantly higher and the latter was significantly lower than control, phase II and phase III; hsa-miR-22-5 p: control 1.063, phase II 1.115, phase III 1.454, phase IV 4.945; hsa-miR-151a-5 p: control1.045, phase II 1.120, phase III 1.035, phase IV 0.6633).
3.3 interpretation of kits
According to the verification result, the markers adopted by the kit for PD diagnosis are hsa-miR-374a-5p and hsa-miR-374b-5p, and the individual is determined as a PD patient according to the relative expression quantity judgment threshold of the markers which is 3, namely, the relative expression quantity is higher than the threshold (the relative expression level of a normal control is about 1, so that the individual can also be judged as the expression).
According to the verification result, the markers adopted by the kit for the stage (stage II) of the PD patient are hsa-miR-199a-3p and hsa-miR-195-5p, and the relative expression determination threshold values of the markers are as follows: 0.3(hsa-miR-199a-3p) and 0.6(hsa-miR-195-5p), namely, if the threshold value is lower, the PD patient is judged to be in stage II.
According to the verification result, the marker adopted by the kit for the stage III of the PD patient is hsa-miR-28-5p, and the threshold value is 5 according to the relative expression quantity of the marker, namely the stage III of the PD patient is judged if the threshold value is higher than the threshold value.
According to the verification result, the marker adopted by the kit for determining the stage (IV stage) of the PD patient can be hsa-miR-22-5p, and the threshold value is determined to be 5 according to the relative expression quantity of the marker, namely the stage of the PD patient is determined to be the IV stage if the relative expression quantity is higher than the threshold value. The marker adopted by the kit for determining the stage (IV stage) of the PD patient can also be hsa-miR-151a-5p, and the relative expression quantity of the marker is used as a judgment threshold value of 0.7, namely the stage of the PD patient is judged to be the IV stage when the relative expression quantity of the marker is lower than the threshold value.
The results show that the kit detection by using the markers can accurately position the PD stage by combining clinical symptom verification, thereby effectively helping clinicians to develop different treatment means according to different disease development stages of PD patients, and providing reliable clinical examination basis for early diagnosis and treatment of PD.
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Claims (10)
1. A PD diagnosis and staging kit, comprising: the kit comprises real-time quantitative PCR primers for detecting the expression level of any one or two of hsa-miR-374a-5p and hsa-miR-374b-5p, and real-time quantitative PCR primers for detecting the expression level of any one or more of hsa-miR-199a-3p, hsa-miR-195-5p, hsa-miR-28-5p, hsa-miR-22-5p and hsa-miR-151a-5 p.
2. A PD diagnostic kit, characterized in that: comprises real-time quantitative PCR primers for detecting the expression level of markers, wherein the markers are hsa-miR-374a-5p and/or hsa-miR-374b-5 p.
3. A PD patient staging kit, comprising: comprises real-time quantitative PCR primers for detecting the expression level of a marker, wherein the marker is hsa-miR-199a-3p and/or hsa-miR-195-5 p.
4. A PD patient staging kit, comprising: the kit comprises a real-time quantitative PCR primer for detecting the expression level of a marker, wherein the marker is hsa-miR-28-5 p.
5. A PD patient staging kit, comprising: comprises real-time quantitative PCR primers for detecting the expression level of a marker, wherein the marker is hsa-miR-22-5p and/or hsa-miR-151a-5 p.
6. The kit according to any one of claims 1 to 5, characterized in that: the marker is extracted from peripheral blood; the sampling amount of the peripheral blood is 5-15 mL per individual.
7. The kit of claim 6, wherein: the marker is extracted from serum exosomes in peripheral blood.
Application of hsa-miR-374a-5p, hsa-miR-374b-5p, hsa-miR-199a-3p, hsa-miR-195-5p, hsa-miR-28-5p, hsa-miR-22-5p and hsa-miR-151a-5p in preparation of PD diagnosis and PD patient staging kits and reagents.
Application of the reverse transcription primers of hsa-miR-374a-5p, hsa-miR-374b-5p, hsa-miR-199a-3p, hsa-miR-195-5p, hsa-miR-28-5p, hsa-miR-22-5p and hsa-miR-151a-5p in preparation of PD diagnosis and PD patient staging kits and reagents.
Application of amplification primers of hsa-miR-374a-5p, hsa-miR-374b-5p, hsa-miR-199a-3p, hsa-miR-195-5p, hsa-miR-28-5p, hsa-miR-22-5p and hsa-miR-151a-5p in preparation of PD diagnosis and PD patient staging kits and reagents.
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| CN104080910A (en) * | 2011-11-03 | 2014-10-01 | 高雄医学大学 | Methods of using microRNA 195 in providing neuroprotection |
| CN111944809A (en) * | 2020-07-28 | 2020-11-17 | 武汉睿健医药科技有限公司 | Diagnostic marker for Parkinson's disease and application thereof |
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| CN104080910A (en) * | 2011-11-03 | 2014-10-01 | 高雄医学大学 | Methods of using microRNA 195 in providing neuroprotection |
| CN111944809A (en) * | 2020-07-28 | 2020-11-17 | 武汉睿健医药科技有限公司 | Diagnostic marker for Parkinson's disease and application thereof |
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