CN109266737B

CN109266737B - Exosome biomarker for auxiliary diagnosis of SCA3/MJD and screening and identifying method thereof

Info

Publication number: CN109266737B
Application number: CN201811222136.1A
Authority: CN
Inventors: 江泓; 侯晓灿; 陈召; 裘嵘; 唐北沙
Original assignee: Xiangya Hospital of Central South University
Current assignee: Xiangya Hospital of Central South University
Priority date: 2018-10-19
Filing date: 2018-10-19
Publication date: 2021-06-29
Anticipated expiration: 2038-10-19
Also published as: CN109266737A

Abstract

The invention discloses an exosome biomarker for auxiliary diagnosis of SCA3/MJD and a screening and identifying method thereof, belonging to the technical field of cell and molecular biology. The invention extracts total RNA from plasma and cerebrospinal fluid exosomes of a SCA3/MJD patient, screens and identifies miRNAs with differential expression by applying Small RNA Sequencing (sRNA seq), and combines with Real-time fluorescent Quantitative PCR (Quantitative Real-time PCR, qPCR) verification to verify that novel-mir-7014 can be used as a potential biomarker for guiding SCA3/MJD clinical typing and reflecting disease severity, so that a novel specific auxiliary diagnosis method is provided for SCA3/MJD, and the method has potential clinical application value in disease monitoring and auxiliary treatment.

Description

Exosome biomarker for auxiliary diagnosis of SCA3/MJD and screening and identifying method thereof

Technical Field

The invention relates to the technical field of cell and molecular biology, in particular to an exosome biomarker for auxiliary diagnosis of SCA3/MJD and a screening and identifying method thereof.

Background

Hereditary spinocerebellar ataxias (SCAs) is a common type of degenerative disease of the nervous system, especially most commonly spinocerebellar ataxia type 3/Machado-Joseph disease (SCA 3/MJD), which accounts for almost 60-70% of SCAs patients in the chinese population. The learners classify SCA3/MJD into the following 5 subtypes, type 1: onset is usually before the age of 20 years, progresses rapidly, and is mainly manifested by pyramidal fascicles (myotonia and myospasm) and extrapyramidal features (motor retardation and dystonia) combined with ataxia; type 2 usually develops between 20 and 50 years of age with ataxia with progressive external ophthalmoplegia and pyramidal tractsThe sign is the main manifestation; later onset of type 3, the onset age is usually between 40-75 years old, and is mainly characterized by ataxia combined with peripheral nerve manifestations such as motor neuropathy and muscular atrophy; in addition, there are type 4 marked by Parkinson's disease and type 5 marked by pure spastic paraplegia. Of these five subtypes, type 2 is most common, followed by the less

common types

3, 1, 4 and 5, which are much rarer. Pathogenic gene of SCA3/MJDATXN3The coding region contains CAG repetitive sequence, the copy number of normal people is about 13-40, and the patient can be amplified to 51-86 due to dynamic mutation. In addition to the variation in copy number of CAG repeats, which accounts for 50-60% of clinical heterogeneity in patients, modifier genes, epigenetics, etc., are involved in regulation.ATXN3The carboxyl terminal of gene coding protein ataxin-3 contains a polyglutamine (polyQ) peptide chain, and ataxin-3 protein containing abnormally expanded polyQ peptide chain can be selectively accumulated in a specific region (cerebellum, brainstem, spinal cord and the like) of a nervous system to form a neuron nuclear inclusion body and cause cell death. Because relevant mechanisms such as the pathogenesis, clinical typing, disease progression and the like are not completely clarified, no specific treatment is available, the lethality rate and the disability rate are high, and heavy burden is brought to patients, families and society.

Exosomes (exosomes) are used as the most concerned intercellular communication medium, and provide a new idea for the study of clinical typing, disease severity, possible pathogenesis and the like of the diseases. The exosome is an extracellular vesicle with the diameter of 40-150nm secreted by various living cells, and the existence of the exosome can be detected in body fluids such as cerebrospinal fluid, plasma, urine and the like. Exosome contains uncoded RNAs (miRNAs, lncRNAs and the like), mRNAs, proteins and other components, and the components can be transported to act on target cells to play corresponding regulation and control functions, so that the exosome is a very important carrier for intercellular substance and information communication. The exosome has small form, is easy to permeate blood brain barrier, has immune exemption, can be transported along with body fluid and is not easy to be phagocytized by phagocyte, has the advantages of small detection damage, good stability, high sensitivity and the like, and prompts the research direction of the exosome as a new biomarker of neurodegenerative diseases and a potential treatment carrier.

In conclusion, the search for an exosome biomarker for assisting in diagnosing SCA3/MJD is of great significance for assisting in diagnosis and early treatment of SCA 3/MJD.

Disclosure of Invention

The key technical problems to be solved by the invention are as follows: overcomes the defects of the prior art, provides an exosome biomarker for assisting in diagnosing SCA3/MJD and a screening and identifying method thereof, and provides support for clinical early diagnosis and early treatment of SCA 3/MJD.

The technical scheme adopted by the invention for solving the technical problems is as follows: an exosome biomarker for auxiliary diagnosis of SCA3/MJD, which is miRNA derived from exosome and containing a nucleotide sequence shown as SEQ ID.1.

Further, the miRNA is novel-mir-7014 derived from exosome.

Further, the screening and identification method of the exosome biomarker for assisting in diagnosing the SCA3/MJD comprises the following steps:

1) collecting plasma and cerebrospinal fluid samples, extracting exosomes and exosome RNAs in the samples, and identifying the exosomes;

2) sequencing the exosome small RNAs extracted in the step 1) by using sRNA seq technology, and screening out miRNAs with differential expression;

3) collecting a plasma sample in an expanding way, and extracting exosome RNAs in the sample;

4) and detecting the relative expression level of the miRNAs by adopting a qPCR analysis method, and performing verification evaluation on the screened miRNAs.

Further, the screening and identification method of the exosome biomarker for assisting in diagnosing the SCA3/MJD further comprises GO annotation and KEGG metabolic pathway analysis on novel-mir-7014 between the steps 2) and 3).

Further, a screening and identifying method for exosome biomarkers for auxiliary diagnosis of SCA3/MJD, wherein the qPCR analysis method in the step 4) specifically comprises the following operations:

a) preparing reaction mixed solution of reverse transcription, and performing reverse transcription on the miRNAs to form cDNAs;

b) designing a Primer by Primer 5;

c) preparing a qPCR reaction system, using U6 as a standardized internal reference, performing real-time fluorescent quantitative PCR analysis, and performing relative expression content analysis of the miRNAs by a-delta CT method.

Further, the sequence of the forward primer of the novel-mir-7014 was CCTGTGTTCCTGAGCCTTG.

Further, the application of the exosome biomarker for assisting in diagnosing the SCA3/MJD in preparing the SCA3/MJD auxiliary diagnosis kit.

The screening and identifying method for the exosome biomarker for auxiliary diagnosis of SCA3/MJD has the beneficial effects that: the novel-mir-7014 is efficiently screened and identified as differentially expressed miRNAs of SCA3/MJD exosome sources, can be used as a potential biomarker for guiding the clinical typing of SCA3/MJD and reflecting the disease severity, provides a new specific auxiliary diagnosis method for the clinical diagnosis of SCA3/MJD, and has potential clinical application value in disease monitoring and auxiliary treatment.

Drawings

FIG. 1 is a schematic flow diagram of a screening and identification method for exosome biomarkers for aiding in the diagnosis of SCA 3/MJD;

FIG. 2-Electron micrograph of exosome identification;

FIG. 3-nanoparticle size diagram for exosome identification;

FIG. 4-is a block diagram of an exosome biomarker for aiding in the diagnosis of SCA 3/MJD;

FIG. 5-is a classification diagram of GO annotated target genes differentially expressing gene novel-mir-7014;

FIG. 6-is a classification diagram of the target gene of KEGG analysis differential expression gene novel-mir-7014;

FIG. 7-is a graph of enrichment of the target gene pathway for the KEGG analysis of the differentially expressed gene novel-mir-7014;

FIG. 8 is a KEGG analysis of the metabolic pathway of protein processing involved in the endoplasmic reticulum of the differentially expressed gene novel-mir-7014;

FIG. 9 is a diagram showing the KEGG analysis of the involvement of the differentially expressed gene novel-mir-7014 in the axon-guided nervous system metabolic pathway;

figure 10-expression profile of qPCR verified mirnas.

Detailed Description

The invention is further illustrated with reference to the following figures and examples, which are not intended to limit the scope of the invention in any way.

Example 1

In this example 1, differential expression miRNAs related to SCA3/MJD were screened by the following procedures:

1. collecting samples: plasma samples of 12 SCA3/MJD patients (including 3 subtypes: 3

cases

1, 6

cases

2, 3 cases 3) and 12 age-gender-matched healthy persons were used as controls; cerebrospinal fluid samples from 12 SCA3/MJD patients (including 3 subtypes: 3

cases

1, 6

cases

2, 3 cases 3) and 10 age, sex matched aneurysm/migraine patients were controls.

Plasma sample: 10ml of whole blood of the SCA3/MJD patient and the control group is collected by an anticoagulation tube, centrifuged at 3000rpm and 4 ℃ for 15min, and plasma is separated for standby and completed within 2 hours.

Cerebrospinal fluid sample: 10-15ml of cerebrospinal fluid of SCA3/MJD patients and control groups is collected by a sterile centrifuge tube for standby, and the collection is completed within 2 hours.

2. Exosome, exosome RNA extraction and exosome identification

(1) Exosomes were extracted using the exoEasy Kit method, and exosome RNA was extracted using the exoRNeasy Serum/Plasma Kit method.

(2) Identification of exosomes: and observing the form of the exosome by adopting an electron microscope, and detecting the particle size distribution by using nanoparticle tracking analysis to identify the exosome.

And (3) observing by an electron microscope: and identifying the form, the size and the like of the sample by scanning with a transmission electron microscope (Tecnai G2 Spirit, FEI, America).

And (3) detecting the particle size distribution: the sample within the field of view cell was subjected to a nano-range Particle scan using a ZetaView (Particle metric, Munich, Germany) instrument.

Referring to FIGS. 2-3, FIG. 2 shows that the extract is in the form of disc-shaped vesicles, FIG. 3 shows that the diameter of the vesicles is between 40-150nm, and the extract can be determined as an exosome by the analysis of the form and the diameter and can be used as a sample for the subsequent Small RNA Sequencing.

3. Sequencing small RNAs by using sRNA seq technology, comparing plasma patients and controls, cerebrospinal fluid patients and controls, 3 plasma subtypes, 3 cerebrospinal fluid subtypes, cerebrospinal fluid and plasma corresponding subtype groups, and screening miRNAs with differential expression, wherein the screening conditions are as follows:P value =0, Q value = 0.

Differentially expressed miRNAs screened for in this example are shown in tables 1-4, where: up-regulated 53 miRNAs expression and down-regulated 20 miRNAs expression in plasma exosomes; 69 miRNAs are up-regulated and 47 are down-regulated in cerebrospinal fluid exosomes; in plasma exosomes, 1 miRNAs were expressed and 1 was down-regulated compared to type 1 and type 2; 1 miRNAs were up-regulated and 8 were down-regulated in type 1 versus type 3; type 2 compared with type 3 miRNAs expression was up-regulated by 1, down-regulated by 10; in cerebrospinal fluid exosomes, 4 miRNAs were up-regulated and 13 were down-regulated in type 1 versus type 2 expression; 1 miRNAs were up-regulated and 12 were down-regulated in type 1 versus type 3; there were 3 miRNAs with up-regulated expression and 10 with down-regulated expression compared to type 2 and type 3.

TABLE 1 MiRNAs differentially expressed in plasma exosomes

TABLE 2 MiRNAs differentially expressed in cerebrospinal fluid exosomes

TABLE 3 pairwise comparison of 1, 2, 3 subtype plasma exosome-differential miRNAs in SCA3/MJD

TABLE 4 pairwise comparison of 1, 2, 3 subtype cerebrospinal fluid exosome-differential miRNAs in SCA3/MJD

In this example, the screened miRNAs with differential expression are novel-mir-7014 with nucleotide sequences shown in SEQ ID.1, and the structural diagram of novel-mir-7014 is shown in FIG. 4, and when compared with plasma exosomes and cerebrospinal fluid exosomes in a control group, novel-mir-7014 has obvious expression abnormality in comparison among 1, 2 and 3 subtype cerebrospinal fluid exosomes, wherein novel-mir-7014 is down-regulated in plasma exosomes, is up-regulated in cerebrospinal fluid exosomes, and is sequentially up-regulated in 1, 2 and 3 subtype cerebrospinal fluid exosomes.

4. To understand the functional properties and participating metabolic pathways of novel-mir-7014, we performed GO annotation and KEGG metabolic pathway analysis on novel-mir-7014, see FIGS. 5-9.

Enrichment and classification of differential genes GO in novel-mir-7014 target genes show that biological process items are mainly found in processes of single organism, metabolism, biological regulation and the like; the cell component items are mainly cells, cell components and organelle membranes; molecular functional terms are most common for binding and catalytic activities.

The differential target gene of novel-mir-7014 is further classified by KEGG, and the first 15 bits of the nervous system bit column group are found.

The KEGG pathway enrichment analysis of the differential target gene aiming at novel-mir-7014 shows that the main enrichment pathway of the target gene is endoplasmic reticulum protein processing and axon guiding, and both pathways are reported to be related to SCA3/MJD pathogenesis.

Example 2

This example used qPCR to verify differentially expressed miRNAs, where the miRNAs tested included novel-miR-9034, novel-miR-7660, novel-miR-5219, novel-miR-8421, novel-miR-769, novel-miR-7014, novel-miR-6628, novel-miR-4682, novel-miR-2738, novel-miR-1643, has-miR-92b-3p, has-miR-486-3p, has-miR-484, has-miR-378g, has-miR-375, has-miR-328-3p, has-miR-3074-5p, has-miR-24-3p, has-miR-206, has-miR-204-5p, has-miR-151a-3p, has-miR-146b-5p, has-miR-142-3p, has-miR-1290, has-miR-1268a, has-miR-122-5p and has-let-7d-3p, wherein the specific operating process of qPCR verification is as follows:

1. collecting samples: plasma samples of 18 SCA3/MJD patients and 16 age, gender-matched healthy persons were controls; 10ml of whole blood of the SCA3/MJD patient and the control group is collected by an anticoagulation tube, centrifuged at 3000rpm and 4 ℃ for 15min, and plasma is separated for standby and completed within 2 hours.

2. The exosome RNAs extraction process was the same as in example 1.

3. Primers were designed by Primer 5, and the Primer information is shown in table 5.

TABLE 5 primer sequence Listing of miRNAs

4. Reverse transcription of miRNAs to synthesize cDNA:

reaction mixture liquid: 4. mu.l of MiScript HiSpec Buffer; 10. mu.l of MiScript nucleic acid Mix 2. mu.l; 2 μ l of miScript Reverse Transcriptase Mix; template RNA 12 ul;

reverse transcription reactions were performed on PCR: respectively reacting at 37 ℃ for 60 min; reacting at 95 ℃ for 5 min; after the reaction was completed, it was put on ice for use.

5. qPCR: a 25uL reaction system was used, comprising: 2 QuantiTect SYBR Green PCR Master Mix: 12.5. mu.l SYBR Green (polymerase chain reaction System); 10. mistcript Universal Primer: 2.5. mu.l (reverse primer); 10. mistript Primer Assay: 2.5. mu.l (forward primer); RNase-free water (ribonuclease-free water): 7 ul; template cDNA: 0.5 ul.

U6 is used as an internal reference gene, a QuantStudio 5 Real-Time PCR instrument is used for detection, and qPCR relative quantitative analysis is carried out on the internal reference gene and each sample.

The qPCR reaction procedure was: pre-denaturation at 95 ℃ for 1min, denaturation at 95 ℃ for 10s, annealing at 55 ℃/60 ℃ for 5s, and extension at 72 ℃ for 10-15 s for 40 cycles. And (3) analyzing data by a-delta CT method, wherein the-delta CT = reference gene CT value-target gene CT value, the larger the delta CT value is, the lower the gene expression level is, and the smaller the delta CT value is, the higher the gene expression level is.

Referring to FIG. 10, qPCR analysis shows that when the expression level of novel-mir-7014 in the experimental group is lower than that in the control group, the delta CT value of the experimental group is greater than that of the control group, and the expression level of novel-mir-7014 in the experimental group is lower than that in the control group, then compared with the control group, novel-mir-7014 is down-regulated in plasma exosomes of patients, and the sequencing result is consistent. The expression level of novel-mir-7014 in cerebrospinal fluid of a patient is higher than that of a control group, and the expression level of novel-mir-7014 in plasma exosomes is lower than that of the control group; and sequencing results show that 1, 2 and 3 subtype cerebrospinal fluid exosomes are sequentially up-regulated compared with the former, the onset age of the three subtypes is sequentially increased and the disease severity is sequentially reduced according to the clinical characteristics of the 1, 2 and 3 subtypes, namely, the expression level of novel-mir-7014 is increased along with the reduction of the disease severity of SCA3/MJD, and the novel-mir-7014 can be used as a potential biomarker for clinical typing of SCA3/MJD and reflecting the disease severity.

In conclusion, the novel-mir-7014 from the exosome can be used as a biomarker for SCA3/MJD clinical early auxiliary diagnosis, guidance of clinical typing and reflection of potential disease severity.

Sequence listing

<110> Hunan ya Hospital of Zhongnan university

<120> exosome biomarker for auxiliary diagnosis of SCA3/MJD and screening and identifying method thereof

<130> 20181019

<141> 2018-10-19

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 15

<212> RNA

<213> Artificial Synthesis ()

<400> 1

gucaguggca cagcc 15

Claims

1. An exosome biomarker for auxiliary diagnosis of SCA3/MJD, which is characterized in that the exosome biomarker is novel-mir-7014 derived from exosome, and the nucleotide sequence of the novel-mir-7014 is shown in SEQ ID NO. 1.

2. The screening and identification method of exosome biomarkers for auxiliary diagnosis of SCA3/MJD according to claim 1, comprising the steps of:

2) sequencing the exosome Small RNAs extracted in the step 1) by using sRNA Seq technology, and screening out differentially expressed miRNAs;

3. The method for screening and identifying exosome biomarkers for auxiliary diagnosis of SCA3/MJD according to claim 2, further comprising GO annotation and KEGG metabolic pathway analysis of novel-mir-7014 between steps 2) and 3).

4. The screening and identification method of exosome biomarkers for auxiliary diagnosis of SCA3/MJD according to claim 2, wherein the qPCR analysis method in step 4) is specifically operated as:

b) designing a Primer by Primer 5;

c) preparing a qPCR reaction system, using U6 as a standardized internal reference, carrying out qPCR analysis, and carrying out relative expression content analysis on the miRNAs by a-delta CT method.

5. The method for screening and identifying exosome biomarkers for auxiliary diagnosis of SCA3/MJD according to claim 4, wherein the sequence of the forward primer of novel-mir-7014 is CCTGTGTTCCTGAGCCTTG.

6. Use of an exosome biomarker for aiding diagnosis of SCA3/MJD as claimed in any of claims 1-5 in the preparation of a SCA3/MJD aiding diagnostic kit.