CN112322719A

CN112322719A - Application of circulating blood exosome miRNA as retinal vein occlusion disease diagnosis marker

Info

Publication number: CN112322719A
Application number: CN202011227575.9A
Authority: CN
Inventors: 池在龙; 张宗端
Original assignee: Wenzhou Medical University
Current assignee: Wenzhou Medical University
Priority date: 2020-11-06
Filing date: 2020-11-06
Publication date: 2021-02-05

Abstract

The invention discloses an application of circulating blood exosome miRNA (micro ribonucleic acid) as a diagnostic marker for retinal vein occlusion diseases, wherein a single miRNA or a plurality of miRNAs in circulating blood exosome miR-378d, miR-146a-5p, miR-148a-3p, miR-24-3p, miR-221-3p, miR-28-3p, miR-652-3p, miR-532-5p, miR-223-5p, miR-148b-3p, miR-143-3p, miR-361-3p, miR-199a-3p, miR-7-5p, miR-382-5p, miR-27b-3p, miR-222-3p, miR-425-5p, miR-152-3p and miR-4326 can be used as a more precise and stable marker, has the characteristics of good stability, convenient material taking, high sensitivity and high specificity.

Description

Application of circulating blood exosome miRNA as retinal vein occlusion disease diagnosis marker

Technical Field

The invention relates to the technical field of molecular diagnosis, in particular to circulating blood exosome miRNA serving as a retinal vein occlusion biomarker and application thereof.

Background

Retinal Vein Occlusion (RVO), a common retinal vascular disease, is a significant cause of vision impairment worldwide. RVOs are classified into Central Retinal Vein Occlusion (CRVO) and Branch Retinal Vein Occlusion (BRVO) according to the location of the occlusion, and their common causes include arteriosclerotic compression, thrombosis induced by vascular endothelial injury, and vascular inflammation. In addition, diseases causing elevation of blood viscosity such as hyperlipidemia, multiple myeloma, and hyperthyroidism, which are altered by hemodynamics, may also be the cause of RVO induction. Pathological changes such as retinal capillary non-perfusion, macular edema and retinal hemorrhage are the main causes of visual impairment. Retinal hemorrhage, cystoid macular edema, effusion, and lint spots are visible in the fundus of RVO patients. It has been shown that RVO causes increase of inflammatory factors such as Vascular Endothelial Growth Factor (VEGF), Placental Growth Factor (PGF), platelet-derived growth factor (PDGF), interleukin 6 (IL-6), interleukin 8 (IL-8), etc., and thus pathological changes such as macular edema, vascular leakage, and iris neovascularization. Although there have been many studies on the pathogenesis of RVO, the exact pathogenesis and molecular mechanism of RVO have not been clearly concluded.

The exosome is a secretory vesicle-like body with phospholipid bilayers, which can be secreted by various cells, the diameter of the exosome is 30-150nm when the exosome is observed under an electron microscope, the exosome is formed by invagination of an outer membrane of a multivesicular body (MVB) in the cell, and the exosome can wrap various intracellular genetic information such as surface protein receptors, lipids, mRNA, microRNA (miRNA) and other non-coding RNA. When the MVB outer membrane is fused with a cell membrane, exosomes in the MVB outer membrane are released from cells, enter intercellular substance, can be absorbed by adjacent receptor cells, or are transported to a far end along with body fluid to act on different cells. Various living cells of different types can secrete exosomes under physiological or pathological states, so the exosomes are widely present in various body fluids of organisms such as blood, urine, saliva, cerebrospinal fluid, ascites, pleural fluid and the like, and the content, the membrane surface marker and the like of the exosomes have the characteristics of donor cells. Due to the phospholipid bilayer structure of exosome and rich RNA, nucleic acid can be transferred into recipient cells more easily, and the protein is a good information transfer tool. The phospholipid bilayer of exosomes makes it difficult to destroy molecules in exosomes, and can exist in body fluid relatively stably, and specific molecular information carried by exosomes can be used as ideal biomarkers.

Blood is the best messenger for all human diseases, and exosome carries intracellular messenger to be released into various body fluids, thereby providing a new idea for body fluid detection. Among the existing clinical markers, blood free mirnas with good specificity are not found. Exosome mirnas have more marker potential than free mirnas. The plasma exosomes of a patient are collected, expression difference of exosome miRNA in RVO diseases is screened through high-throughput sequencing and bioinformatics analysis, expression level is further verified through real-time fluorescent quantitative PCR, and the possibility of using the exosome miRNA as a biomarker is discussed.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a more accurate and convenient way for diagnosing retinal vein occlusion by taking miRNA of a circulating blood exosome as a retinal vein occlusion biomarker and application thereof.

The invention aims to provide a miRNA marker of a circulating blood exosome related to auxiliary diagnosis of retinal vein occlusion.

The invention also aims to provide the circulating blood exosome miRNA and application of the primer thereof in preparing a retinal vein occlusion auxiliary diagnosis kit.

Still another object of the present invention is to provide an auxiliary diagnostic kit for retinal vein occlusion.

The purpose of the invention can be realized by the following technical scheme:

a miRNA marker of circulating blood exosome related to auxiliary diagnosis of retinal vein occlusion, the marker is one or at least 2 combinations of miR-378d, miR-146a-5p, miR-148a-3p, miR-24-3p, miR-221-3p, miR-28-3p, miR-652-3p, miR-532-5p, miR-223-5p, miR-148b-3p, miR-143-3p, miR-361-3p, miR-199a-3p, miR-7-5p, miR-382-5p, miR-27b-3p, miR-222-3p, miR-425-5p, miR-152-3p and miR-4326.

The circulating blood exosome miRNA marker is applied to auxiliary diagnosis of retinal vein occlusion.

The circulating blood exosome miRNA marker is applied to preparation of a retinal vein occlusion auxiliary diagnosis kit.

A circulating blood exosome miRNA marker primer related to auxiliary diagnosis of retinal vein occlusion, the primer comprises one or more miRNA primers of miR-378d, miR-146a-5p, miR-148a-3p, miR-24-3p, miR-221-3p, miR-28-3p, miR-652-3p, miR-532-5p, miR-223-5p, miR-148b-3p, miR-143-3p, miR-361-3p, miR-199a-3p, miR-7-5p, miR-382-5p, miR-27b-3p, miR-222-3p, miR-425-5p, miR-152-3p and miR-4326;

the primer is applied to auxiliary diagnosis of retinal vein occlusion or preparation of an auxiliary diagnosis kit for retinal vein occlusion.

A retinal vein occlusion auxiliary diagnosis kit contains one or more miRNA primers of miR-378d, miR-146a-5p, miR-148a-3p, miR-24-3p, miR-221-3p, miR-28-3p, miR-652-3p, miR-532-5p, miR-223-5p, miR-148b-3p, miR-143-3p, miR-361-3p, miR-199a-3p, miR-7-5p, miR-382-5p, miR-27b-3p, miR-222-3p, miR-425-5p, miR-152-3p and miR-4326.

The kit also comprises reagents commonly used in PCR technology.

The kit can also comprise common reagents for PCR reaction, such as reverse transcriptase, buffer solution, dNTPs, MgCl2, DEPC water, Taq enzyme and the like; standards and/or controls may also be included.

The miRNA markers miR-378d, miR-146a-5p, miR-148a-3p, miR-24-3p, miR-221-3p, miR-28-3p, miR-652-3p, miR-532-5p, miR-223-5p, miR-148b-3p, miR-143-3p, miR-361-3p, miR-199a-3p, miR-7-5p, miR-382-5p, miR-27b-3p, miR-222-3p, miR-425-5p, miR-152-3p and miR-4326 related to the diagnosis of retinal vein occlusion have the disclosed sequences of each miRNA, the sequence is shown in SEQ ID NO.1-20, the corresponding relation is shown in Table 1,

TABLE 1

SEQ ID NO.1	hsa-miR-7-5p
		SEQ ID NO.2	hsa-miR-652-3p
SEQ ID NO.3	hsa-miR-532-5p
		SEQ ID NO.4	hsa-miR-425-5p
SEQ ID NO.5	hsa-miR-382-5p
		SEQ ID NO.6	hsa-miR-378d
SEQ ID NO.7	hsa-miR-361-3p
		SEQ ID NO.8	hsa-miR-28-3p
SEQ ID NO.9	hsa-miR-27b-3p
		SEQ ID NO.10	hsa-miR-24-3p
SEQ ID NO.11	hsa-miR-223-5p
		SEQ ID NO.12	hsa-miR-222-3p
SEQ ID NO.13	hsa-miR-221-3p
		SEQ ID NO.14	hsa-miR-199a-3p
SEQ ID NO.15	hsa-miR-152-3p
		SEQ ID NO.16	hsa-miR-148a-3p
SEQ ID NO.17	hsa-miR-148b-3p
		SEQ ID NO.18	hsa-miR-146a-5p
SEQ ID NO.19	hsa-miR-143-3p
		SEQ ID NO.20	hsa-miR-4326

The combination of the miRNA markers as auxiliary diagnosis markers of retinal vein occlusion requires creative work of the technicians in the field. Amplification primers of all miRNA markers can be purchased from the market, and the primers of the circulating blood exosome miRNA markers used in the embodiment of the invention are specific miRNA stem-loop PCR primers synthesized and produced by Guangzhou multifunctional gene Co.

The invention has the beneficial effects that: compared with the traditional detection of free miRNA in peripheral blood, the exosome as a representative of a novel biomarker has the characteristics of good stability, high sensitivity and high specificity. The phospholipid bilayer of the exosome enables molecules in the exosome not to be easily damaged, can exist in body fluid relatively stably, is low in external interference, and can be used as a marker which is more accurate and stable. In addition, exosomes are produced by the endoplasmic reticulum of donor cells, and are not produced by cell death disruption, and thus are more latent for markers than are blood free RNA. The application carries out rigorous and multistage verification and evaluation on the differential expression miRNA in the retinal vein occlusion and the serum and the plasma of a control group, and confirms the reliability and the repeatability of the group of miRNA as a noninvasive marker for diagnosing the retinal vein occlusion, the detection material of the peripheral blood exosome is convenient, the detection cost is low, and the miRNA can be used as an ideal auxiliary diagnosis method. The invention provides a new research direction for diagnosing retinal vein obstruction.

Drawings

FIG. 1: plasma exosome miRNA expression, wherein compared with a control group, the plasma exosome miRNA expression of the RVO group patients is different;

FIG. 2: bioinformatics analysis of 302 candidate target genes differentially expressing mirnas, a: the GO biological process analysis enrichment degree, wherein gray represents the background gene of the functional region in the species, and black represents the gene enrichment quantity (P is less than 0.01) of the functional region of the target sample; b: according to the KEGG analysis result, the abscissa (Rich factor) represents the proportion of the candidate target genes in the channel background represented by the ordinate, the size of the point represents the number of the candidate target genes, and the color depth represents the size of a P value;

FIG. 3: and (4) detecting the expression quantity of 20 genes which appear at high frequency in real time by fluorescence qPCR (independent sample t test, P is less than 0.05, P is less than 0.01, P is less than 0.001, and P is less than 0.0001).

Detailed Description

The invention will be further described in the following with reference to the drawings and examples, without limiting the invention thereto.

Collecting the detailed case data of patients, which are in an affiliated eye vision hospital of Wenzhou medical university and are 40-80 years old and confirmed to be RVO through clinical examination, with or without secondary macular edema, and peripheral blood of 10 mL;

meanwhile, collecting age-matched simple cataract cases as a control group;

the peripheral blood is collected by using a disposable EDTA vacuum blood collection tube, centrifuging at 2000 Xg and 4 ℃ for 10 minutes, separating plasma and blood cells, and respectively freezing and storing in an ultra-low temperature refrigerator at-80 ℃.

Wherein the diagnostic criteria for RVO are: fundus manifestation: the retinal vein is circuitous and dilated with or without flame-like hemorrhage, and part of the retinal vein is visible and grey white cotton wool spots; FFA: the blocked veins in the arteriovenous phase have tortuosity and slow filling, the vessel wall is colored, the fluorescence leaks, the fluorescence of a macular region is enhanced, and a part of patients can see a non-perfusion region; OCT: macular edema, fluid accumulation in the retina, and the like.

RVO collection cases excluded cases in several cases:

firstly, the eye drops suffer from other eyeground diseases, such as Diabetic Retinopathy (DR), age-related macular degeneration (AMD), retinal detachment, macular holes, retinitis pigmentosa, optic neuritis, uveitis and the like;

② serious complications appear, such as vitreous hemorrhage, serious cataract, neovascular glaucoma, etc.;

thirdly, the vitreous cavity medicine injection operation of the retina photocoagulation or glucocorticoid is already performed;

fourthly, the patients have special hereditary diseases such as Marfan syndrome and the like or special systemic diseases such as leukemia, hyperthyroidism, multiple myeloma and the like;

infectious diseases such as viral hepatitis, syphilis or AIDS and the like;

sixthly, the patient has serious cardiovascular and cerebrovascular diseases, serious liver and kidney insufficiency or blood coagulation dysfunction.

Sample processing

Plasma of 8 RVO patients was drawn and plasma exosomes were isolated:

(1) removing cell debris: after thawing the frozen plasma on ice, the plasma was centrifuged at 2000g at 4 ℃ for 10 minutes and the supernatant was transferred to a new centrifuge tube. 10000 Xg, centrifuge at 4 ℃ for 40 minutes, and the supernatant is transferred to a new centrifuge tube. For ease of handling, a 1.5mL centrifuge tube is used, and the volume of plasma used generally does not exceed 1.2 mL.

(2) And (3) separating exosomes: an Exosome Isolation Reagent manufactured by the company Guangzhou acute biology was added thereto in an amount of 1/3 plasma volume, and after being sufficiently mixed by blowing with a pipette, the mixture was left to stand at 4 ℃ for 30 minutes. Violent shaking is not suitable to avoid influencing the yield.

(3) Collecting exosomes: centrifuging at 15000 Xg and 4 deg.C for 15 min, discarding supernatant, and precipitating to obtain exosome.

Total RNA extraction of plasma exosomes of RVO patients

Total RNA extraction from plasma exosomes E.Z.N.A. produced by Omega Bio-tek was used

Total RNA Kit。

(1) Cracking: 1mL of RNA-containing protein was added to the exosome pellet

And (4) reagent, fully grinding by using a grinder, standing for 1-2 minutes at room temperature, and fully cracking.

(2) And (3) extraction: to the homogenate was added 200. mu.L of chloroform, the mixture was thoroughly mixed by turning upside down, and then allowed to stand on ice for 10 minutes. 12000 Xg, 4 ℃ centrifugal 15 minutes, the sample will be divided into three layers.

(3) Column passing: 70% of the upper colorless transparent aqueous phase liquid is transferred to a new centrifugal tube to avoid sucking the middle layer impurities and the lower layer liquid. Adding equal volume of 70% ethanol, and mixing by turning upside down. Adding the mixed liquid

Centrifuging RNA Column at room temperature for 1 min at 12000 × g in a volume of no more than 700 μ L each time, discarding the liquid in the collection tube, continuing to use Column, adding the rest mixed solution into Column again, centrifuging, and repeating for multiple times until the mixed solution passes through the Column completely.

(4) Washing the membrane: wash buffer I500. mu.L, 12000 Xg, was added to the Column, centrifuged at room temperature for 30 seconds, and the liquid in the collection tube was discarded. Add Wash buffer II with pre-proportioned absolute ethanol 500. mu.L, 12000 Xg to Column, centrifuge at room temperature for 30 seconds, discard the liquid in the collection tube, repeat twice.

(5) Spin-drying: centrifuging at 20000 Xg for two minutes at room temperature, and drying the washing solution.

(6) Dissolving: adding 40 μ L DEPC water near the filtration membrane, incubating at room temperature for 5min, replacing the collection tube, centrifuging at maximum speed at room temperature for 1 min, and collecting the filtered liquid.

(7) And (3) detecting the concentration: detecting the concentration and purity of RNA by using an enzyme-labeling instrument, wherein the detection result of the purity of RNA is as follows: the ratio of the absorbance 260/280 was between 1.8 and 2.1. If the concentration is too low, the filtered liquid can be added into the filter membrane again for incubation for 5min, and the RNA concentration and purity are detected after centrifugation and collection again. Or pre-heating DEPC water in advance to increase RNA solubility.

RNA sequencing of plasma exosomes of RVO patients

RNA in plasma exosomes of 8 RVO patients is collected, reverse transcription is carried out to obtain cDNA, then non-specific PCR amplification is carried out, then Illumina HiSeq 2500 is used for miRNA high-throughput sequencing, a linker used in reverse transcription is removed, and a fragment of less than 17nt (provided by Guangzhou pluripotent Gene Co., Ltd.) is filtered. And comparing the obtained sequencing result with the existing miRNA sequencing data of the plasma exosomes of the cataract patient in a laboratory to obtain the miRNA with differential expression. The comparison results are tested by using an edgeR test tool, and the results show that the expression of 302 genes has significant difference with | log (fold change) | > 1 bit threshold value and P < 0.05, wherein the expression of 115 genes is up-regulated miRNA and the expression of 187 genes is down-regulated miRNA, and the results are shown in figure 1.

Prediction of target genes for differentially expressed mirnas

The action site of the miRNA is reversely predicted by complementary pairing of seed sequences of the miRNA, and then the detection of a predicted target is carried out by referring to the conservation type in miRNA species and the heat stability of miRNA-mRNA double strands. To reduce false positive results, the target gene sites were predicted using four tools, miRTarBase (carolina. imins. athena-innovation. gr/diana _ tool), Targetscan (Targetscan. org/vert _72), miRWALK (miRWALK. umun.uni-heidelberg. de), and miRDB (miRDB. org), and the target gene predicted site results of different software were mapped to Venn, and the target genes covered by at least three predicted results were taken as the target gene candidates for the miRNA. The candidate target genes are annotated for biological functions through Gene Ontology (GO) (FIG. 2A), and the high-frequency functional annotation of the candidate target genes relative to the background is analyzed to predict the biological effect caused by differential expression. The results show that the major functional regions of the action sites of the RVO patients differentially expressing mirnas compared to the control group are: apoptosis process, RNA synthesis process, cell proliferation regulation, cell migration under the stimulation of growth factors, endocytosis, Wnt pathway, cytokines and the like. In addition, there is some gene enrichment in nerve growth and new blood vessels. Meanwhile, enrichment analysis is carried out on the KEGG channel annotation to obtain a signal transduction and disease channel with statistical significance relative to the background, so that distribution information of a gene set possibly related to diseases on the KEGG type is predicted (fig. 2B). The results show that the several paths with the highest correlation are: axon guidance, biological metabolism, cancer, Wnt pathway, insulin resistance pathway, neurotrophic factor signaling pathway, and stem cell pluripotency modulating signals. In addition, AMPK signaling pathway and insulin receptor signaling pathway are also more gene-rich. The miRNA differentially expressed by the RVO patient is mainly involved in processes of apoptosis, endocytosis, cell proliferation, migration, angiogenesis and the like, which are consistent with pathological changes of the RVO.

Screening of exosome mirnas as RVO biomarkers

Selecting 20 miRNAs with high frequency occurrence from miRNAs related to RVO possible related biological processes and disease pathways, carrying out next screening verification, wherein 19 miRNAs comprise miR-146a-5p, miR-148a-3p, miR-24-3p, miR-221-3p, miR-28-3p, miR-652-3p, miR-532-5p, miR-223-5p, miR-148b-3p, miR-143-3p, miR-361-3p, miR-199a-3p, miR-7-5p, miR-382-5p, miR-27b-3p, miR-222-3p, miR-425-5p, miR-152-3p and miR-4326 are subjected to down-regulation, miR-378d expression is up-regulated.

We performed fluorescent quantitative PCR detection validation on these 20 differential mirnas. In peripheral blood samples of other RVO patients, total RNA of 20 RVO disease patients and 20 control groups of plasma exosomes are randomly extracted, and the 20 miRNA are quantitatively verified. Patient case baseline as shown in table 2, two sets of baseline were not different (t-test, p-0.5518). The results show that the quantitative PCR verification results of 20 miRNAs are consistent with the trend of the sequencing results and have significant differences (figure 3), and the quantitative PCR verification results can well distinguish cataract patients from RVO patients and can be used as potential biomarkers for RVO diagnosis.

TABLE 2 basic data of patients

The research shows that single or a plurality of miRNAs in circulating blood exosomes miR-378d, miR-146a-5p, miR-148a-3p, miR-24-3p, miR-221-3p, miR-28-3p, miR-652-3p, miR-532-5p, miR-223-5p, miR-148b-3p, miR-143-3p, miR-361-3p, miR-199a-3p, miR-7-5p, miR-382-5p, miR-27b-3p, miR-222-3p, miR-425-5p, miR-152-3p and miR-4326 can be combined to be used as an RVO biomarker.

The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above-mentioned embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may occur to those skilled in the art without departing from the principle of the invention, and are considered to be within the scope of the invention.

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Claims

1. The application of the reagent for detecting the circulating blood exosome miRNA expression level in preparing a retinal vein occlusion diagnostic kit is characterized in that: the miRNA markers comprise:

one or at least 2 combinations of miR-378d, miR-146a-5p, miR-148a-3p, miR-24-3p, miR-221-3p, miR-28-3p, miR-652-3p, miR-532-5p, miR-223-5p, miR-148b-3p, miR-143-3p, miR-361-3p, miR-199a-3p, miR-7-5p, miR-382-5p, miR-27b-3p, miR-222-3p, miR-425-5p, miR-152-3p and miR-4326.

2. Use of the reagent for detecting the expression level of miRNA in circulating blood exosomes according to claim 1 for preparing a retinal vein occlusion diagnostic kit, wherein the retinal vein occlusion comprises central retinal vein occlusion and branch retinal vein occlusion.

3. The application of the reagent for detecting the miRNA expression level of circulating blood exosomes in the preparation of a retinal vein occlusion diagnostic kit according to claim 1, wherein the kit comprises at least one specific amplification miR-378d, miR-146a-5p, miR-148a-3p, miR-24-3p, miR-221-3p, miR-28-3p, miR-652-3p, miR-532-5p, miR-223-5p, miR-148b-3p, miR-143-3p, miR-361-3p, miR-199a-3p, miR-7-5p, miR-382-5p, miR-27b-3p, miR-222-3p, Primers of miR-425-5p, miR-152-3p and miR-4326.

4. The use of the reagent for detecting the miRNA expression level of circulating blood exosomes according to claim 3 in the preparation of a retinal vein occlusion diagnostic kit,the kit also comprises reverse transcriptase, buffer solution, dNTPs and MgCl₂DEPC water, fluorescent probes, rnase inhibitors, and Taq enzyme.