CN112442534A

CN112442534A - Application of circulating blood exosome miR-146a-5p as retinal vein occlusion treatment and prognosis evaluation marker

Info

Publication number: CN112442534A
Application number: CN202011228925.3A
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: 2021-03-05
Anticipated expiration: 2040-11-06
Also published as: CN112442534B

Abstract

The invention discloses application of circulating blood exosome miR-146a-5p as a retinal vein occlusion treatment and prognosis evaluation marker, changes of candidate genes in a disease treatment process are verified through animal experiments, the relation between miRNA and RVO anti-VEGF treatment is further explored, and the up-regulation of the expression quantity of plasma exosome miR-146a-5p is confirmed to be used as an auxiliary index for predicting good treatment effect, so that a new research direction is provided for diagnosing retinal vein occlusion.

Description

Application of circulating blood exosome miR-146a-5p as retinal vein occlusion treatment and prognosis evaluation marker

Technical Field

The invention relates to the technical field of molecular diagnosis, in particular to application of a circulating blood exosome miR-146a-5p as a retinal vein occlusion treatment and prognosis evaluation marker.

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.

Of the complications of RVO, macular edema is the most common and most affecting central vision, and its cause is related to the disturbance of blood circulation, increased permeability of vascular endothelial cells after RVO. In the treatment of RVO complicated by macular edema, anti-VEGF medicament treatment is a widely applied method at present, and commonly used medicaments comprise ranibizumab, combaici, and the like. 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.

Plasma exosomes of RVO patients before and after anti-VEGF drug treatment are collected, expression difference of exosome miRNA in RVO diseases is screened through high-throughput sequencing and bioinformatics analysis, expression level is verified through real-time fluorescence quantitative PCR, application feasibility of the exosome miRNA in disease treatment effect and prognosis evaluation is discussed, and new diagnosis and treatment ideas and theoretical bases can be provided for RVO diseases.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to protect the new application of the circulating blood exosome miR-146a-5p as a marker for retinal vein occlusion treatment and prognosis evaluation, and provides a more accurate and convenient mode for the retinal vein occlusion prognosis effect.

The invention aims to provide a miRNA marker of circulating blood exosome related to retinal vein occlusion treatment and auxiliary evaluation of prognosis effect.

The invention also aims to provide application of the circulating blood exosome miRNA and the primer thereof in preparation of a kit for retinal vein occlusion treatment or prognosis evaluation.

It is still another object of the present invention to provide a kit for retinal vein occlusion treatment or prognosis evaluation.

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

a miRNA marker of circulating blood exosome related to retinal vein occlusion treatment and prognosis effect auxiliary evaluation is miR-146a-5 p.

The circulating blood exosome miR-146a-5p marker is applied to retinal vein occlusion treatment and auxiliary evaluation of prognosis effect.

The circulating blood exosome miR-146a-5p marker is applied to preparation of a retinal vein occlusion treatment and prognosis effect auxiliary evaluation kit.

A primer of a miR-146a-5p marker of circulating blood exosome related to retinal vein occlusion treatment and prognosis effect auxiliary evaluation;

the primer is applied to the auxiliary evaluation kit for retinal vein occlusion treatment and prognosis effect.

An auxiliary evaluation kit for retinal vein occlusion treatment and prognosis effect, which contains miR-146a-5p primer.

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.

Sequences of circulating blood exosomes miR-146a-5p related to the invention are disclosed, and the sequences are shown in SEQ ID NO. 1. Amplification primers of the miR-146a-5p marker can be obtained by market purchase, and the primers of the circulating blood exosome miR-146a-5p used in the embodiment of the invention are specific miRNA stem-loop PCR primers provided by Guangzhou multifunctional gene company Limited (GeneCopoeia).

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 analyzes the relationship between miRNA and anti-VEGF treatment effect by comparing the differential expression of miRNA of circulating blood exosomes before and after anti-VEGF treatment and combining clinical manifestations of patients before and after treatment. Animal experiments verify the expression change of candidate genes in the disease treatment process, and further explore the relation between miRNA and RVO and anti-VEGF treatment, so that the up-regulation of miR-146a-5p expression in plasma exosomes can be used as an auxiliary index for predicting good treatment effect, and the invention provides a new research direction for diagnosing the retinal vein occlusion treatment prognosis effect.

Drawings

FIG. 1: RVO patients received changes in peripheral plasma exosome mirnas before and after anti-VEGF treatment. A: compared with the pre-treatment (RVO), the expression difference of plasma exosome source miRNA of the anti-VEGF treatment (RVO-t) patients is significant, and the difference is expressed by | log (fold change) | > 1 threshold, and P < 0.05; b: 12 miRNA expression differential heatmaps likely associated with anti-VEGF treatment; c: the expression level of miRNA-146a-5P in peripheral blood plasma exosomes in the same patient 7 days before and after anti-VEGF treatment (one-way ANOVA, n 17, P < 0.001, P < 0.0001); d: the expression level of miRNA-96-5p in peripheral blood plasma exosomes of the same patient before anti-VEGF treatment and 7 days after anti-VEGF treatment; FIG. 2: observation of therapeutic effects of anti-VEGF treatment on RVO secondary macular edema. A: when a patient is admitted, an OCT image of a macular region shows that obvious cystoid macular edema exists, a fovea structure disappears, the serous fluid of retina is separated, and CFT is 1201 mu m; b: the same patient rechecks the OCT image of the macular area 1 month after anti-VEGF treatment, the retina is flat, the central fovea structure of the macula is recovered, and the CFT is 164 mu m. C: patients had a significant decrease in post-treatment CFT measured by OCT before and after anti-VEGF treatment, i.e. macular edema was significantly reduced, compared to pre-treatment (paired t-test, n-17, P < 0.01). D: BCVA of a patient by a pre-and post-VEGF-resistant logarithmic record method has obviously improved vision after VEGF-resistant treatment compared with that before treatment (n is 17 and P is less than 0.05 in a paired t test); FIG. 3: the relation between the expression quantity of plasma exosome miRNA and the change of CFT and BCVA before and after VEGF-resistant RVO secondary macular edema treatment is characterized in that ROC curve analysis is carried out on the expression quantity of peripheral blood exosome miR-146a-5p after treatment of a patient and the comparison before treatment by taking the obvious reduction of CFT and the obvious regression of macular edema of the patient as judgment standards with good treatment effects, and the AUC is 0.918;

FIG. 4: RVO animal models and the effects of intravitreal anti-VEGF drug injections; a: mouse fundus imaging. B-C: fundus imaging after model making of the RVO model shows that the vein injury part is obviously narrowed, the proximal blood vessel is contracted, and the distal end is expanded in a tortuosity way. D: retinal vascular circulation time was visualized by fundus fluorescein angiography 7 days after mouse RVO modeling (paired t-test, n-4, P < 0.001). E: the mouse model RVO has VEGF content in the vitreous humor (t test, n is 4, P is less than 0.001). F: VEGF content in mouse RVO model retinas (t-test, n-4, P < 0.01). G: mouse RVO model and retina miRNA-146a-5p expression after anti-VEGF treatment. Control: no treatment group; RVO: performing temporary operation treatment on vitreous cavity by injecting medicine 7 days after RVO molding; anti-VEGF: after 7 days of RVO molding, the anti-VEGF medicine is injected into the vitreous cavity, and the injection is carried out for 7 days. Compared with the control group, the retina miR-146a-5P in the RVO group is slightly reduced, and the retina in the anti-VEGF treatment group is obviously increased (single-factor analysis of variance, n is 5, P is less than 0.05).

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 information of a patient with secondary macular edema, which is diagnosed as RVO through clinical examination and is in an affiliated eye vision hospital of Wenzhou medical university, the age of 40-80 years, and 10mL of peripheral blood before anti-VEGF treatment and 10mL of peripheral blood after 7 days of intravitreal injection of anti-VEGF medicines;

the peripheral blood is collected by using a disposable EDTA anticoagulant 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

Peripheral blood of the same RVO patient before anti-VEGF treatment and 7 days after intravitreal injection of anti-VEGF drug were 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.

anti-VEGF pre-and post-treatment RVO patient plasma exosome total RNA extraction

Total RNA extraction from plasma exosomes was all produced using Omega Bio-tek

Total RNA Kit。

(1) Cracking: 1mL of exosome 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.

anti-VEGF pre-and post-treatment RVO patient plasma exosome RNA sequencing

RNA in plasma exosomes of 8 RVO patients before and after anti-VEGF treatment is collected, is subjected to non-specific PCR amplification after being subjected to reverse transcription into cDNA, then miRNA high-throughput sequencing is performed by using Illumina HiSeq 2500, a linker used during reverse transcription is removed, and a fragment of less than 17nt (provided by Guangzhou energy-recovering gene Co., Ltd.) is filtered. Compared with the results before treatment, 452 genes were obtained with differential expression, and the expression difference of 63 miRNAs in total was considered significant from 36 up-and 27 down-regulated genes by using the edgeR test tool, and the results are shown in FIG. 1A.

Screening of exosome miRNA as anti-VEGF treatment prognosis effect biomarker of RVO patients is carried out by verifying 12 miRNAs (figure 1B) with higher expression quantity and larger difference one by one in 63 miRNAs with different expressions, and the applicant verifies the regulation and control functions of miR-146a-5p and miR-96-5p in the anti-VEGF treatment process of RVO in the application. We further investigated its expression in RVO diseases and treatment for miR-146a-5p and miR-96-5 p.

Peripheral blood samples from 17 additional cases before and after anti-VEGF treatment were examined and the clinical data were as follows: 9 men and 8 women aged 60.8 and 10.3 years (46-74 years). At the same time, 20 cataract patients' peripheral blood was collected as a control, and the basic data was: 12 men and 8 women, 62.65 + -7.5 years (46-71 years), were tested to be indistinguishable (t-test, P0.8727). And then detecting the miR-146a-5p and miR-96-5p expression quantity of the 17 groups of peripheral blood sample exosomes before and after anti-VEGF treatment and the miR-146a-5p and miR-96-5p expression quantity of the control group. Compared with a control group, the expression level of the peripheral blood exosomes miR-146a-5p of the patients in the RVO affected group is slightly reduced, the expression level of the peripheral blood exosomes miR-146a-5p of the patients in the same group is obviously increased after the patients are subjected to anti-VEGF treatment for 7 days (figure 1C), the expression difference of the peripheral blood exosomes miR-146a-5p of the patients in the RVO affected group is larger than that of the miR-96-5p of the control group, the expression level of the peripheral blood exosomes miR-96-5p of the patients in the RVO affected group is slightly reduced, and the expression level of the peripheral blood exosomes miR-96-5p of the patients in the same group is not obviously different after the patients are.

Relation between plasma exosome miR-146a-5p expression quantity of RVO patient and anti-VEGF treatment prognosis effect of RVO patient

The 17 cases were collected and examined for best corrected vision with OCT before and after one month of treatment against VEGF. OCT images of macular edema secondary to RVO can be divided into three types: retinal spongiform swelling, macular cystoid edema, or retinal neuroepithelial serous detachment. As shown in FIG. 2A, the patient before treatment had high macular edema, indistinguishable central foveal structures, cystoid fluid space within the retina, and fluid space between the neuroepithelium and the pigment epithelium. After a patient receives anti-VEGF treatment for one month, the structure of yellow spots is recovered by rechecking OCT, the liquid cavity in the retina disappears, the retina is flat, and the structures of all layers are clear, as shown in figure 2B.

According to the judgment standard that the treatment effect is good, the thickness of an OCT (optical coherence tomography) macular region of a patient is obviously reduced and macular edema is obviously relieved after treatment, ROC curve analysis is carried out on the expression quantity of a peripheral blood exosome miR-146a-5p of the patient after treatment (figure 3), and the result shows that the prediction accuracy of the miR-146a-5p serving as a marker with good treatment effect is very good (AUC is 0.918).

The result shows that after the RVO patient is treated by resisting VEGF, the up-regulation of miR-146a-5p expression quantity in plasma exosome can be used as an auxiliary index for predicting good treatment effect.

Modification of miR-146a-5p in RVO animal model

In order to study whether the change of the expression level of miR-146a-5p in a patient is actually caused by anti-VEGF treatment, the retinal vessels are damaged by laser photocoagulation under the assistance of a micro IV fundus imaging system, and a disease model of RVO is simulated in a mouse body, so that compared with before photocoagulation (figure 4A), the damaged vessels are obviously narrowed, and the far blood vessels expand and fill and the near blood vessels contract (figures 4B-C). After the laser light stares at retinal blood vessels for 7 days, the FFA detects the retinal blood vessel circulation time, verifies whether the retinal blood vessels are blocked and whether the model is effectively established (figure 4D). The results show that the retinal vascular circulation time of the molded eye is significantly prolonged, indicating that the model is successfully established (paired t test, n is 4, and p is 0.0008).

Thereafter, we examined the VEGF content in the eyes of the mouse model RVO by ELISA (fig. 4E-F), and the results showed that the VEGF content in the vitreous humor of the RVO-model was significantly higher than that of the control group (n-4, P-0.0006), the trend was the same in the retina, and the VEGF content was higher than that in the vitreous humor (n-4, P-0.0012). It was demonstrated that RVO modeling caused an increase in the expression level of VEGF in the vitreous humor and in the retina in the eyes of mice.

Then, after 7 days from molding, the group treated with 2. mu.L of Campsip intravitreal injection and RVO was subjected to a pseudo-surgical procedure, i.e., only Hamilton needle insertion without injection of the drug. The mouse retinas were taken 7 days after the drug injection into the vitreous cavity to detect the expression level of miR-146a-5p (FIG. 4G). The results show that the plasma exosomes miR-146a-5p have the same tendency of change after RVO patients and anti-VEGF treatment, miR-146a-5p in the retinas of the RVO model mice is slightly down-regulated compared with a control group, and miR-146a-5p expression is obviously up-regulated after anti-VEGF treatment (one-factor analysis of variance, n is 5, and p is 0.0122).

The result shows that the expression quantity of miR-146a-5p in the retina of a mouse can be changed by injecting the anti-VEGF medicament into the vitreous cavity after the RVO pathological process and the RVO.

We combine and analyze the therapeutic effect result of the anti-VEGF treatment of the RVO patient and the detection result of miR-146a-5p in plasma exosomes, and find that the change of miR-146a-5p can be used as an auxiliary analysis index for predicting good clinical therapeutic effect in the anti-VEGF treatment process of the RVO patient, and the method has specificity and sensitivity. In the research results, the expression quantity of miR-146a-5p of RVO patients is reduced, and the expression of miR-146a-5p is obviously increased after receiving anti-VEGF treatment, which shows that miR-146a-5p plays an important role in the generation, development and treatment processes of RVO.

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.

Sequence listing

<110> Wenzhou university of medical science

<120> application of circulating blood exosome miR-146a-5p as retinal vein occlusion treatment and prognosis evaluation marker

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<212> RNA

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Claims

1. Application of a reagent for detecting the expression level of circulating blood exosome miR-146a-5p in preparation of a kit for retinal vein occlusion treatment or prognosis evaluation.

2. The use of the reagent for detecting the expression level of circulating blood exosome miR-146a-5p according to claim 1 in the preparation of a kit for retinal vein occlusion treatment or prognosis evaluation, wherein the retinal vein occlusion comprises central retinal vein occlusion and branch retinal vein occlusion.

3. The application of the reagent for detecting the expression level of the circulating blood exosome miR-146a-5p in preparing a kit for retinal vein occlusion treatment or prognosis evaluation according to claim 1, wherein the retinal vein occlusion treatment is anti-VEGF treatment.

4. The application of the reagent for detecting the expression level of the circulating blood exosome miR-146a-5p in preparing the kit for retinal vein occlusion treatment or prognosis evaluation according to claim 1, wherein the kit further comprises reverse transcriptase, buffer solution, dNTPs, MgCl and dNTPs₂DEPC water, fluorescent probes, rnase inhibitors, and Taq enzyme.