CN116536416A - Application of serum-derived exosome marker miR-150-5p in preparation of depression serum diagnostic product - Google Patents

Abstract

The invention discloses an application of serum-derived exosome marker miR-150-5p in preparation of a depression serum diagnosis product, and belongs to the technical field of biological medicines. The invention detects and verifies the mixed serum sample and individual serum sample of the depression through the high-throughput RNA sequencing and qRT-PCR individual verification technology, and discovers that miR-150-5p presents specific high expression in the depression serum exosome, and the miRNA can be used as a serum exosome marker for diagnosing the depression.

Description

Application of serum-derived exosome marker miR-150-5p in preparation of depression serum diagnostic product

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of serum-derived exosome marker miR-150-5p in preparation of a depression serum diagnosis product.

Background

Depression (Depressive Disorder, DD) is a high-risk mental disorder characterized by a major disturbance of low mood and loss of pleasure that severely jeopardizes human health. Epidemiological investigation data show that depression is one of the major disease burden problems in serious threat to human health worldwide, and the prevalence rate varies from 3% to 17% worldwide, and has great influence on physical and mental health and social life of patients. The etiology of depression is extremely complex, involving psychological, genetic, epigenetic, neuroendocrine, and neuroimmune factors. Currently, clinical diagnosis of depression includes medical history evaluation, mental examination, and the like, and common evaluation scales include: hamilton depression scale (HAMD), montgomery depression scale (MADRS), rapid depressive symptom self-questionnaire, and the like. Scale assessment often depends on the diagnostic level of the clinician, is highly subjective, and lacks objective, quantifiable biological indicators in the clinic to aid in the diagnosis of depression. Therefore, the research on the accurate molecular mechanism of occurrence and development of the depression, and the search of new specific diagnostic markers have important clinical significance for early diagnosis and treatment of the depression and improvement of the life quality of patients.

Exosomes (exosomes) are nanoscale extracellular vesicles secreted by eukaryotic cells, about 30-150nm in diameter, with a typical "teacup" like morphology. Exosomes are widely present in biological fluids such as saliva, plasma, urine, ascites, milk and bile, carry biologically active substances such as nucleic acids, proteins, lipids and the like, and play a role in the physiological and pathological processes of the body. Exosomes are rich in biomarkers for disease diagnosis and prognosis, and currently exosome markers have made a certain progress in the fields of cancer, cardiovascular diseases and the like. For example, sandfeld et al have found that the exosome protein CD151 is highly expressed in lung cancer patients; hannafon et al found that exosomes miR-1246 and miR-21 are highly enriched in exosomes derived from breast cancer cells; corsten et al have found that exosomes miR-499, miR-133 are upregulated in patients with acute myocardial infarction. Exosome mirnas are the most commonly used biomarkers with tissue specificity, and the membrane structure of exosomes can enhance the stability of miRNA molecules, enhancing their potential as disease biomarkers. Therefore, differential expression analysis is carried out on serum exosome miRNA of depression patients by a high-throughput RNA sequencing technology, and clinical individual verification is carried out by combining a qRT-PCR technology, so that the depression serum-derived exosome markers with better sensitivity and specificity are screened and analyzed, and the blank of clinical blood diagnosis indexes of depression can be filled.

miR-150-5p is found to be closely related to colorectal cancer, nasopharyngeal cancer, breast cancer and the like in the previous report. For example, cheng et al have found through research that miR-150-5p inhibits tumor progression by targeting Vascular Epithelial Growth Factor A (VEGFA) in regulated colorectal cancer; li and the like find that miR-150-5p inhibits nasopharyngeal carcinoma by inhibiting pyrroline-5-carboxylic acid reductase-1 (PYCR 1); sugita et al found through research that miR-150-5p was associated with Triple Negative Breast Cancer (TNBC), and artificial manipulation of miR-150-5p overexpression could regulate proliferation, cloning, migration and drug resistance of TNBC cells. miR-150-5p can also participate in multiple biological processes such as vesicle transport in synapses, negative regulation of transferase activity, cycle of synapse vesicles, regulation of neuronal projection development and the like. However, no report on miR-150-5p in respect of depression diagnosis is available at present.

Disclosure of Invention

The invention aims to provide an application of a serum-derived exosome marker miR-150-5p in preparation of a depression serum diagnosis product.

In order to achieve the above purpose, the invention is realized by adopting the following technical scheme:

the invention discloses an application of a serum-derived exosome marker miR-150-5p in preparation of a depression serum diagnosis product.

Preferably, the product is a diagnostic reagent or a diagnostic kit.

Preferably, serum-derived exosome miR-150-5p expression is detected by high-throughput RNA sequencing, qRT-PCR.

Preferably, the serum-derived exosome marker miR-150-5p is significantly highly expressed in depression serum.

The invention also discloses application of the reagent for detecting the serum-derived exosome marker miR-150-5p in preparation of a serum diagnostic product for depression.

Preferably, the product is a diagnostic reagent or a diagnostic kit.

Preferably, serum-derived exosome miR-150-5p expression is detected by high-throughput RNA sequencing, qRT-PCR techniques.

Preferably, the serum-derived exosome marker miR-150-5p is significantly highly expressed in depression serum.

The invention also discloses a depression serum diagnostic kit, which comprises a reagent for detecting the exosome marker miR-150-5p in a serum sample of depression to be detected.

Compared with the prior art, the invention has the following beneficial effects:

the invention applies high-throughput RNA sequencing to screen miRNA (micro ribonucleic acid) which is differentially expressed by a vs healthy control group of a depressive disorder group, namely miR-150-5p, and discloses a new application of a depression serum source exosome marker miR-150-5p in preparation of a depression diagnosis product for the first time, and the high-throughput RNA sequencing and qRT-PCR individual verification technology is used for detecting and verifying depression mixed serum samples and individual serum samples, so that the result shows that miR-150-5p is remarkably and highly expressed in depression crowds (p is less than 0.001). ROC curve analysis shows that the AUC value of miR-150-5p is 0.958, the maximum index is 0.86, the 95% credible interval is (0.914,1), the optimal critical value is 1.9541, the sensitivity is 90.00%, the specificity is 96.00%, and the miR-150-5p is shown to be specifically and highly expressed in depression serum exosomes.

Drawings

FIG. 1 is a transmission electron microscopy image (1 μm) of the exosomes of the depressive group;

FIG. 2 is a transmission electron microscopy image (100 nm) of the exosomes of the depressive disorder group;

FIG. 3 is a transmission electron microscope image (1 μm) of healthy control exosomes;

FIG. 4 is a transmission electron microscope image (100 nm) of healthy control exosomes;

FIG. 5 is a schematic of serum exosome concentrations in the depressed group;

FIG. 6 is a schematic diagram of serum exosomes particle size in the depressive group;

FIG. 7 is a schematic of serum exosome concentration in healthy control group;

FIG. 8 is a schematic diagram of serum exosomes particle sizes in healthy control groups;

FIG. 9 is a schematic illustration of nano-flow fluorescence detection of depressive disorder groups;

FIG. 10 is a schematic diagram of a nano-flow fluorescence assay for a healthy control group;

FIG. 11 is a high throughput RNA sequencing analysis result (differential expression miRNA volcanic pattern) of serum exosomes from 3 cases of serum mix samples from the depression group and 3 cases of serum mix samples from the healthy control group;

FIG. 12 is a high throughput RNA sequencing analysis result (differential expression miRNA cluster) of serum exosomes from 3 cases of serum mix samples from the depression group and 3 cases of serum mix samples from the healthy control group;

FIG. 13 is a graph showing the expression level of the serum exosome difference miR-150-5p verified by qRT-PCR. qRT-PCR assays were performed in 50 depressed patients and 50 healthy controls, with the Y-axis showing miRNA expression levels as log ₁₀ 2 ^-ΔCt The dots represent outliers and represent P<0.001；

FIG. 14 is a graph of the diagnostic value of ROC curve analysis miR-150-5p in depression.

Detailed Description

In order that those skilled in the art will better understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.

It should be noted that the terms "first," "second," and the like in the description and the claims of the present invention and the above figures are used for distinguishing between similar objects and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used may be interchanged where appropriate such that the embodiments of the invention described herein may be implemented in sequences other than those illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

The invention is described in further detail below with reference to the attached drawing figures:

1. collection and grouping of serum samples

Samples were collected from 60 (32 men; 28 women) depressed patients in the university of Yan Yangyang Hospital psychiatric department (9 months 2020 to 3 months 2022), 60 normal healthy people (34 men; 26 women). The samples consider factors such as age, sex, time of collection, consistency of storage conditions, presence or absence of underlying disease, etc. The person to be collected takes blood in an empty stomach in the morning, and takes 5mL of whole blood by a vacuum blood taking tube (yellow cap and isolation glue), and stands for 30min at room temperature; centrifuging at room temperature for 5min (3000 g), packaging the upper serum into 100 μl/tube, and immediately storing at-80deg.C to avoid repeated freeze thawing. Serum samples of the depression group and the healthy control group were mixed in an equal amount per 20 cases (25. Mu.L per case) of the group, and were divided into 6 mixed samples of depression groups 1 to 3 (B1, B2, B3) and healthy control groups 1 to 3 (H1, H2, H3) for high-throughput RNA sequencing. The remaining samples were stored separately for individual verification.

2. Screening of MiRNAs differentially expressed by serum exosomes in depressive groups and healthy control groups

2.1 extraction of serum exosomes

ExoQuick kit based on polymer precipitation (Exosome Precipitation Solution, system Biosciences, SBI) was used to isolate exosomes:

(1) 0.5mL of serum sample was taken, 0.5mL of DBS-2 buffer containing protease inhibitor and phosphatase inhibitor, no calcium and magnesium was added, and the mixture was homogenized, and centrifuged at 1500g for 20min at 4 ℃.

(2) The supernatant was taken and added with 252. Mu.L of Exoquick reagent and incubated for 1h at 4 ℃.

(3) Centrifuging at 4deg.C for 30min at 1500g, removing supernatant, and collecting precipitate to obtain exosome particles. 150. Mu.L of DBS-2 buffer was added to the pellet for resuspension, either directly or frozen in liquid nitrogen.

2.2 identification of serum exosomes

(1) And (3) transmission electron microscope observation: the exosome mixture was pipetted into 10 μl and deposited on a copper mesh for 1min, and the supernatant was blotted off with filter paper. The uranyl acetate 10 mu L is dripped on a copper net to precipitate for 1min, the floating liquid is sucked by filter paper, and the filter paper is dried for a plurality of minutes at normal temperature. And (5) performing electron microscope detection imaging at 100kv to obtain a transmission electron microscope imaging result. Under a transmission electron microscope, both sets of exosomes were in a "teacup" like structure (fig. 1-4).

(2) Particle size analysis: the exosome mixture was diluted to 30. Mu.L by taking out 5. Mu.L. Firstly, testing the performance of an instrument by using a standard substance, and loading an exosome sample after the exosome sample is qualified, wherein gradient dilution is needed to avoid the sample from blocking a sample injection needle. And (3) obtaining the information of the particle size and concentration of the exosomes detected by a particle size analyzer (NanoFCM, N30E) after the sample is detected. The results show that: the particle size concentration of extract of exosomes in depression group is 2.01E+10particles/mL (see figure 5), the diameter is mainly distributed in 30-150nm (average 80.71 nm), and the diameter is consistent with the size of exosomes (see figure 6); the particle size concentration of the exosome extraction of the healthy control group is 2.01E+10particles/mL (figure 7), the diameter is mainly distributed at 30-150nm (average 83.07 nm), and the diameter is consistent with the size of the exosome (figure 8);

(3) Exosome sample fluorescent label and nano-flow detection

mu.L of the exosomes were diluted to 60. Mu.L, and 30. Mu.L of the diluted exosomes were added to 20. Mu.L of fluorescence-labeled antibodies CD9 (FITC MouseAnti-Human CD9, BD: 555371) and CD81 (FITC MouseAnti-Human CD81, BD: 551108), mixed well, incubated at 37℃in the absence of light for 30min, and subjected to gradient dilution and then on-line, and nanoparticle tracking analyzers (PARTICLE METRIX, zetaVIEW) were used to obtain the instrument detection protein index results (Table 1, FIGS. 9 to 10).

TABLE 1 expression Positive Rate of exosome protein

2.3 exosome RNA extraction

200. Mu.L of exosome mixture was used to extract total exosome RNA using EXOsome RNA isolation kit (Norgen) kit.

(1) 300. Mu.L of lysi buffer A and 37.5. Mu.L of lysi buffer B were added to 200. Mu.L of the resuspension containing purified exosomes.

(2) Fully mixing and incubating for 10min at room temperature;

(3) 500 μl of 100% ethanol was added, and vortexed for 10s;

(4) Transferring the liquid into Mini Spin Columns, and centrifuging at 6000rpm at normal temperature for 1min;

(5) Repeating the fourth step until all the liquid is transferred to the adsorption column;

(6) 600. Mu.L of eluent A is added into an adsorption column and centrifuged at 6000rpm for 30s at normal temperature;

(7) Repeating the step six, and washing twice;

(8) Idling the adsorption column, and centrifuging at the normal temperature of 14000rpm for 60s; transferring the mixture into another eluting tube, adding 50 mu L of eluent, and centrifuging at normal temperature for 60s and 120s at 1800rpm and 8000rpm respectively;

(9) To obtain maximum recovery efficiency, the eluted buffer was transferred back to the adsorption column, allowed to stand at room temperature for 2min, centrifuged at 2000rpm for 1min at room temperature and at 5000 rpm for 2min.

2.4 screening of differentially expressed miRNAs

High throughput RNA sequencing was performed on 6 pooled serum exosomes from the depressive group 1-3 (B1, B2, B3) and the healthy control group 1-3 (H1, H2, H3), and the variation in the expression profile of serum exosomes miRNAs from the depressive group and the healthy control group was analyzed to screen 146 total miRNAs differentially expressed in the depressed patient exosomes, with up-regulated expression of 89 and down-regulated expression of 57 (FIGS. 11-12). According to the principle that the difference is most obvious and the function of neurons is related, miR-150-5p is selected as a candidate marker, and clinical samples prove that the miR-150-5p is consistent with a sequencing result.

3. Verification of miR-150-5p expression in clinical samples of depression group and healthy control group

3.1 reverse transcription

RNA was reverse transcribed into cDNA using reverse transcription kit SweScript RT I First Strand cDNA Synthesis Kit (G3330).

(1) The reverse transcription reaction system was prepared (20. Mu.L, see Table 2, using the stem-loop reverse transcription method).

TABLE 2 20. Mu.L reverse transcription reaction System

(2) Mix gently and centrifuge.

(3) Reverse transcription procedure set (Table 3), reverse transcription was performed on a conventional PCR apparatus.

TABLE 3 reverse transcription procedure settings

3.2 real-time fluorescent quantitative PCR

Quantitative expression detection was performed on a PCR instrument using a fluorescent quantitative kit 2 x SYBR Green qPCR Master Mix (None ROX) (G3320), and 3 multiplex wells were set. 2 ^-ΔΔCt The method analyzes miRNA expression change (U6 is an internal reference). Mature sequence of hsa-miR-150-5p (MIMAT 0000451) was obtained in the MIRBase database (https:// www.mirbase.org): 5'-UCUCCCAACCCUUGU ACCAGUG-3'. The sequence of the designed miRNA primer is shown in Table 4.

TABLE 4miRNA primer sequences

(1) A reaction system (15. Mu.L, see Table 5) was prepared by taking 0.1ml of a PCR reaction plate, and 3 tubes were prepared for each reverse transcription product. And finishing the spot sample by using the PCR sealing plate film and matching with a film sealing instrument.

Table 5 real-time fluorescent quantitative PCR reaction system

(2) PCR amplification was performed on a fluorescent quantitative PCR apparatus, and the procedure was set (see Table 6).

TABLE 6PCR amplification program settings

3.3 results

Detection of 50 depressed patients and 50 healthy controls by qRT-PCR revealed that the expression level of miR-150-5P in the serum exosomes of the depressed group was significantly increased with significant statistical differences (P < 0.001) (FIG. 13).

3.4 subject work curve (ROC) correlation analysis and biomarker evaluation index calculation

And evaluating the predictive ability of the screened apo miRNA closely related to the depression according to the collected content of the serum apo miR-150-5p of the depression group and the healthy control group. The ROC curve analysis results show (fig. 14), in the independent serum samples, the area under the curve (AUC) value of serum exosome miR-150-5p is 0.958, the maximum log index is 0.86, the 95% credible interval is (0.914,1), the optimal critical value is 1.9541, the sensitivity is 90.00%, and the specificity is 96.00%, which indicates that the above method for detecting the relative expression amount of the exosome miRNA shows good judgment capability and fitting performance for depression.

In conclusion, the invention detects and verifies the mixed serum sample and individual serum sample of the depression through the high-throughput RNA sequencing and qRT-PCR individual verification technology, and discovers that miR-150-5p presents specific high expression in the serum exosome of the depression, so that miR-150-5p is used as a serum exosome marker for diagnosing the depression, and important reference can be provided for early diagnosis of the depression.

The above is only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited by this, and any modification made on the basis of the technical scheme according to the technical idea of the present invention falls within the protection scope of the claims of the present invention.

Claims (9)

1. Application of serum-derived exosome marker miR-150-5p in preparation of depression serum diagnostic products.

2. The use according to claim 1, wherein the product is a diagnostic reagent or a diagnostic kit.

3. The use of claim 1, wherein serum-derived exosome miR-150-5p expression is detected by high throughput RNA sequencing, qRT-PCR.

4. The use of claim 1, wherein the serum-derived exosome marker miR-150-5p is significantly elevated in depressive serum.

5. Application of reagent for detecting serum-derived exosome marker miR-150-5p in preparation of serum diagnostic product for depression.

6. The use according to claim 5, wherein the product is a diagnostic reagent or a diagnostic kit.

7. The use of claim 5, wherein serum-derived exosome miR-150-5p expression is detected by high throughput RNA sequencing, qRT-PCR techniques.

8. The use of claim 5, wherein the serum-derived exosome marker miR-150-5p is significantly elevated in depressive serum.

9. A depression serum diagnostic kit is characterized by comprising a reagent for detecting an exosome marker miR-150-5p in a serum sample of depression to be detected.