Application of plasma exosome let-7b-5p as normal intraocular pressure glaucoma diagnostic marker

Abstract

The invention discloses an application of an exosome let-7b-5p from circulating blood plasma as a diagnostic marker of normal intraocular pressure glaucoma. The invention discloses application of a reagent for detecting the expression quantity of hsalet-7b-5p in preparation of a reagent for auxiliary diagnosis of normal intraocular pressure type glaucoma. Through high-throughput sequencing and quantitative PCR analysis in nearly 225 patients, it is found that let-7b-5p in plasma-derived exosomes can effectively distinguish normal intraocular pressure type glaucoma patients from normal people and cataract or other common ophthalmic disease patients, the sensitivity is high, the specificity is good and is far higher than that of an existing detection method, and the kit is suitable for clinical application. And the diagnosis performance is extremely excellent. An important basis is provided for diagnosis of the normal intraocular pressure type glaucoma, and the method has significance for prevention and treatment of the normal intraocular pressure type glaucoma in China.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and molecular diagnosis technology, and in particular to the field of normal intraocular pressure glaucoma diagnostic markers. Background Art

[0002] Glaucoma is a degenerative disease of the optic nerve characterized by optic atrophy and visual field loss. It is the world's leading irreversible blinding eye disease. Pathological increase in intraocular pressure is its main feature, but retinal ganglion cell (RGC) loss and axonal damage can also occur in individuals with normal intraocular pressure.

[0003] Normal-tension glaucoma (NTG) patients often have delayed diagnosis due to the lack of obvious symptoms in the early stages. Therefore, it is of great clinical significance to find non-invasive biomarkers for early diagnosis and disease assessment of normal-tension glaucoma.

[0004] As a medium for intercellular signal transmission, exosomes exist between tissue cells and in body fluids. They participate in intercellular communication by transmitting bioactive molecules such as proteins, lipids and nucleic acids in donor cells, and play an important role in the occurrence and development of diseases. The biomolecules carried by these exosomes can partially reflect the state of donor cells, which varies greatly due to different cell types, physiological and pathological conditions and other environments. Exosomes have a lipid bilayer membrane structure, which can effectively protect the biological activity of nucleic acids and proteins and prevent degradation; exosomes can easily pass through biological barriers, such as the blood-brain barrier and the blood-retinal barrier, and are widely present in various body fluids. A large number of studies have shown that exosome RNA can be used as a biomarker for various complex diseases such as neurodegenerative diseases and tumors, and has shown good prospects for translational applications. The purpose of this study is to further explore the characteristic circulating blood exosome RNAs of normal tension glaucoma (NTG) and explore its feasibility as a biomarker. Summary of the invention

[0005] The purpose of the present invention is to provide a new RNA that can be used as a biomarker for diagnosing and evaluating normal-tension glaucoma disease, and in particular, a new plasma exosome exosome RNA that can be used as a biomarker for diagnosing and evaluating normal-tension glaucoma disease.

[0006] A use of a biomarker in preparing a kit or an evaluation model for diagnosing normal-tension glaucoma, wherein the biomarker comprises let-7b-5p.

[0007] Preferably, let-7b-5p is derived from patient plasma exosomes.

[0008] Preferably, the biomarker also includes its combination with mitochondrial mtRNA in plasma exosomes.

[0009] Preferably, the mitochondrial mtRNA in the plasma exosomes includes one or more of ND1, ND2, ND3, ND4, ND4L, ND5, ND6, COX1, COX2, COX3, and CYTB.

[0010] Preferably, the kit for diagnosing or evaluating normal-tension glaucoma is a kit for quantitatively detecting the plasma exosome let-7b-5p molecular marker.

[0011] Preferably, the kit is used to assess the severity of normal-tension glaucoma.

[0012] Preferably, the kit is used to evaluate the degree of visual field loss in normal-tension glaucoma.

[0013] Preferably, the kit is used to evaluate the degree of optic nerve damage in normal-tension glaucoma.

[0014] Preferably, the internal reference for biomarker standardization is miR-30a-5p.

[0015] Preferably, the normal-tension glaucoma diagnostic assessment model includes input variables, which include the expression level of let-7b-5p and the patient's age.

[0016] Through experimental verification of the embodiments, the present invention provides a method for diagnosing and evaluating normal-tension glaucoma based on non-invasive detection technology, which has high sensitivity and high specificity in early normal-tension glaucoma, and further, when combined with age as a diagnostic model, has high sensitivity and good specificity, and has extremely superior diagnostic performance. It provides an important basis for the diagnosis of normal-tension glaucoma, and is of great significance for the prevention and treatment of normal-tension glaucoma in my country. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A is the TEM identification image of exosomes; 1B is the NTA particle size distribution image of exosomes; 1C is the characteristic protein markers of exosomes detected by WB: ALIX, Tsg101, Syntenin are positive markers, and Calnexin is a negative marker;

[0018] Figure 2A is an analysis of the changes in the exosome mRNA expression profile of the NTG group compared with the control group (blue dots represent downregulation when log2FC<-1; red dots represent upregulation when log2FC>1); 2B is a GO and KEGG analysis of differentially expressed mRNA; 2C is a Venn diagram analysis of neurodegenerative disease-related signaling pathways, with red representing genes that participate in these pathways; 2D is a result of verifying the expression of mitochondrial-related genes on mixed samples;

[0019] Figure 3 A is the distribution diagram of the proportion of different types of RNA in the whole transcriptome of plasma exosomes; 3B is the distribution diagram of the expression of differential RNA in plasma exosomes of NTG patients;

[0020] Figure 4 A is a graph showing the expression of candidate markers for preliminary verification on mixed samples; 4B is a graph showing the expression of miR-30a-5p on individual samples (n=20);

[0021] Figure 5 A is a graph showing the expression of let-7b-5p in the control group (n=23) and the NTG group (n=23) in Example 3; 5B is a graph showing the ROC curve analysis of the two models in Example 3;

[0022] Figure 6 A is the expression result diagram of let-7b-5p in cataract (n=27), PRK (n=20), and NTG group (n=27) in Example 4; 6B is the expression trend diagram of let-7b-5p in all control group samples (n=70) and NTG (n=50) samples in the training set and validation set;

[0023] Figure 7 This is a ROC curve analysis diagram of the disease diagnosis ability of different models in the validation set in Example 4;

[0024] Figure 8 To verify the expression trend of let-7b-5p in different disease groups;

[0025] Fig. 9 A is the comparison of let-7b-5p expression levels in different disease stages; 9B is the ROC curve showing that let-7b-5p has better diagnostic ability for severe patients. DETAILED DESCRIPTION

[0026] As mentioned above, in view of the defects of the prior art, the inventor of this case has proposed the technical solution of the present invention after long-term research and extensive practice. The technical solution of the present invention will be further disclosed and explained in conjunction with examples below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. It should be pointed out that the embodiments described below are intended to facilitate the understanding of the present invention and do not have any limiting effect on it. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0027] Example 1: Patient specimen collection

[0028] A total of 225 patients from Wenzhou Medical University Eye and Optometry Hospital were selected, including 70 NTG patients, 70 cataract patients, 20 PRK patients, and 65 patients with other diseases. The basic clinical characteristics of these subjects are shown in Table 1.

[0029] Table 1 Demographics and basic clinical characteristics of patients

[0030]

[0031] The plasma of 20 NTG patients and 20 senile cataract patients were mixed in equal proportions as mixed samples, and their exosomes were extracted for whole transcriptome high-throughput sequencing. The training set cohort included 23 NTG patients and 23 cataract patients for preliminary verification of the candidate RNA screened. 27 NTG patients, 27 cataract patients, 20 PRK and 65 other disease groups were included in the validation set cohort to further verify the applicability and stability of the RNA screened in the early stage as a biomarker. Other disease groups included 20 cases of POAG, 15 cases of DR, 10 cases of AMD, 10 cases of BRVO, and 10 cases of uveitis.

[0032] Example 2: Analysis of transcriptome characteristics of plasma exosomes in NTG patients

[0033] 2.1 Phenotypic identification of plasma exosomes

[0034] Take advantage of Ribo TM Exosome separation reagent combined with differential centrifugation separation method was used to separate total exosomes from plasma. Through transmission electron microscopy, it was found that the exosomes had a typical cup-shaped structure ( Figure 1 A). NanoSight shows that the main peak particle size of the separated exosomes is 85.7nm, which is consistent with the size of plasma exosomes, and the percentage of the main peak is 100% ( Figure 1 B). Immunoblotting experiments verified the exosome-specific positive and negative proteins: Alix, Tsg101, Syntenin and Calnexin. Figure 1 As shown in C, Alix, Tsg101, and Syntenin were significantly enriched in plasma exosomes, while Calnexin was not detected in the exosome samples, proving that the extracted samples were exosomes. High-throughput sequencing was used to analyze the transcriptome data in plasma exosomes of mixed samples. Next, bioinformatics technology was used to analyze the high-throughput sequencing results and screen candidate RNAs that may serve as biomarkers.

[0035] 2.2 Bioinformatics analysis of plasma exosome mRNA

[0036] In order to clarify the reliability of the sequencing results, we first performed bioinformatics analysis on the mRNA in the high-throughput sequencing results. The volcano plot showed the changes in the expression profiles of mRNA in the sequencing results of the control group and the NTG group, among which there were 435 up-regulated genes and 2069 down-regulated genes ( Figure 2 A).

[0037] KEGG pathway analysis showed that it was significantly enriched in multiple neurodegenerative disease-related pathways, and gene annotation analysis results also showed that it was significantly enriched in mitochondrial-related biological processes ( Figure 2 B). Next, we selected genes involved in these neurodegenerative disease-related pathways and further analyzed their commonalities. Through Venn diagram analysis, we found that the mRNAs involved in these neurodegenerative disease pathways were all related to mitochondrial function, and most of them were mitochondrial genomes ( Figure 2 C). High-throughput sequencing results showed that compared with the cataract group, the expression of mitochondrial mRNA in plasma exosomes of patients in the NTG group was significantly upregulated. The expression levels of these mitochondrial RNAs were initially verified in mixed samples, and the results were consistent with the sequencing results ( Figure 2 D) There was a significant up-regulation in the NTG group.

[0038] Combined with the clinical characteristics of NTG disease, its significant clinical feature is optic nerve degeneration, so mitochondrial function is closely related to the occurrence of NTG disease. The present invention newly discovered that mitochondrial RNA associated with common neurodegenerative diseases in plasma exosomes of NTG patients is significantly upregulated. This result further strengthens the significance of circulating plasma exosome detection in clinical diagnosis and accurately reflects disease characteristics.

[0039] 2.3 Bioinformatics analysis of plasma exosomal miRNA

[0040] The differentially expressed RNA in the plasma exosome transcriptome sequencing results was analyzed by category proportion, and it was found that the largest proportion of RNA was miRNA, followed by lncRNA, pseudogene and mRNA ( Figure 3A). The differentially expressed miRNAs are sorted from high to low according to the expression level. The total counts of the top 20 miRNAs account for 98% of all miRNA readings. We believe that these miRNAs are likely to play a major role ( Figure 3 B). To further explore the potential functions of non-coding RNAs, in the following analysis, we used bioinformatics techniques to predict target genes for the top 20 miRNAs and performed KEGG and GO enrichment analysis on their target genes.

[0041] Example 3. Biomarker Development

[0042] Two up-regulated and three down-regulated plasma exosomal miRNAs were selected as candidate biomarkers, and their relative expression levels in mixed samples were verified using quantitative PCR ( Figure 4 A). The validation results showed that the expression trends of miR-423-5p, miR-320a-3p and miR-146a-5p were inconsistent with the sequencing results, so they were eliminated; the expression trends of miR-186-5p and let-7b-5p were consistent with the sequencing results, but the Ct values ​​of miR-186-5p in both groups were greater than 30, indicating that its actual expression level was low. When performing quantitative PCR validation, the stability of the results was low. Based on the above considerations, let-7b-5p, which had the most significant expression difference and higher expression level, was finally selected to further expand the validation of samples at the individual level.

[0043] Using miR-30a-5p as an internal reference gene to normalize the target gene, it was verified that there was no statistical difference in the Ct value of miR-30a-5p between the control group and the NTG group ( Figure 4 B), can be used as a stable internal reference.

[0044] The expression trend of let-7b-5p was further verified at the individual level. Compared with the control group (23 cataract patients), let-7b-5p was significantly downregulated in the NTG group (23 cases). Figure 5A). Due to the particularity of the disease, patients in the control group did not undergo visual field and optical coherence tomography (OCT) examinations, and lacked relevant indicators. This study only used the common indicators of the two groups, namely gender, age and biomarker expression levels, as risk factors to construct a disease prediction model. These characteristics of the patients were categorized and numbered (age was a binary variable: 0 for age less than 40 and 1 for age greater than or equal to 40, the value of the biomarker expression level after logarithmic transformation (base 2) was a continuous variable, and gender was a binary variable: 0 for male and 1 for female). Logistic regression was used to establish a disease prediction model, and it was found that age and let-7b-5p expression level were the main risk factors. ROC analysis was then used to evaluate the diagnostic ability of let-7b-5p for the disease. The expression level of Let-7b-5p and other variables (gender and age) were converted into a predicted probability of 0-1 through a logistic regression model. The logistic regression model of the receiver operating characteristic (AUC-ROC) performed disease prediction and diagnostic analysis on single factors (expression level) and multiple factors (expression level combined with age). The AUC values ​​of the two models were compared ( Figure 5 B), the single factor prediction model (biomarker expression), its AUC was 0.735 (95% CI, 0.584-0.887); the multi-factor prediction model, which combined the biomarker expression level and patient age, had an AUC of 0.813 (95% CI, 0.678-0.947). This result shows that when the patient's age is taken into consideration, the diagnostic ability of this marker for NTG is greatly improved. Next, we will further expand the sample size to verify the diagnostic efficiency and clinical application value of this biomarker.

[0045] Example 4. Biomarker Validation and Specificity Analysis

[0046] 4.1PRK as a validation set for healthy control group

[0047] The average age of cataract patients is older than that of NTG patients. These elderly patients may have potential age-related chronic diseases, but they may not be aware of them, so it cannot be completely ruled out. Based on this possibility, we recruited another group of patients with refractive error (PRK) (average age of 24.8 years) as a healthy control group to rule out the possible effects of potential age-related diseases.

[0048] In the validation cohort, NTG patients (n=27), cataract patients (n=27), and PRK patients (n=20) from different hospitals were selected as a validation set to further verify the diagnostic ability of let-7b-5p. The quantitative PCR method was used to verify the biomarker expression levels of individual samples in this sample set. The results showed that compared with cataract or PRK, the expression of let-7b-5p in the NTG group showed a significant downregulation trend, and there was a statistically significant difference (P<0.0001) ( Figure 6 A). In order to clarify the diagnostic ability of let-7b-5p for NTG, we summarized the total data of all control groups and calculated the relative expression of let-7b-5p in the NTG group ( Figure 6 B), the results are consistent with the previous statistical results, both showing that let-7b-5p was statistically significantly downregulated in the NTG group. Logistic regression analysis also showed that the expression level of let-7b-5p and the patient's age were the main risk factors in both the training cohort and the validation cohort. This result is consistent with the epidemiological results of NTG, which shows that most NTG patients are over 40 years old.

[0049] ROC analysis was performed on different prediction models ( Figure 7 ), when cataract was used as the control group, the AUC value of the gene expression diagnostic model was 0.883 (95% CI, 0.797-0.970), the sensitivity was 88.9%, and the specificity was 74.1%; when the gene expression was combined with age, the AUC value was 0.923 (95% CI, 0.851-0.996), the sensitivity was 81.5%, and the specificity was 92.6%. With PRK as the healthy control group, the AUC of the simple gene expression diagnostic model was 0.868 (95% CI, 0.785-0.951); the AUC of the gene expression combined with age diagnostic model was 0.934 (95% CI, 0.876-0.991); the above results show that whether cataract or PRK is used as the control group, the diagnostic model combining let-7b-5p expression level with age has better diagnostic and predictive ability for NTG disease, which is consistent with the results of the training cohort, indicating the stability and reliability of the biomarker. These results demonstrate that let-7b-5p has good diagnostic ability for NTG regardless of whether cataract or PRK is used as the control group, and also prove the feasibility of clinical transformation of this biomarker.

[0050] 4.2 Validation sets of different diseases as control groups

[0051] To further verify the specificity of the biomarker, 65 patients with other eye diseases were included. The results showed that let-7b-5p was upregulated in the diabetic retinopathy (DR) and open-angle primary glaucoma (POAG) groups, and had no difference with the age-related macular degeneration (AMD), branch retinal vein occlusion (BRVO) and uveitis groups ( Figure 8 ). Let-7b-5p was significantly downregulated only in NTG patients. Through logistic regression and ROC curve analysis, we can conclude that this biomarker has good stability and reliability for diagnosing NTG disease, especially when the patient is older than 40 years old, the diagnostic ability of this marker for the disease is significantly improved.

[0052] Example 5. Correlation between plasma exosome let-7b-5p expression and clinical indicators

[0053] 5.1 Correlation analysis between Let-7b-5p and clinical indicators

[0054] To further analyze the correlation between biomarkers and clinical characteristics in the NTG group. Logistic regression results showed that age was a significant risk factor. Based on this result, we divided NTG patients into two groups. The first group included all NTG patients; the second group included only NTG patients over 40 years old. Spearman correlation analysis was used to describe the correlation between let-7b-5p expression levels and visual field (MD, PSD, VFI) and retinal nerve fiber layer (RNFL) in NTG patients. Each index was selected from the examination index of eyes diagnosed with normal-tension glaucoma. If both eyes of the patient were diagnosed with normal-tension glaucoma, the average of the indexes of both eyes was used for analysis. The analysis results showed that let-7b-5p expression levels were significantly correlated with patients' MD, PSD, VFI, and RNFL (Table 2), especially when the patient was older than 40 years old, the correlation was stronger. This part of the results is consistent with the results of the previous logistic regression model, indicating that in the middle-aged and elderly population, when the patient is older than 40 years old, this biomarker has better representativeness and correlation with the severity of NTG disease.

[0055] Table 2 Spearman correlation analysis between Let-7b-5p expression level and clinical indicators (MD, PSD, VFI, RNFL) of patients in the NTG group

[0056]

[0057] Note: Group 1 means that NTG patients of all ages are included in the analysis; Group 2 means that only patients over 40 years old are included in the analysis.

[0058] 5.2 Let-7b-5p's ability to assess NTG disease grading

[0059] In order to fully explore the clinical evaluation ability of this biomarker in the diagnosis of NTG, the average relative expression of let-7b-5p in the NTG group was used as the critical value, and the NTG patients were divided into a low expression group and a high expression group, and the relationship between the clinical characteristics of the two groups was analyzed (Table 3). According to the analysis results, we found that the age of patients in the low expression group was significantly older than that of patients in the high expression group, and there was a statistical difference; among the relevant indicators of visual field examination, the MD and VFI values ​​of the low expression group were significantly lower than those of the high expression group, and the PSD value was significantly higher. After statistical non-parametric t-test, it was found that there were statistical differences, indicating that patients in the low expression group had more severe visual field defects than those in the high expression group. The RNFL value reflects the degree of optic nerve damage in patients. From the results, it was found that the low expression group had more severe optic nerve damage than the high expression group. These results all illustrate the correlation between this biomarker and clinical indicators, and indicate that this biomarker can be used to assess the severity of NTG disease.

[0060] Table 3 NTG patients were divided into two groups according to the expression level of let-7b-5p, and the relationship between the clinical indicators of the two groups was analyzed

[0061]

[0062] Note: The average expression level (log2FC) of the biomarker in the NTG group is taken as the critical value. The values ​​greater than this value are the high expression group, and the values ​​lower than this value are the low expression group.

[0063] In the visual field examination, patients with mean deviation (MD)>-6dB are clinically considered to be mild glaucoma patients, and patients with MD≤-6dB are considered to be moderate to severe glaucoma patients. According to this clinical grading standard for glaucoma, we divided the patients in the NTG group into two groups: when the MD of both eyes or one eye was>-6dB, they were defined as mild NTG patients; when the MD of at least one eye was ≤-6dB, they were defined as moderate to severe NTG patients. First, the expression level of let-7b-5p between the two groups was analyzed. The higher the severity of NTG, the lower the expression level of let-7b-5p. The difference between the two groups was statistically significant, P=0.0182( Fig. 9 A). ROC curve was used to analyze the diagnostic ability of let-7b-5p in NTG disease of different stages ( Fig. 9 B). The AUC of the mild group was 0.761 (95% CI, 0.647-0.874); the AUC of the moderate-to-severe group was 0.882 (95% CI, 0.772-0.991), indicating that let-7b-5p was more conducive to the diagnosis of moderate-to-severe NTG patients than the mild group.

[0064] Based on all the above results, we concluded that the biomarker let-7b-5p can be used as a potential biomarker to assess the severity of NTG or as a biomarker for screening the occurrence of NTG in age-related risk populations. This study provides a solid foundation for the clinical transformation of plasma exosome biomarkers. We will further increase the number of samples in multiple centers to clarify the clinical transformation application prospects of this biomarker.

[0065] Finally, it is necessary to explain here that the above embodiments are only used to further illustrate the technical solution of the present invention in detail and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by technicians in this field based on the above content of the present invention all fall within the scope of protection of the present invention.

Claims

1. Use of a biomarker in the preparation of a diagnostic kit or evaluation model for normal intraocular pressure glaucoma, characterized in that: The biomarkers include let-7b-5p.

2. The use according to claim 1, characterized in that: let-7b-5p comes from patient plasma exosomes.

3. The use according to claim 2, characterized in that: The biomarkers also include their association with mitochondrial mtRNA in plasma exosomes.

4. The use according to claim 3, characterized in that: The mitochondrial mtRNA in the plasma exosomes includes one or more combinations of ND1, ND2, ND3, ND4, ND4L, ND5, ND6, COX1, COX2, COX3, and CYTB.

5. The use according to claim 2, characterized in that: The kit for diagnosing or evaluating normal-tension glaucoma is a kit for quantitatively detecting the plasma exosome let-7b-5p molecular marker.

6. The use according to claim 2, characterized in that: The kit is used for evaluating the severity of normal-tension glaucoma.

7. The use according to claim 6, characterized in that: The kit is used for evaluating the degree of visual field loss in normal-tension glaucoma.

8. The use according to claim 6, characterized in that: The kit is used for evaluating the degree of optic nerve damage in normal-tension glaucoma.

9. The use according to claim 2, characterized in that: The internal reference for the biomarker normalization was miR-30a-5p.

10. The use according to claim 2, characterized in that: The normal-tension glaucoma diagnostic assessment model includes input variables including the expression level of let-7b-5p and the patient's age.

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