Molecular marker for diagnosing primary open-angle glaucoma and application thereof

Abstract

The present application relates to a kind of molecular markers for early diagnosis of glaucoma, the molecular marker is one or more of miRNA and piRNA, the miRNA is selected from one or more of hsa-miR-451a and hsa-miR-142-5p, the piRNA is selected from one or more of piR-hsa-767596 and piR-hsa-731834.The present application finds that relative to healthy control group, POAG group patient in vivo piR-hsa-767596, piR-hsa-731834 and hsa-miR-451a expression is significantly down-regulated, its AUC value in ROC curve is 0.657, 0.736 and 0.696 respectively, and diagnosis performance is good, indicate that the above-mentioned miRNAs and piRNAs can be used as early diagnosis POAG molecular marker and be applied to clinic.

Description

Technical Field

[0001] This invention relates to the field of biological detection, specifically to a molecular marker for diagnosing primary open-angle glaucoma and its application. Background Technology

[0002] Primary open-angle glaucoma (POAG) is a chronic, progressive optic neuropathy characterized by elevated intraocular pressure and morphological changes in the optic disc and retinal nerve fiber layer, but without other eye diseases or congenital abnormalities. The pathogenesis of POAG is complex, involving multiple factors including elevated intraocular pressure, optic nerve axonal damage, oxidative stress, and retinal ganglion cell apoptosis.

[0003] Currently, the main treatments for glaucoma involve lowering intraocular pressure through laser therapy, topical medications (such as drugs to lower intraocular pressure), and surgical intervention. However, these methods only slow the progression of vision loss and do not completely prevent it. Furthermore, glaucoma is often asymptomatic in its early stages, which frequently leads to delayed diagnosis until significant and often irreversible vision impairment occurs. The lack of effective non-invasive diagnostic methods is a major challenge for the early detection and intervention of glaucoma, thus necessitating the research of new molecular biomarkers.

[0004] SncRNAs, defined as non-coding RNAs shorter than 200 nucleotides, are increasingly recognized for their crucial roles in gene regulation and disease pathogenesis. Among sncRNAs, miRNAs are the most extensively studied and have been identified as potential diagnostic biomarkers for a variety of diseases. Several studies have reported aberrant expression of certain miRNAs in the aqueous humor, retina, and serum of patients with POAG22–27. These miRNAs are thought to regulate key pathological processes, including apoptosis, inflammation, and intraocular pressure (IOP) homeostasis. Furthermore, aberrant expression of piRNAs has been reported under various pathological conditions, including tumors, cardiovascular diseases, and neurodegenerative diseases. These findings provide new insights into the molecular mechanisms of POAG; however, research on miRNAs and piRNAs in the context of POAG remains limited. Summary of the Invention

[0005] To solve the above-mentioned technical problems, the present invention includes the following aspects:

[0006] A first aspect of the present invention provides a molecular marker for the early diagnosis of glaucoma, said molecular marker being one or more of miRNA and piRNA.

[0007] Preferably, the piRNA is selected from one or more of piR-hsa-767596 (SEQ ID NO.5), piR-hsa-731834 (SEQ ID NO.6), piR-hsa-1800335 (SEQ ID NO.7), piR-hsa-123803 (SEQ ID NO.8), piR-hsa-2835291 (SEQ ID NO.9), piR-hsa-148612 (SEQ ID NO.10), piR-hsa-2545121 (SEQ ID NO.11), piR-hsa-125110 (SEQ ID NO.12), and piR-hsa-729882 (SEQ ID NO.13).

[0008] More preferably, the piRNA is selected from one or more of piR-hsa-767596 (SEQ ID NO.5) and piR-hsa-731834 (SEQ ID NO.6).

[0009] More preferably, the piRNA is piR-hsa-731834 (SEQ ID NO.6).

[0010] Preferably, the miRNA is selected from one or more of hsa-miR-142-5p (SEQ ID NO.14), hsa-miR-451a (SEQ ID NO.15), hsa-miR-142-3p (SEQ ID NO.16), hsa-miR-101-3p (SEQ ID NO.17), hsa-miR-144-5p (SEQ ID NO.18), hsa-miR-126-3p (SEQ ID NO.19), hsa-miR-16-5p (SEQ ID NO.20), hsa-miR-1246 (SEQ ID NO.21), and hsa-let-7g-5p (SEQ ID NO.22).

[0011] More preferably, the miRNA is selected from one or more of hsa-miR-142-5p (SEQ ID NO.14) and hsa-miR-451a (SEQ ID NO.15).

[0012] More preferably, the miRNA is hsa-miR-451a (SEQ ID NO.15).

[0013] Preferably, the molecular marker is a combination of miRNA and piRNA. More preferably, the molecular marker is a combination of hsa-miR-451a (SEQ ID NO.15) and piR-hsa-767596 (SEQ ID NO.5), or a combination of hsa-miR-451a (SEQ ID NO.15) and piR-hsa-731834 (SEQ ID NO.6), or a combination of piR-hsa-767596 (SEQ ID NO.5) and piR-hsa-731834 (SEQ ID NO.6).

[0014] More preferably, the molecular marker is a combination of hsa-miR-451a (SEQ ID NO.15) and piR-hsa-731834 (SEQ ID NO.6).

[0015] More preferably, the molecular markers are a combination of hsa-miR-451a (SEQ ID NO.15), piR-hsa-767596 (SEQ ID NO.5) and piR-hsa-731834 (SEQ ID NO.6).

[0016] Preferably, the glaucoma is primary open-angle glaucoma.

[0017] A second aspect of the present invention provides a kit for the early diagnosis of glaucoma, the kit comprising reagents for detecting the level of a molecular marker in a sample to be tested, the molecular marker being one or more of miRNA and piRNA.

[0018] Preferably, the piRNA is selected from one or more of piR-hsa-767596 (SEQ ID NO.5), piR-hsa-731834 (SEQ ID NO.6), piR-hsa-1800335 (SEQ ID NO.7), piR-hsa-123803 (SEQ ID NO.8), piR-hsa-2835291 (SEQ ID NO.9), piR-hsa-148612 (SEQ ID NO.10), piR-hsa-2545121 (SEQ ID NO.11), piR-hsa-125110 (SEQ ID NO.12), and piR-hsa-729882 (SEQ ID NO.13).

[0019] More preferably, the piRNA is selected from one or more of piR-hsa-767596 (SEQ ID NO.5) and piR-hsa-731834 (SEQ ID NO.6).

[0020] More preferably, the piRNA is piR-hsa-731834 (SEQ ID NO.6).

[0021] Preferably, the miRNA is selected from one or more of hsa-miR-142-5p (SEQ ID NO.14), hsa-miR-451a (SEQ ID NO.15), hsa-miR-142-3p (SEQ ID NO.16), hsa-miR-101-3p (SEQ ID NO.17), hsa-miR-144-5p (SEQ ID NO.18), hsa-miR-126-3p (SEQ ID NO.19), hsa-miR-16-5p (SEQ ID NO.20), hsa-miR-1246 (SEQ ID NO.21), and hsa-let-7g-5p (SEQ ID NO.22).

[0022] More preferably, the miRNA is selected from one or more of hsa-miR-142-5p (SEQ ID NO.14) and hsa-miR-451a (SEQ ID NO.15).

[0023] More preferably, the miRNA is hsa-miR-451a (SEQ ID NO.15).

[0024] Preferably, the molecular marker is a combination of miRNA and piRNA. More preferably, the molecular marker is a combination of hsa-miR-451a (SEQ ID NO.15) and piR-hsa-767596 (SEQ ID NO.5), or a combination of hsa-miR-451a (SEQ ID NO.15) and piR-hsa-731834 (SEQ ID NO.6), or a combination of piR-hsa-767596 (SEQ ID NO.5) and piR-hsa-731834 (SEQ ID NO.6).

[0025] More preferably, the molecular marker is a combination of hsa-miR-451a (SEQ ID NO.15) and piR-hsa-731834 (SEQ ID NO.6).

[0026] More preferably, the molecular markers are a combination of hsa-miR-451a (SEQ ID NO.15), piR-hsa-767596 (SEQ ID NO.5) and piR-hsa-731834 (SEQ ID NO.6).

[0027] Preferably, the glaucoma is primary open-angle glaucoma.

[0028] Preferably, the reagent includes a piR-hsa-731834 gene-specific forward primer and a reverse primer. The sequence of the forward primer is shown in SEQ ID NO.1. The reverse primer is a universal reverse primer from a miRNA first-strand cDNA synthesis kit (tailing method), which was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0029] Preferably, the reagent includes a piR-hsa-767596 gene-specific forward primer and a reverse primer. The sequence of the forward primer is shown in SEQ ID NO.2. The reverse primer is a universal reverse primer from a miRNA first-strand cDNA synthesis kit (tailing method), which was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0030] Preferably, the reagent includes an hsa-miR-451 gene-specific forward primer and a reverse primer. The sequence of the forward primer is shown in SEQ ID NO.3. The reverse primer is a universal reverse primer from a miRNA first-strand cDNA synthesis kit (tailing method), which was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0031] Preferably, the reagent includes an hsa-miR-142-5p gene-specific forward primer and a reverse primer. The sequence of the forward primer is shown in SEQ ID NO.4. The reverse primer is a universal reverse primer from a miRNA first-strand cDNA synthesis kit (tailing method), which was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0032] Preferably, the test sample is selected from one or more of the subject's tissues, whole blood, plasma, serum, saliva, sputum, pleural effusion, bronchoalveolar lavage fluid, and urine. More preferably, the test sample is selected from one or more of the subject's whole blood, plasma, and serum.

[0033] Preferably, the subject is a mammal. More preferably, the subject is a human.

[0034] A third aspect of the present invention provides the use of a reagent for detecting miRNA and / or piRNA in the preparation of a kit for the early diagnosis of glaucoma.

[0035] Preferably, the piRNA is selected from one or more of piR-hsa-767596 (SEQ ID NO.5), piR-hsa-731834 (SEQ ID NO.6), piR-hsa-1800335 (SEQ ID NO.7), piR-hsa-123803 (SEQ ID NO.8), piR-hsa-2835291 (SEQ ID NO.9), piR-hsa-148612 (SEQ ID NO.10), piR-hsa-2545121 (SEQ ID NO.11), piR-hsa-125110 (SEQ ID NO.12), and piR-hsa-729882 (SEQ ID NO.13).

[0036] More preferably, the piRNA is selected from one or more of piR-hsa-767596 (SEQ ID NO.5) and piR-hsa-731834 (SEQ ID NO.6).

[0037] More preferably, the piRNA is piR-hsa-731834 (SEQ ID NO.6).

[0038] Preferably, the miRNA is selected from one or more of hsa-miR-142-5p (SEQ ID NO.14), hsa-miR-451a (SEQ ID NO.15), hsa-miR-142-3p (SEQ ID NO.16), hsa-miR-101-3p (SEQ ID NO.17), hsa-miR-144-5p (SEQ ID NO.18), hsa-miR-126-3p (SEQ ID NO.19), hsa-miR-16-5p (SEQ ID NO.20), hsa-miR-1246 (SEQ ID NO.21), and hsa-let-7g-5p (SEQ ID NO.22).

[0039] More preferably, the miRNA is selected from one or more of hsa-miR-142-5p (SEQ ID NO.14) and hsa-miR-451a (SEQ ID NO.15).

[0040] More preferably, the miRNA is hsa-miR-451a (SEQ ID NO.15).

[0041] Preferably, the molecular marker is a combination of miRNA and piRNA. More preferably, the molecular marker is a combination of hsa-miR-451a (SEQ ID NO.15) and piR-hsa-767596 (SEQ ID NO.5), or a combination of hsa-miR-451a (SEQ ID NO.15) and piR-hsa-731834 (SEQ ID NO.6), or a combination of piR-hsa-767596 (SEQ ID NO.5) and piR-hsa-731834 (SEQ ID NO.6).

[0042] More preferably, the molecular marker is a combination of hsa-miR-451a (SEQ ID NO.15) and piR-hsa-731834 (SEQ ID NO.6).

[0043] More preferably, the molecular markers are a combination of hsa-miR-451a (SEQ ID NO.15), piR-hsa-767596 (SEQ ID NO.5) and piR-hsa-731834 (SEQ ID NO.6).

[0044] Preferably, the detection reagent includes a piR-hsa-731834 gene-specific forward primer and a reverse primer. The sequence of the forward primer is shown in SEQ ID NO.1. The reverse primer is a universal reverse primer from a miRNA first-strand cDNA synthesis kit (tailing method), which was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0045] Preferably, the detection reagent includes a piR-hsa-767596 gene-specific forward primer and a reverse primer. The sequence of the forward primer is shown in SEQ ID NO.2. The reverse primer is a universal reverse primer from a miRNA first-strand cDNA synthesis kit (tailing method), which was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0046] Preferably, the detection reagent includes an hsa-miR-451 gene-specific forward primer and a reverse primer. The sequence of the forward primer is shown in SEQ ID NO.3. The reverse primer is a universal reverse primer from a miRNA first-strand cDNA synthesis kit (tailing method), which was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0047] Preferably, the detection reagent includes an hsa-miR-142-5p gene-specific forward primer and a reverse primer. The sequence of the forward primer is shown in SEQ ID NO.4. The reverse primer is a universal reverse primer from a miRNA first-strand cDNA synthesis kit (tailing method), which was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0048] Preferably, the glaucoma is primary open-angle glaucoma.

[0049] The technical effects of this invention are as follows:

[0050] This invention used Pandora sequencing to systematically analyze the expression profiles of miRNAs and piRNAs in PBMCs of POAG patients and healthy controls, revealing significant changes in multiple miRNAs and piRNAs in POAG patients compared to the healthy control group. Further qRT-PCR analysis of two DE miRNAs and two DE piRNAs in the validation set showed downregulated expression of piR-hsa-767596 (p<0.05), piR-hsa-731834 (p<0.01), and hsa-miR-451a (p<0.05) in the POAG group. The AUC values ​​of piR-hsa-767596, piR-hsa-731834, and hsa-miR-451a in the receiver operating characteristic (ROC) curves were 0.657, 0.736, and 0.696, respectively, indicating good diagnostic performance. This suggests that these miRNAs and piRNAs can be used as molecular markers for the early diagnosis of POAG in clinical practice. Attached Figure Description

[0051] Figure 1 Heatmap of differentially expressed piRNAs (A) and miRNAs (B) in POAG;

[0052] Figure 2 The results of functional enrichment analysis of target genes associated with piRNAs in POAG are as follows: Figure 2 The GO pie chart for A shows the enrichment biological processes, molecular functions, and cellular components of target genes corresponding to differentially expressed piRNAs. Figure 2 B's KEGG bar plot shows the top 10 enriched target gene pathways associated with differentially expressed piRNAs;

[0053] Figure 3 The results of functional enrichment analysis of target genes associated with miRNAs in POAG are as follows: Figure 3 The GO pie chart for A shows the enrichment biological processes, molecular functions, and cellular components of target genes corresponding to differentially expressed miRNAs. Figure 3 B's KEGG bar plot shows the top 10 enriched target gene pathways associated with differentially expressed miRNAs;

[0054] Figure 4 qRT-PCR validation results for selected sncRNAs in healthy controls and POAG patients, among which Figure 4 A represents the expression level of piR-hsa-767596. Figure 4 B represents the expression level of piR-hsa-731834. Figure 4 C represents the expression level of hsa-miR-451a. Figure 4 D represents the expression level of hsa-miR-142-5p (data are expressed as mean ± SEM, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001);

[0055] Figure 5 ROC curve analysis of sncRNA for diagnosing POAG, where Figure 5 A represents the ROC curves for the differential expression of piR-hsa-767596, piR-hsa-731834, and hsa-miR-451a. Figure 5 B represents the ROC curves of different sncRNA combinations (combination 1: piR-hsa-767596 and piR-hsa-731834, combination 2: piR-hsa-767596 and hsa-miR-451a, combination 3: piR-hsa-731834 and hsa-miR-451a). Detailed Implementation

[0056] The present invention will be further described below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods.

[0057] Experimental Example 1: Study on tsRNAs biomarkers for early diagnosis of primary open-angle glaucoma.

[0058] 1. Test Methods

[0059] 1.1 Patients and Samples

[0060] All patients with porcine POAG and healthy controls were recruited at the People's Hospital Affiliated to Ningbo University between May 2024 and February 2025. This study was approved by the Ethics Committee of the People's Hospital Affiliated to Ningbo University, and all participants provided written informed consent in accordance with the principles of the Declaration of Helsinki. Ten patients with porcine POAG and ten healthy controls were selected for Pandora sequencing, and the validation cohort included 30 patients with porcine POAG and 30 healthy controls. Each participant provided 5 ml of peripheral blood for testing. All enrolled participants underwent comprehensive ophthalmological examinations and detailed medical history analysis. Ophthalmological examinations included best-corrected visual acuity testing, IOP measurement, gonioscopy, slit-lamp examination, fundus examination, visual field testing, optical coherence tomography (OCTA) analysis, and OCTA analysis.

[0061] POAG was diagnosed by an experienced glaucoma specialist based on the following criteria: (1) open anterior chamber angle confirmed by gonioscopy; (2) secondary causes of glaucoma, including pigment dispersion, pseudo-detachment, or other anterior segment abnormalities; and (3) evidence of optic nerve head injury in glaucoma, such as increased cup-to-disc ratio, thinning of the neuroretinal margin, notch, or localized retinal nerve fiber layer defects, with or without corresponding visual field loss. To exclude normal-tension glaucoma, POAG patients had untreated intraocular pressure exceeding 21 mmHg. All samples were from participants without significant comorbidities (such as diabetes, cancer, or chronic obstructive pulmonary disease) and no history of other major eye diseases (excluding glaucoma and cataracts). Clinical data of the subjects are shown in Table 1.

[0062] Table 1. Baseline demographic and clinical characteristics of POAG patients and healthy controls in the validation cohort.

[0063]

[0064]

[0065] Note: 1 Pearson's χ² test; 2 Independent samples t-test; 3 Mann-Whitney U test, after continuity correction; 4 Independent samples t-test, corrected for variance. IOP: intraocular pressure; BCVA: best corrected visual acuity; ACD: anterior chamber depth; CCT: central corneal thickness; AL: axial length; POAG: primary open-angle glaucoma; HC: healthy controls.

[0066] 1.2 PBMC Cell Isolation

[0067] PBMCs were isolated from 5 ml of whole blood using density gradient centrifugation. The blood was diluted with an equal volume of PBS and plated onto a Fi-coll-Paque (Cytiva, USA) plate. After centrifugation at 1500 rpm for 40 minutes, the PBMC layer at the interface was collected. The cells were washed twice with PBS and resuspended in 1 ml of Trizol (Thermo Fisher Scientific, USA). The isolated PBMCs were stored at -80°C for later use.

[0068] 1.3 Pandora Sequencing and Data Analysis

[0069] PANDORA-seq (panoramic RNA visualization sequencing by overcoming RNA modification-induced abortion) and raw data analysis were performed by Guangzhou Epigenetics Technology Co., Ltd. T4 polynucleotide kinase was used to convert 3'-P and 2',3'-cP to 3'-OH and add 5'-P. Specific RNA methylation modifications were removed using AlkB. RNA sequences of 15–45 nucleotides were then annotated using SPORTS 1.1 software. Reference databases included the miRNA database miRBase 21 and the piRNA database piRBase.

[0070] TPM was used to normalize RNA expression levels in the samples. Subsequently, differentially expressed genes were identified using the DEGseq R package based on fold change and p-value (fold change ≥1, p < 0.05). Furthermore, target gene prediction algorithms (miRanda and RNAhybrid) were used to predict regulatory targets of microRNAs. Further analysis was conducted to determine whether target genes were enriched in the KEGG and GO pathways.

[0071] 1.4 sncRNA extraction and reverse transcription real-time PCR (qRT-PCR)

[0072] Total RNA was extracted from PBMC cells using Trizol and analyzed using Thermo Scientific. TM μDrop TMRNA concentration and purity were determined using a Thermo Fisher Scientific (USA) plate and a Thermo Scientific Varioskan LUX (Thermo Fisher Scientific, USA). Subsequently, template RNA was reverse transcribed into cDNA using a miRNA first-strand cDNA synthesis kit (tailing method) (Sangon Biotech (Shanghai) Co., Ltd.) according to the manufacturer's instructions. The resulting cDNA was then analyzed by real-time quantitative PCR using Taq Pro Universal SYBR qPCR Master Mix (Vazyme) with a Thermo Scientific Varioskan LUX. Two [units / items / etc.] were used. -ΔΔCT The expression levels of relevant sncRNAs were calculated, with U6 as an internal reference gene. The reverse primers for U6 and sncRNAs were universal reverse primers from the miRNA first-strand cDNA synthesis kit (tailing method) (Sangon Biotech (Shanghai) Co., Ltd.). The gene-specific forward primers for sncRNAs were synthesized by Shanghai Bioengineering Co., Ltd., and their sequences are shown in the table below.

[0073]

[0074] 1.5 Statistical Analysis using Statis 4.5

[0075] Statistical analysis was performed using GraphPad Prism 9 and SPSS 26.0. Normally distributed data were expressed as mean ± standard deviation (mean ± SD), and independent samples t-tests were used for comparisons between groups. Non-normally distributed data were expressed as median and interquartile range [M(Q1,Q3)], and Mann-Whitney U tests were used for comparisons between groups. Fisher's exact test was used for comparisons of unordered categorical data among groups. A p-value < 0.05 was considered statistically significant.

[0076] 2. Test Results

[0077] 2.1 Differential expression of tsRNAs in POAG patients

[0078] Differential expression of small non-coding RNAs (sncRNAs) was screened based on thresholds of |log2FC|≥1 and p<0.05, identifying piRNAs and miRNAs with high expression levels (Tables 2 and 3). Heatmaps show significant differential expression of piRNAs and miRNAs between POAG and the control group. Figure 1 ).

[0079] Table 2 Differentially expressed piRNA sequences

[0080]

[0081]

[0082] Table 3 Differentially expressed miRNA sequences

[0083]

[0084] 2.2 DE sncRNA functional enrichment analysis

[0085] To elucidate the biological functions of DE sncRNA target genes, this study performed GO and KEGG pathway enrichment analyses. The results showed that the target genes of piRNAs and miRNAs were significantly enriched in biological processes (BP), cellular components (CC), and molecular functions (MF). Specifically, the target genes of piRNAs were associated with extracellular matrix organization (BP), collagen trimer (CC), and antigen binding (MF). Figure 2 A) miRNA target genes are mainly associated with cellular responses to insulin stimulation (BP), proteasome cofactor complex (CC), and pseudouracil synthase activity (MF). Figure 3 A). Furthermore, KEGG pathway analysis revealed that the enriched pathways primarily included propionic acid metabolism, valine, leucine, and isoleucine biosynthesis, and protein digestion and absorption pathways. Figure 2 (B and 3B).

[0086] 2.3 qRT-PCR Validation of DE sncRNAs in POAG

[0087] To validate the aforementioned experimental results, differentially expressed piRNAs and miRNAs were screened based on thresholds of |log2FC|≥1 and p<0.05. Two differentially expressed piRNAs and two differentially expressed miRNAs were selected from the sequences with high expression levels for qRT-PCR analysis. The validation set included 30 controls and 30 patients with poxonitis-associated acute exacerbation (POAG). qRT-PCR results showed that piR-hsa-767596 (p<0.05), piR-hsa-731834 (p<0.01), and hsa-miR-451a (p<0.05) expression was downregulated, consistent with sequencing results, while hsa-miR-142-5p (p=0.3824) expression showed no significant change between the two groups. Figure 4 ).

[0088] 2.4 ROC curve analysis for diagnostic efficacy evaluation

[0089] To further evaluate the potential of these sncRNAs as diagnostic markers for POAG, receiver operating characteristic (ROC) curves were plotted, and the area under the curve (AUC) was calculated. The results showed that the AUC values ​​of piR-hsa-767596, piR-hsa-731834, and hsa-miR-451a were 0.657, 0.736, and 0.696, respectively, indicating good diagnostic performance. Figure 5 A). Furthermore, combined analysis showed improved diagnostic accuracy, particularly the combination of piR-hsa-731834 and hsa-miR-451a, which increased the AUC to 0.814, indicating their potential as diagnostic biomarkers for POAG. Figure 5 B).

[0090] Although specific embodiments of the invention have been described, those skilled in the art will recognize that various changes and modifications can be made to the invention without departing from its scope or spirit. Therefore, the invention is intended to cover all such changes and modifications falling within the scope of the appended claims and their equivalents.

Claims

1. The application of a reagent for detecting the expression levels of the combination of hsa-miR-451a and piR-hsa-731834 in the preparation of a kit for early diagnosis of glaucoma; wherein the glaucoma is primary open-angle glaucoma.

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