Application of tsRNA as a molecular marker for diagnosing primary open-angle glaucoma

Abstract

The present application relates to a kind of molecular markers for early diagnosis of glaucoma, the molecular marker is tsRNA, the tsRNA is selected from one or more of tsRNA-5009b-ValCAC and tsRNA-5003c-GlyGCC.The present application finds that relative to healthy control group, the expression of tsRNA-5009b-ValCAC in POAG group patient is significantly down-regulated, its AUC value in ROC curve is 0.717, and diagnosis performance is good, indicating that the tsRNAs can be used as early diagnosis of POAG molecular marker and applied to clinical.

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] Glaucoma is an eye disease characterized by progressive degeneration of retinal ganglion cells (RGCs) and is a leading cause of irreversible vision loss worldwide. According to the World Health Organization (WHO), the global prevalence of glaucoma exceeds 80 million cases. This number is projected to increase to 110 million by 2040, with a significant impact on the aging population. Among the various types of glaucoma, primary open-angle glaucoma (POAG) is the most common, accounting for more than 70% of all cases.

[0003] 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. POAG has a high incidence rate, which gradually increases with age. Due to its insidious onset, POAG is difficult to diagnose; current ophthalmic imaging techniques typically only detect the disease after significant loss of retinal ganglion cells (RGCs).

[0004] Recent advances in high-throughput sequencing have significantly expanded the scope of small non-coding RNAs (sncRNAs), including microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), small interfering RNAs (siRNAs), and trna-derived small RNAs (tsRNAs). SncRNAs, defined as non-coding RNAs shorter than 200 nucleotides, are increasingly recognized for their crucial roles in gene regulation and disease pathogenesis. Emerging evidence suggests that dysregulation of sncRNAs plays a key role in a variety of diseases, including cancer, cardiovascular disease, immune dysfunction, and neurodegenerative diseases. Newly emerging dual markers in liquid biopsies, particularly miRNAs (such as miR-143-3p and miR-27a-3p), have demonstrated potential for glaucoma diagnosis. However, the diagnostic functions of other types of sncRNAs, including piRNAs and tsRNAs, remain largely unexplored.

[0005] tsRNAs have emerged as a novel regulatory molecule with potential links to neurodegenerative diseases. tRF-5, derived from the precise 5' cleavage of mature tRNA, has been reported as a key regulator of various biological processes, including protein translation, cellular metabolism, and epigenetic regulation. tRF-5 (such as tRF5-GlyGCC and tRF5-GluCTC) have been reported to be upregulated in Alzheimer's disease, and NSun2 deficiency induces the accumulation of 5'-tRF in neurons, triggering stress responses and apoptosis, highlighting their potential neurobiological relevance.

[0006] Currently, there are no reports of tsRNA being used as a molecular marker for diagnosing primary open-angle glaucoma. Summary of the Invention

[0007] To solve the above-mentioned technical problems, the present invention includes the following aspects:

[0008] A first aspect of the present invention provides a molecular marker for the early diagnosis of glaucoma, said molecular marker being tsRNA.

[0009] Preferably, the tsRNA is selected from one or more of tsRNA-5009b-ValCAC (SEQ ID NO.3), tsRNA-5016c-CysGCA (SEQ ID NO.4), tsRNA-5003c-GlyGCC (SEQ ID NO.5), tsRNA-3017a-ValTAC (SEQ ID NO.6), tsRNA-5026b-ValAAC (SEQ ID NO.7), tsRNA-5023b-LeuCAG (SEQ ID NO.8), tsRNA-5009c-ValCAC (SEQ ID NO.9), tsRNA-5030c-GluCTC (SEQ ID NO.10), and tsRNA-5003c-GlyGCC (SEQ ID NO.11).

[0010] Preferably, the tsRNA is selected from one or more of tsRNA-5009b-ValCAC (SEQ ID NO.3) and tsRNA-5003c-GlyGCC (SEQ ID NO.11).

[0011] More preferably, the tsRNA is tsRNA-5009b-ValCAC (SEQ ID NO.3).

[0012] Preferably, the glaucoma is primary open-angle glaucoma.

[0013] 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 tsRNA.

[0014] Preferably, the tsRNA is selected from one or more of tsRNA-5009b-ValCAC (SEQ ID NO.3), tsRNA-5016c-CysGCA (SEQ ID NO.4), tsRNA-5003c-GlyGCC (SEQ ID NO.5), tsRNA-3017a-ValTAC (SEQ ID NO.6), tsRNA-5026b-ValAAC (SEQ ID NO.7), tsRNA-5023b-LeuCAG (SEQ ID NO.8), tsRNA-5009c-ValCAC (SEQ ID NO.9), tsRNA-5030c-GluCTC (SEQ ID NO.10), and tsRNA-5003c-GlyGCC (SEQ ID NO.11).

[0015] Preferably, the tsRNA is selected from one or more of tsRNA-5009b-ValCAC (SEQ ID NO.3) and tsRNA-5003c-GlyGCC (SEQ ID NO.11).

[0016] More preferably, the tsRNA is tsRNA-5009b-ValCAC (SEQ ID NO.3).

[0017] Preferably, the glaucoma is primary open-angle glaucoma.

[0018] Preferably, the reagent includes a tsRNA gene-specific forward primer and a reverse primer. The sequence of the forward primer is shown in SEQ ID NO.1, and 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.

[0019] Preferably, the reagent includes a tsRNA 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 the miRNA first-strand cDNA synthesis kit (tailing method), which was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0020] 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.

[0021] Preferably, the subject is a mammal. More preferably, the subject is a human.

[0022] A third aspect of the present invention provides the use of a reagent for detecting tsRNA in the preparation of a kit for the early diagnosis of glaucoma.

[0023] Preferably, the tsRNA is selected from one or more of tsRNA-5009b-ValCAC (SEQ ID NO.3), tsRNA-5016c-CysGCA (SEQ ID NO.4), tsRNA-5003c-GlyGCC (SEQ ID NO.5), tsRNA-3017a-ValTAC (SEQ ID NO.6), tsRNA-5026b-ValAAC (SEQ ID NO.7), tsRNA-5023b-LeuCAG (SEQ ID NO.8), tsRNA-5009c-ValCAC (SEQ ID NO.9), tsRNA-5030c-GluCTC (SEQ ID NO.10), and tsRNA-5003c-GlyGCC (SEQ ID NO.11).

[0024] Preferably, the tsRNA is selected from one or more of tsRNA-5009b-ValCAC (SEQ ID NO.3) and tsRNA-5003c-GlyGCC (SEQ ID NO.11).

[0025] More preferably, the tsRNA is tsRNA-5009b-ValCAC (SEQ ID NO.3).

[0026] Preferably, the glaucoma is primary open-angle glaucoma.

[0027] Preferably, the reagent for detecting tsRNA includes a tsRNA gene-specific forward primer and a reverse primer. The sequence of the forward primer is shown in SEQ ID NO.1, and 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.

[0028] Preferably, the reagent for detecting tsRNA includes a tsRNA gene-specific forward primer and a reverse primer. The sequence of the forward primer is shown in SEQ ID NO.2, and 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] The technical effects of this invention are as follows:

[0030] This invention uses Pandora sequencing to systematically analyze the expression profiles of tsRNAs in PBMCs of POAG patients and healthy controls, revealing significant changes in multiple tsRNAs in POAG patients compared to healthy controls. Further qRT-PCR analysis of two DE tsRNAs in the validation set showed downregulated expression of tsRNA-5009b-ValCAC in POAG patients (p<0.05). The AUC value of tsRNA-5009b-ValCAC in the receiver operating characteristic (ROC) curve was 0.717, indicating good diagnostic performance and suggesting that this tsRNA can be used clinically as a molecular marker for the early diagnosis of POAG. Attached Figure Description

[0031] Figure 1 Heatmap of differentially expressed tsRNAs in POAG;

[0032] Figure 2 Functional enrichment analysis of target genes associated with DE sncRNAs in POAG, including 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 tsRNAs. Figure 2 B's KEGG bar plot shows the top 10 enriched target gene pathways associated with differentially expressed tsRNAs;

[0033] Figure 3 qRT-PCR validation results for selected snncRNAs in healthy controls and POAG patients, among which Figure 3 A represents the expression level of tsRNA-5009b-ValCAC. Figure 3 B represents the expression level of tsRNA-5003c-GlyGCC (data are expressed as mean ± SEM, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001).

[0034] Figure 4 ROC curve analysis of tsRNA-5009b-ValCAC for diagnosing POAG. Detailed Implementation

[0035] 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.

[0036] Experimental Example 1: Study on tsRNAs biomarkers for early diagnosis of primary open-angle glaucoma patients

[0037] 1. Test Methods

[0038] 1.1 Patients and Samples

[0039] 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.

[0040] 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.

[0041] Table 1. Baseline demographic and clinical characteristics of POAG patients and healthy controls in the validation cohort.

[0042]

[0043] Note: 1 Pearson's χ² test; 2 Independent samples t-test; 3 Mann-Whitney U test, after continuity correction; 4Independent 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.

[0044] 1.2 PBMC Cell Isolation

[0045] 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.

[0046] 1.3 Pandora Sequencing and Data Analysis

[0047] PANDORA-seq (panoramic RNA display 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. The tsRNA database tRFdb was used as the reference database.

[0048] RNA expression levels in the samples were normalized using TPM. Differentially expressed genes were then 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 microRNA regulatory targets. Further analysis was conducted to determine whether target genes were enriched in the KEGG and GO pathways.

[0049] 1.4 sncRNA extraction and reverse transcription real-time PCR (qRT-PCR)

[0050] 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.

[0051]

[0052] 1.5 Statistical Analysis using Statis 4.5

[0053] 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.

[0054] 2. Test Results

[0055] 2.1 Differential expression of tsRNAs in POAG patients

[0056] Differential expression of small non-coding RNAs (sncRNAs) was screened based on thresholds of |log2FC|≥1 and p<0.05, identifying nine tsRNAs with high expression levels (Table 2). A heatmap of differentially expressed tsRNAs showed significant clustering between POAG and control samples. Figure 1 ).

[0057] Table 2 Differentially expressed tsRNA sequences

[0058]

[0059] 2.2 DE sncRNA functional enrichment analysis

[0060] To elucidate the biological functions of DE sncRNA target genes, this study performed GO and KEGG pathway enrichment analyses. The results showed that tsRNA target genes were significantly enriched in biological processes (BP), cellular components (CC), and molecular functions (MF). Specifically, tsRNA target genes were enriched in magnesium ion homeostasis (BP), hemoglobin complex (CC), and internal deoxyribonuclease activity (MF). Figure 2 A). Furthermore, KEGG pathway analysis revealed that the enriched pathways primarily included the propionic acid metabolic pathway, the glycine, serine, and threonine metabolic pathways, and the biosynthetic pathways of valine, leucine, and isoleucine. Figure 2 B).

[0061] 2.3 qRT-PCR Validation of DE sncRNAs in POAG

[0062] To validate the results of the aforementioned screening experiment, differentially expressed tsRNAs were screened based on thresholds of |log2FC|≥1 and p<0.05. Two tsRNAs (tsRNA-5009b-ValCAC and tsRNA-5003c-GlyGCC) were selected from the high-expression tsRNA sequences and subjected to qRT-PCR analysis in the validation set, which included 30 controls and 30 patients with porcine angina pectoris (POAG). The qRT-PCR results showed that tsRNA-5009b-ValCAC expression was downregulated in the POAG group (p<0.05), consistent with the sequencing results. Figure 3 A), while tsRNA-5003c-GlyGCC (p=0.3611) expression showed no significant change between the two groups. Figure 3 B).

[0063] 2.4 ROC curve analysis for diagnostic efficacy evaluation

[0064] 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 tsRNA-5009b-ValCAC had an AUC of 0.717, indicating good diagnostic performance. Figure 4 ).

[0065] 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 tsRNA in the preparation of a kit for diagnosing glaucoma, characterized in that, The tsRNA is tsRNA-5009b-ValCAC, the glaucoma is primary open-angle glaucoma, and the nucleotide sequence of the tsRNA-5009b-ValCAC is shown in SEQ ID NO. 3.

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