KR102692739B1 - miRNA biomarker for diagnosis of normal tension glaucoma and uses thereof - Google Patents

Abstract

The present invention relates to a miRNA biomarker for diagnosing normal-tension glaucoma and its use. In the normal-tension glaucoma group compared to the normal group, the expression level of 12 types of miRNAs of the present invention is up- or down-regulated by more than 2-fold, so it can be used to diagnose normal-tension glaucoma. It can be used effectively.

Description

miRNA biomarker for diagnosis of normal tension glaucoma and uses thereof {miRNA biomarker for diagnosis of normal tension glaucoma and uses thereof}

The present invention relates to a miRNA biomarker for diagnosing normal tension glaucoma and its use.

Glaucoma is one of the world's three major blindness diseases, along with macular degeneration and diabetic retinopathy, and is the second most important disease causing irreversible blindness. According to the National Health Insurance Corporation, 440,000 patients received treatment for glaucoma in 2010. It increased by 73.1% (324,000 people) over 5 years from 4,000 to 768,000 in 2015. The total medical expenses for glaucoma patients increased from KRW 87.7 billion in 2010 to KRW 171.7 billion in 2015.

Among primary open-angle glaucoma, the prevalence of normal tension glaucoma (NTG), in which the intraocular pressure is not as high, is higher in Koreans and other Asians compared to other races, which appears to be a distinct epidemiological characteristic among races. In an epidemiological survey in Korea, normal tension glaucoma accounts for the majority (77%) among primary open-angle glaucoma, and in an epidemiological survey of all Asians, the average rate of normal tension glaucoma accounts for the majority at 76.3%.

Although the intraocular pressure is not high in normal tension glaucoma, the cause of glaucoma is not yet well known, and it is also unclear why it is more prevalent in Asians in particular. Glaucoma is a multifactorial disorder whose pathogenesis is not yet clearly known.

Because intraocular pressure is the most important risk factor for glaucoma and the only modifiable factor, lowering intraocular pressure is currently established as the primary treatment for glaucoma. The goal is to maintain some degree of visual function by slowing the progression of glaucoma by lowering intraocular pressure, and irreversible optic nerve damage cannot be restored. microRNAs regulate hundreds of different target mRNAs, and one target is regulated by multiple microRNAs. It is reasonable and important to study miRNAs in glaucoma, which has diverse clinical manifestations and the disease itself shows incomplete gene penetrance. Aqueous humor contains not only genes but also various bioregulatory factors and diagnostic indicators, and aqueous humor is a widely used specimen not only for anterior segment diseases, but also for vitreous body, retinal diseases, glaucoma, and optic nerve diseases at the back of the eye.

Meanwhile, Korean Patent No. 1585794 discloses a technology for the prevention or treatment of ocular diseases using miRNA, which is expressed in the aqueous humor of patients with exfoliation glaucoma or primary open-angle glaucoma. Although microRNA is disclosed in Human Molecular Genetics (2018, Vol. 27, No. 7 p1263-1275), the miRNA biomarker for diagnosing normal tension glaucoma of the present invention and its use have not yet been disclosed.

The present invention was developed in response to the above-mentioned needs. The present invention provides a miRNA biomarker for diagnosing normal tension glaucoma and its use, and the expression level of the miRNA of the present invention is more than twice that in the normal tension glaucoma group compared to the normal group. By confirming whether it was up- or down-regulated, the present invention was completed.

In order to achieve the above object, the present invention provides hsa-let-7a-5p, hsa-let-7c-5p, hsa-let-7f-5p, hsa-miR-192-5p, hsa-miR-10a-5p, hsa-miR-10b-5p, hsa-miR-375, hsa-miR-4510, hsa-miR-4639-5p, hsa-miR-6777-5p, hsa-let-7b-3p and hsa-miR-222- Provided is a biomarker composition for diagnosing normal tension glaucoma containing one or more microRNAs selected from 3p as an active ingredient.

In addition, the present invention is hsa-let-7a-5p, hsa-let-7c-5p, hsa-let-7f-5p, hsa-miR-192-5p, hsa-miR-10a-5p, hsa-miR-10b One or more selected from -5p, hsa-miR-375, hsa-miR-4510, hsa-miR-4639-5p, hsa-miR-6777-5p, hsa-let-7b-3p and hsa-miR-222-3p A composition for diagnosing normal tension glaucoma comprising an agent capable of detecting microRNA is provided.

Additionally, the present invention provides a kit for diagnosing normal tension glaucoma comprising the composition.

In addition, the present invention provides hsa-let-7a-5p, hsa-let-7c-5p, hsa-let-7f-5p, hsa-miR-192-5p, hsa-miR-10a-5p, hsa-miR-10b-5p, hsa-miR-375, hsa-miR-4510, hsa-miR-4639-5p, hsa-miR-6777-5p, hsa-let-7b-3p and hsa-miR-222- Measuring the expression level of one or more microRNAs selected from 3p; and

It provides a method of providing information for the diagnosis of normal tension glaucoma, including the step of comparing the expression level of the miRNA with the expression level of the corresponding miRNA in a normal control sample.

The present invention relates to a miRNA biomarker for diagnosing normal-tension glaucoma and its use. In the normal-tension glaucoma group compared to the normal group, the expression level of 12 types of miRNAs of the present invention is up- or down-regulated by more than 2-fold, so it can be used to diagnose normal-tension glaucoma. It can be used effectively.

Figure 1 shows the results of screening the expression level of miRNAs to select 12 types of miRNAs with large expression level changes in the normal tension glaucoma patient group among the miRNAs present in the aqueous humor.
Figure 2 shows the results confirming the increased expression level of the miRNA hsa-let-7c-5p in the normal tension glaucoma patient group (NTG) compared to the control group (CTL). *** indicates that the increase in the expression level of the miRNA hsa-let-7c-5p in the normal tension glaucoma patient group (NTG) compared to the control group (CTL) is statistically significant, p < 0.001.

The present invention is hsa-let-7a-5p, hsa-let-7c-5p, hsa-let-7f-5p, hsa-miR-192-5p, hsa-miR-10a-5p, hsa-miR-10b-5p , hsa-miR-375, hsa-miR-4510, hsa-miR-4639-5p, hsa-miR-6777-5p, hsa-let-7b-3p and hsa-miR-222-3p. Provided is a biomarker composition for diagnosing normal tension glaucoma that includes the active ingredient.

The hsa-let-7a-5p, hsa-let-7c-5p, hsa-let-7f-5p, hsa-miR-192-5p, hsa-miR-10a-5p, hsa-miR-10b-5p, hsa -miR-375, hsa-miR-4510, hsa-miR-4639-5p, hsa-miR-6777-5p, hsa-let-7b-3p and hsa-miR-222-3p are of SEQ ID NOs: 1 to 12, respectively. It is preferable that it consists of a base sequence, but it is not limited to this.

In the present invention, micro RNA (miRNA) refers to 21 to 23 non-coding RNAs that post-transcriptionally regulate gene expression by promoting degradation of target RNA or inhibiting their translation. Not only miRNAs represented by specific base sequences, but also precursors (pre-miRNAs, pri-miRNAs) of the above-mentioned miRNAs, miRNAs with equivalent biological functions, such as homologs (i.e. homologs or orthologs), variants such as genetic polymorphisms, and derivatives.

'Diagnosis' in the present invention refers to determining the susceptibility of an object, that is, a test subject, to a specific disease or condition, determining whether an object currently has a specific disease or condition, and determining whether an object currently has a specific disease or condition. A concept that includes determining the prognosis of an affected subject or therametrics (e.g., monitoring the condition of the subject to provide information about treatment efficacy).

In addition, the present invention is hsa-let-7a-5p, hsa-let-7c-5p, hsa-let-7f-5p, hsa-miR-192-5p, hsa-miR-10a-5p, hsa-miR-10b One or more selected from -5p, hsa-miR-375, hsa-miR-4510, hsa-miR-4639-5p, hsa-miR-6777-5p, hsa-let-7b-3p and hsa-miR-222-3p It relates to a composition for diagnosing normal tension glaucoma containing an agent capable of detecting microRNA.

The hsa-let-7a-5p, hsa-let-7c-5p, hsa-let-7f-5p, hsa-miR-192-5p, hsa-miR-10a-5p, hsa-miR-10b-5p, hsa -miR-375, hsa-miR-4510, hsa-miR-4639-5p, hsa-miR-6777-5p, hsa-let-7b-3p and hsa-miR-222-3p are of SEQ ID NOs: 1 to 12, respectively. It is preferable that it consists of a base sequence, but it is not limited to this.

The agent is an agent that can recognize the microRNA by binding complementary to it or amplify the microRNA, and a specific example is preferably an antisense oligonucleotide, primer, or probe that can specifically detect the miRNA. It is not limited.

The preparation may be directly or indirectly labeled to measure the expression level of the microRNA. Specifically, the label may include, but is not limited to, ligands, beads, radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent substances, chemiluminescent substances, magnetic particles, haptens, and dyes. It is not limited to this. As specific examples, the ligands include biotin, avidin, and streptoavidin, the enzymes include luciferase, peroxidase, and beta galactosidase, and the fluorescent substances include fluorescein, coumarin, rhodamine, Includes, but is not limited to, phycoerythrin and sulforodamic acid chloride (Texas red). Most of the known labels can be used as these detectable labels, and a person skilled in the art will be able to select an appropriate label to suit the purpose of the invention.

The term "marker or diagnosis marker" used in the present invention refers to a substance that can diagnose an individual with normal tension glaucoma by distinguishing it from normal cells or normal individuals. Normal tension glaucoma progresses or develops compared to normal cells. It includes organic biomolecules such as polypeptides, proteins, or nucleic acids (e.g., mRNA, etc.), lipids, glycolipids, glycoproteins, or sugars (monosaccharides, disaccharides, oligosaccharides, etc.) that show an increase or decrease in cells or organisms. For the purpose of the present invention, the normal tension glaucoma diagnostic marker of the present invention is a miRNA or a fragment of the corresponding miRNA that specifically shows a difference in expression level in cells of the normal tension glaucoma group compared to cells of normal cells or tissues.

As the term of the present invention, "primer" is a base sequence with a short free 3' terminal hydroxyl group, it can form a base pair with a complementary template and is used for copying the template strand. A short sequence that serves as a starting point. In the present invention, the primers used for amplifying the mRNA of the gene are used in an appropriate buffer under appropriate conditions (e.g., four different nucleotide triphosphates and a polymerization agent such as DNA, RNA polymerase or reverse transcriptase) and appropriate temperature conditions. It can be a single-stranded oligonucleotide that can act as a starting point for template-directed DNA synthesis, and the appropriate length of the primer may vary depending on the purpose of use. The primer sequence does not need to be completely complementary to the polynucleotide of the miRNA or its complementary polynucleotide, but can be used as long as it is sufficiently complementary to hybridize.

The term "probe" as used in the present invention refers to a nucleic acid fragment such as RNA or DNA that is as short as a few bases or as long as several hundreds of bases and can form a specific binding to a gene or mRNA, and is called an oligonucleotide. It can be manufactured in the form of a probe, single stranded DNA probe, double stranded DNA probe, RNA probe, etc., and can be labeled for easier detection. Primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method or other well-known methods. These nucleic acid sequences can be modified using many means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution of a native nucleotide with one or more homologues, and modifications between nucleotides, such as uncharged linkages (e.g., methyl phosphonate, phosphotriester, phosphoronucleotide). amidates, carbamates, etc.) or charged linkages (e.g. phosphorothioate, phosphorodithioate, etc.).

Considering mutations with biologically equivalent activity, the base sequence used in the present invention is interpreted to include sequences showing substantial identity with the sequences listed in the sequence list. The term 'substantial identity' means that when the sequence of the present invention and any other sequence are aligned to the maximum extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art, the minimum It refers to a sequence that exhibits 60% homology, more specifically 70% homology, even more specifically 80% homology, and most specifically 90% homology. Therefore, a base sequence having high homology to the base sequences represented by SEQ ID NOs: 1 to 12, for example, having a homology of 70% or more, specifically 80% or more, and more specifically 90% or more. Base sequences should also be interpreted as being included within the scope of the present invention.

Additionally, the present invention relates to a kit for diagnosing normal tension glaucoma containing the composition of the present invention.

The kit of the present invention may be for RT-PCR, real-time PCR, isothermal PCR, Northern blotting, RNA protection assay, or microarray chip, but is not limited thereto.

The kit of the present invention uses the composition for diagnosing normal tension glaucoma as hsa-let-7a-5p, hsa-let-7c-5p, hsa-let-7f-5p, hsa-miR-192-5p, hsa- miR-10a-5p, hsa-miR-10b-5p, hsa-miR-375, hsa-miR-4510, hsa-miR-4639-5p, hsa-miR-6777-5p, hsa-let-7b-3p and Normal tension glaucoma can be diagnosed by measuring the expression level of one or more microRNAs selected from hsa-miR-222-3p. Specifically, the kit may be an RT-PCR kit, but is not limited thereto. As a specific example, the kit may be a kit containing essential elements required to perform RT-PCR. For example, the RT-PCR kit is hsa-let-7a-5p, hsa-let-7c-5p, hsa-let-7f-5p, hsa-miR-192-5p, hsa-miR-10a-5p , hsa-miR-10b-5p, hsa-miR-375, hsa-miR-4510, hsa-miR-4639-5p, hsa-miR-6777-5p, hsa-let-7b-3p and hsa-miR-222 In addition to each primer specific for one or more microRNAs selected from -3p, a test tube or other suitable container, reaction buffer (pH and magnesium concentration are varied), deoxynucleotides (dNTPs), dideoxynucleotides (ddNTPs), Taq-polymer. It may contain enzymes such as enzymes such as enzymes and reverse transcriptases, DNase, RNAse inhibitors, DEPC-water, sterilized water, etc., and may contain primer pairs specific for DNA, RNA or miRNA used as quantitative controls. You can.

In addition, the present invention provides hsa-let-7a-5p, hsa-let-7c-5p, hsa-let-7f-5p, hsa-miR-192-5p, hsa-miR-10a-5p, hsa-miR-10b-5p, hsa-miR-375, hsa-miR-4510, hsa-miR-4639-5p, hsa-miR-6777-5p, hsa-let-7b-3p and hsa-miR-222- Measuring the expression level of one or more microRNAs selected from 3p; and

It relates to a method of providing information for the diagnosis of normal tension glaucoma, including comparing the expression level of the miRNA with the expression level of the corresponding miRNA in a normal control sample.

The expression level was determined by next generation sequencing (NGS), polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (Real-time PCR), and RNase protection assay. It is preferable to measure using one or more methods selected from (RNase protection assay; RPA), microarray, and northern blotting, but is not limited thereto.

Hereinafter, the present invention will be described in more detail using examples. These examples are only for illustrating the present invention in more detail, and it is obvious to those skilled in the art that the scope of the present invention is not limited thereto.

1. Adoption of patient and aqueous humor samples

The aqueous humor sample used in the present invention was collected from patients who had undergone cataract surgery and had no problems after obtaining prior consent.

Six patients with normal tension glaucoma (NTG) were managed stably using only topical medications, and seven age-matched control subjects who agreed to participate in the present invention were adopted.

Before cataract surgery, approximately 80 to 120 μl of aqueous humor was obtained using a 30-gauge needle. The process of acquiring aqueous humor was performed in all subjects without trauma, thus ruling out the possibility of contamination with cell remnants or blood. All collected samples (aqueous humor) were immediately transferred to liquid nitrogen and stored in a rapidly frozen state.

Clinical data were retrieved from electronic medical records and collected in a completely anonymized manner. Clinical data collected included age, gender, eye orientation, baseline IOP, topical eye drops used, and ocular comorbidities (Table 1).

The clinical trial conducted in the present invention was conducted in accordance with the principles of the Declaration of Helsinki, was approved by the institutional review board of Gyeongsang National University Changwon Hospital College of Medicine (GNUCH-2019-06-001-002), and received prior approval from all subjects registered in the present invention. A consent form was received. All methods were performed in accordance with relevant guidelines and regulations.

Patient and control information # group age gender Eye Laterality Baseline IOP,
mmHg Topical Medication One NTG 56 F Left 13 Latanoprost 2 NTG 57 M Left 13 Latanoprost 3 NTG 55 M Left 17 Dorzolamide/timolol 4 NTG 77 F Right 14 Dorzolamide/timolol 5 NTG 72 F Right 15 Latanoprost 6 NTG 76 F Right 17 Tafluprost 7 NTG 76 F Right 14 Latanoprost 8 NTG 74 F Left 12 Dorzolamide/timolol 9 Control 75 F Left 14 None 10 Control 57 F Right 14 None 11 Control 76 F Right 15 None 12 Control 54 M Left 18 None 13 Control 53 M Right 16 None 14 Control 71 F Right 17 None 15 Control 61 M Right 19 None

IOP; Intraocular pressure, NTG; Normal tension glaucoma, Phaco+PCL; Phacoemulsification and posterior intraocular lens insertion

2. RNA isolation

Total RNA was extracted using Trizol LS reagent (Invitrogen, USA) according to the manufacturer's instructions. The quality of RNA was assessed with an Agilent 2100 bioanalyzer using an RNA 6000 Pico Chip (Agilent Technologies, Netherlands), and quantification of RNA was performed using a NanoDrop 2000 spectrophotometer system (Thermo Fisher Scientific, USA).

3. Library preparation and RNA sequencing

Libraries were prepared from RNA from the control and patient groups using the NEBNext Multiplex Small RNA Library Prep kit (New England BioLabs, Inc., Ipswich, MA, USA) according to the manufacturer's instructions.

180 pg of total RNA from each sample was used to ligate 1 μg of adapter, and then cDNA was synthesized using specific primers and reverse transcriptase for each adapter. To amplify the library, expression patterns of microRNA were analyzed in aqueous humor collected from normal tension glaucoma patients and controls using RNA sequencing, a Next Generation Sequencing (NGS) technique.

As a result, as shown in Figure 1 and Table 2, among the miRNAs present in the aqueous humor, 12 types of miRNAs with a large change in expression level in the normal tension glaucoma patient group compared to the control group were selected, and the expression levels of the 12 selected types of miRNAs were as follows. As shown in Table 2, it was finally confirmed that the expression level increased or decreased by more than twofold in the normal tension glaucoma patient group compared to the control group.

miRNAs whose expression levels increased or decreased in patients with normal tension glaucoma compared to the normal group miRNA Sequence (5'->3') order
number Accession Number Fold change (Log2) p-value manifestation
change hsa-let-7a-5p UGAGGUAGGUAGGUUGUAUAGUU One MIMAT0000062 2.744 0.009 up hsa-let-7c-5p UGAGGUAGGUAGGUUGUAUGGUU 2 MIMAT0000064 6.081 0.018 up hsa-let-7f-5p UGAGGUAGUAGAUUGUAUAGUU 3 MIMAT0000067 4.570 0.044 up hsa-miR-192-5p CUGACCUAUGAAUUGACAGCC 4 MIMAT0000222 6.564 0.031 up hsa-miR-10a-5p UACCCUGUAGAUCCGAAUUUGUG 5 MIMAT0000253 21.733 0.015 up hsa-miR-10b-5p UACCCUGUAGAACCGAAUUUGUG 6 MIMAT0000254 201.495 0.033 up hsa-miR-375 CCCCGCGACGAGCCCCUCGCACAAACCGGACCUGAGCGUUUUGUUCGUUCGGCUCGCGUGAGGC 7 MI0000783 234.233 0.005 up hsa-miR-4510 GUGUAUGUGAGGGAGUAGGAUGUAUGGUUGUUAGAUAGACAACUACAAUCUUUUCUCACAACAGACAG 8 MI0016876 3.083 0.012 up hsa-miR-4639-5p UUGCUAAGUAGGCUGAGAUUGA 9 MIMAT0019697 0.161 0.040 down hsa-miR-6777-5p ACGGGGAGUCAGGCAGUGGUGGA 10 MIMAT0027454 0.208 0.045 down hsa-let-7b-3p CUAUACAACCUACUGCCUUCCC 11 MIMAT0004482 2.522 0.043 up hsa-miR-222-3p AGCUACAUCUGGCUACUGGGGU 12 MIMAT0000279 12.412 0.049 up

4. Quantitative real-time PCR analysis

cDNA synthesis and real-time PCR analysis were performed on a miScript PCR system. cDNA was synthesized from 357 pg of RNA using the miScript II RT Kit with HiSpec buffer according to the manufacturer's instructions.

As an example, amplification was performed using the primer pair hsa-let-7c-5p (5'-UGAGGUAGUAGGUUGUAUGGUU-3', MS00003129) and the internal control hsa-U6 (Hs_RNU6-2_11, MS00033740), and real-time PCR was performed according to the manufacturer's instructions. was performed on a StepOnePlus real-time PCR system (Foster City, CA, USA) using QuantiTect SYBR Green PCR Master mix and MiScript primer assays. Conditions were 95°C for 15 minutes, 94°C for 15 seconds, 55°C for 30 seconds, and 70°C for 30 seconds for 40 cycles. Data analysis was performed using StepOne software v2.2.2 (Applied Biosystems). The expression level of each microRNA was normalized to the median Ct value and calculated using the 2- ^ΔΔCT method.

Through this analysis, the miRNAs listed in Table 3 were selected based on the expression level changes and p values of the final selected miRNAs.

As shown in Figure 2, the expression level of miRNA (hsa-let-7c-5p) in the normal tension glaucoma patient group compared to the control group (CTL) was statistically significantly increased.

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Claims (9)

hsa-let-7a-5p, hsa-let-7c-5p, hsa-let-7f-5p, hsa-miR-192-5p, hsa-miR-10a-5p, hsa-miR-10b-5p, hsa- One or more microRNAs selected from among miR-375, hsa-miR-4510, hsa-miR-4639-5p, hsa-miR-6777-5p, hsa-let-7b-3p, and hsa-miR-222-3p as an active ingredient. A biomarker composition for diagnosing normal tension glaucoma, comprising: The method of claim 1, wherein the hsa-let-7a-5p, hsa-let-7c-5p, hsa-let-7f-5p, hsa-miR-192-5p, hsa-miR-10a-5p, hsa-miR -10b-5p, hsa-miR-375, hsa-miR-4510, hsa-miR-4639-5p, hsa-miR-6777-5p, hsa-let-7b-3p and hsa-miR-222-3p, respectively. A biomarker composition for diagnosing normal tension glaucoma, characterized in that it consists of the base sequences of SEQ ID NOs: 1 to 12. hsa-let-7a-5p, hsa-let-7c-5p, hsa-let-7f-5p, hsa-miR-192-5p, hsa-miR-10a-5p, hsa-miR-10b-5p, hsa- Can detect one or more microRNAs selected from miR-375, hsa-miR-4510, hsa-miR-4639-5p, hsa-miR-6777-5p, hsa-let-7b-3p and hsa-miR-222-3p A composition for diagnosing normal tension glaucoma comprising an agent. The method of claim 3, wherein the hsa-let-7a-5p, hsa-let-7c-5p, hsa-let-7f-5p, hsa-miR-192-5p, hsa-miR-10a-5p, hsa-miR -10b-5p, hsa-miR-375, hsa-miR-4510, hsa-miR-4639-5p, hsa-miR-6777-5p, hsa-let-7b-3p and hsa-miR-222-3p, respectively. A composition for diagnosing normal tension glaucoma, characterized in that it consists of the base sequences of SEQ ID NOs: 1 to 12. The composition for diagnosing normal tension glaucoma according to claim 3, wherein the agent is an antisense oligonucleotide, primer, or probe that binds complementary to the microRNA. A kit for diagnosing normal tension glaucoma, comprising the composition of any one of claims 3 to 5. The kit for diagnosing normal tension glaucoma according to claim 6, wherein the kit is for RT-PCR, real-time PCR, isothermal PCR, Northern blotting, RNA protection assay, or microarray chip. In whole blood or serum, hsa-let-7a-5p, hsa-let-7c-5p, hsa-let-7f-5p, hsa-miR-192-5p, hsa-miR-10a-5p, hsa-miR-10b One or more selected from -5p, hsa-miR-375, hsa-miR-4510, hsa-miR-4639-5p, hsa-miR-6777-5p, hsa-let-7b-3p and hsa-miR-222-3p Measuring the expression level of microRNA; and
A method of providing information for the diagnosis of normal tension glaucoma, comprising: comparing the expression level of the miRNA with the expression level of the corresponding miRNA in a normal control sample. A method for providing information for diagnosing normal tension glaucoma, characterized in that in claim 8, the expression level is measured by at least one method selected from next generation sequencing (NGS), polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (real-time PCR), RNase protection assay (RPA), microarray, and northern blotting.