Exosome-derived microRNA biomarkers and their use for the diagnosis of premature ovarian insufficiency (POI)

Abstract

Use of the biomarkers of the present invention enables early diagnosis of premature ovarian failure, enables diagnosis that distinguishes premature ovarian failure from premature ovarian failure caused by Turner syndrome, and provides a method for screening therapeutic agents for premature ovarian failure through changes in expression of the biomarkers.

Description

[Technical field]

[0001] 2. Background of the Invention 1. Field of the invention The present invention relates to exosome-derived microRNA biomarkers and uses thereof for the diagnosis of premature ovarian insufficiency (POI). [Background technology]

[0002] 2. Description of Related Art The ovaries are the female reproductive organs that mature eggs and secrete sex hormones such as estrogen, progesterone, and testosterone. They maintain the balance of the hormone production system, which plays an important role in maintaining the health of the female reproductive system and maintaining the quality of life of women. Ovarian function can be confirmed by measuring the changes in the levels of follicle-stimulating hormone (FSH), estradiol, or anti-Mullerian hormone (AMH), and reduced ovarian function can lead to hormone imbalance, premature ovarian failure, polycystic ovarian syndrome, and infertility.

[0003] Among these, premature ovarian failure (POI) is a pathological condition in which ovarian function is exhausted before the age of 40, and manifests itself through symptoms of amenorrhea or oligomenorrhea, decreased estrogen, and elevated serum follicle-stimulating hormone (FSH) levels. More specifically, premature ovarian failure is diagnosed when a woman has had no menstruation for more than 6 months before the age of 40 and has a blood level of follicle-stimulating hormone (FSH) greater than 40 mIU / mL measured twice, at least one month apart.

[0004] The prevalence of premature ovarian failure is approximately 1%, and patients with premature ovarian failure generally present with symptoms seen in menopausal women. Premature ovarian failure increases the likelihood of hot flashes, sweating, sleep disorders, and chronic urogenital atrophy due to estrogen deficiency, osteoporosis, coronary artery disease, and Alzheimer's disease. These associated symptoms reduce the quality of life of patients, and survival rates decrease due to increased incidence of other diseases and complications.

[0005] The causes of premature ovarian failure are known to be mainly due to exogenous factors, karyotypic abnormalities, and genetic factors, but in most cases, the cause is unknown (Shelling An, 2010). Some cases of premature ovarian failure may be due to autoimmune diseases or genetic disorders such as fragile X syndrome and Turner syndrome. Although many studies have been conducted on mutations in various genes that affect the ovarian maturation process to determine the cause of premature ovarian failure, the exact mechanism of premature ovarian failure has yet to be revealed. Therefore, the development of premature ovarian failure is a highly heterogeneous and unknown phenomenon. In actual clinical trials, the cause of premature ovarian failure is unknown in approximately 90% of cases.

[0006] Once premature ovarian failure is diagnosed, the ovaries rarely respond to attempts to overstimulate them, and the chances of a live birth are extremely low. Therefore, it is important to have a test that can screen prospective patients who will be diagnosed with premature ovarian failure in advance, but no specific clinical symptoms have been reported that precede premature ovarian failure. Therefore, unless there is a specific medical history (chemotherapy and radiation therapy, ovarian cystectomy, etc.), it is difficult to predict the likelihood of developing premature ovarian failure.

[0007] Therefore, in order to diagnose premature ovarian failure early and find its etiology, various tests are performed, such as chromosome tests, Glazed X syndrome carrier tests, thyroid disease, pituitary disease, autoimmune disease, previous diagnosis and surgery. However, there is very little information on the treatment and prognosis of patients according to the cause, and none of them are used as screening tests for early diagnosis. In addition, most of the current treatment methods for patients with premature ovarian failure are treatment and management after diagnosis. In patients with premature ovarian failure, pregnancy attempts are performed through in vitro activation (IVA), in which whole ovaries or ovarian tissues / follicles are harvested and reimplanted after in vitro culture, but this method is known to have poor results in patients whose ovaries have already been irreparably damaged. Therefore, in reality, there are few tests that can predict abnormal decline in ovarian function in advance and recommend advance preparation.

[0008] It is recommended to determine whether there is a high probability of developing premature ovarian failure and to freeze eggs or try to become pregnant before premature ovarian failure occurs. However, there is currently no clear marker or test method to detect premature ovarian failure at an early stage, which makes early diagnosis extremely difficult.

[0009] Meanwhile, Turner syndrome refers to the loss or partial deletion of one of two X chromosomes in women, or structural defects in X chromosome, resulting in ovarian dysfunction.50% of Turner syndrome patients have karyotype 45X, missing one of two X chromosomes, and the rest have mosaic or partial deletion karyotype.Therefore, karyotype testing is used to analyze Turner syndrome.

[0010] Turner syndrome is known to be one of the genetic causes of premature ovarian failure, therefore, it is necessary to find diagnostic markers that can distinguish premature ovarian failure caused by Turner syndrome from premature ovarian failure caused by other causes.

[0011] Exosomes are tiny vesicles that contain material that carries cellular RNA and proteins. Exosomes are membrane-structured vesicles secreted from various types of cells and are known to play various roles, including binding to other cells and tissues and delivering membrane components, proteins, and RNA. Originally, exosomes were discovered during the final stage of red blood cell maturation, when intracellular proteins are released and removed, leaving only hemoglobin in red blood cells. In addition, in electron microscopy studies, it was observed that exosomes are not directly separated from the cell membrane, but rather originate from a specific intracellular compartment called multivesicular body (MVB) and are released and secreted outside the cell. When multivesicular body fuses with the plasma membrane, the vesicle is released into the extracellular environment, called exosome.

[0012] In recent years, exosomes have received a great deal of attention as markers for disease diagnosis and as intercellular signaling carriers. Exosome isolation and analysis methods are one of the liquid biopsy methods selected by MIT as the top 10 promising future technologies in 2015. Liquid biopsy is recognized as a next-generation treatment strategy because it allows detailed observation of the development and spread of diseases simply by non-invasively examining blood or body fluids. In addition, exosomes are known to be secreted from various cells and circulate through plasma, cerebrospinal fluid, and urine. Exosomes are small and stable endoplasmic reticulum (30-200 nm) structures wrapped in a phospholipid bilayer and contain the same genetic and protein information as the parent cell. Notably, exosomes contain microRNAs (miRNAs), which can be used as useful markers for molecular diagnostics such as early diagnosis of cancer. Exosomes also play an important role in signaling between cells. Therefore, exosomes may act as carriers of factors that cause premature ovarian failure and may modulate the growth and differentiation processes of surrounding cells by entering not only granulosa cells but also other surrounding cells, such as endometrial stromal cells.

[0013] In biology, the role of miRNAs is an area of ​​intense interest, as they have been extensively studied in determining changes in protein function in various diseases, such as cancer. miRNAs are present in mammalian ovaries, and their expression patterns change throughout ovarian development and follicle formation, and are expected to play a functional role in ovarian cycle. Although miRNAs have been reported to play a role in diseases, such as cancer and neurological disorders, as regulators that suppress gene expression, the profile of premature ovarian failure-related miRNAs isolated from premature ovarian failure patients and their functional role in premature ovarian failure have not been identified.

[0014] Existing inventions for diagnosing premature ovarian failure include a method for diagnosing through VEGF (vascular endothelial growth factor), an angiogenesis gene (Korean Patent Registration No. 10-01301968), and a method for diagnosing using a microarray with DNA specific to premature menopause patients (Korean Patent Registration No. 10-0861493). However, all of the above methods have the disadvantage that the target is DNA or blood, which makes early diagnosis difficult or impossible. Summary of the Invention

[0015] Therefore, the present inventors confirmed the miRNA expression profile in exosomes isolated from urine of patients with premature ovarian failure, and identified the miRNAs related to premature ovarian failure caused by Turner syndrome and premature ovarian failure caused by factors other than Turner syndrome, and screened and verified the miRNAs that can be used as diagnostic markers for premature ovarian failure.In addition, the present inventors completed the present invention by confirming the expression of miRNA in a mouse model of premature ovarian failure and by carrying out protein expression analysis to determine the mechanism of action of miRNA in premature ovarian failure.

[0016] Summary of the Invention It is an object of the present invention to provide a composition for diagnosing premature ovarian failure. Another object of the present invention is to provide a kit for diagnosing premature ovarian failure.

[0017] Another object of the present invention is to provide a method for diagnosing premature ovarian failure. Another object of the present invention is to provide a method for screening therapeutic agents for premature ovarian failure.

[0018] In order to achieve the above-mentioned object, the present invention provides a composition for diagnosing premature ovarian failure, comprising an agent capable of measuring the expression level of one or more miRNAs selected from the group consisting of miR-4516, miR-29a-3p, and miR-30b-5p.

[0019] The present invention also provides a kit for diagnosing premature ovarian failure, comprising the diagnostic composition.

[0020] The present invention also provides a method for diagnosing premature ovarian failure comprising the steps of: 1) obtaining a biological sample from an individual, isolating exosomes from the sample, and isolating RNA from the exosomes; and 2) measuring the expression level of one or more miRNAs selected from the group consisting of miR-4516, miR-29a-3p, and miR-30b-5p in the isolated RNA, and comparing the expression level with the expression level of the gene in a normal control group.

[0021] In addition, the present invention provides a method for screening a therapeutic agent for premature ovarian failure, comprising the steps of: 1) treating a cell having a base sequence encoding one or more miRNAs selected from the group consisting of miR-4516, miR-29a-3p, and miR-30b-5p in vitro with a test substance; and 2) Analyzing the expression of miRNA in the isolated cells treated with the test substance and comparing the miRNA expression pattern with the miRNA expression pattern in isolated control cells not treated with the test substance.

[0022] Beneficial Effects Use of the biomarkers of the present invention enables early diagnosis of premature ovarian failure, enables diagnosis that distinguishes premature ovarian failure from premature ovarian failure caused by Turner syndrome, and provides a method for screening therapeutic agents for premature ovarian failure through changes in expression of the biomarkers. [Brief description of the drawings]

[0023] [Figure 1] FIG. 1 is a series of photographs showing exosomes isolated from urine collected from a patient with premature ovarian failure and viewed under an electron microscope. [Diagram 2] FIG. 2 is a series of photographs showing exosomal markers CD9, CD63, and TSG101 in exosomes isolated from urine. [Diagram 3] FIG. 3 is a graph showing the size of exosomes isolated from urine.

[0024] [Figure 4] FIG. 4 is a heat map showing the changes in expression of miRNA genes in patients with premature ovarian failure and normal controls. [Diagram 5] FIG. 5 is a graph showing the number of target genes of 33 miRNAs that were differentially expressed in patients with premature ovarian failure, counted by the category to which they belong. [Figure 6] FIG. 6 is a graph showing the results of using miRNA sequencing and qRT-PCR to confirm the expression of 12 selected miRNAs that showed differential expression between premature ovarian failure without Turner syndrome patients and normal control patients in the premature ovarian failure patient group.

[0025] [Figure 7] FIG. 7 is a graph showing the results of using miRNA sequencing and qRT-PCR to confirm the expression of 12 selected miRNAs that showed differential expression between the Turner syndrome premature ovarian failure patient group and the normal control patient group of premature ovarian failure patients. [Figure 8] FIG. 8 is a graph showing the expression level of miR-4516 for each group (Control: normal control group, Turner: premature ovarian failure patient group with Turner syndrome, POI: premature ovarian failure patient group without Turner syndrome).

[0026] [Figure 9] FIG. 9 is a graph showing the expression level of miR-29a-3p for each group (Control: normal control group, Turner: premature ovarian failure patient group with Turner syndrome, POI: premature ovarian failure patient group without Turner syndrome). [Figure 10] FIG. 10 is a graph showing the expression level of miR-30b-5p for each group (Control: normal control group, Turner: premature ovarian failure patient group with Turner syndrome, POI: premature ovarian failure patient group without Turner syndrome).

[0027] [Figure 11A] FIG. 11a is a series of photographs showing the ovaries of a mouse model of premature ovarian failure injected with cyclophosphamide (CTX) and busulfan (BUS) (bottom), and a control mouse (top). [Figure 11B] FIG. 11b is a graph comparing the sizes of the ovaries in FIG. 11a. [Figure 12A] FIG. 12a is a series of photographs showing ovarian sections stained with hematoxylin and eosin from cyclophosphamide (CTX) and busulfan (BUS) injected premature ovarian failure model mice and control mice. [Figure 12B] FIG. 12b is a graph comparing the number of follicles in FIG. 12a. [Figure 12C]FIG. 12c is a graph comparing the ratio of atretic follicles to all follicles in FIG. 12a.

[0028] [Figure 13] FIG. 13 is a series of photographs showing ovarian sections stained with Sirius red from cyclophosphamide (CTX) and busulfan (BUS) injected premature ovarian failure model mice and control mice. [Figure 14A] FIG. 14a is a series of photographs showing ovarian sections stained with TUNEL from cyclophosphamide (CTX) and busulfan (BUS) injected premature ovarian failure model mice and control mice. [Figure 14B] FIG. 14b is a graph comparing the proportion of TUNEL positive (+) cells to the total cells in FIG. 14a.

[0029] [Figure 15] FIG. 15 is a series of photographs showing ovarian sections stained with Ki67 from cyclophosphamide (CTX) and busulfan (BUS)-injected premature ovarian failure model mice and control mice. [Figure 16A] FIG. 16a is a graph comparing the expression of miR-4516 in the ovaries of premature ovarian failure model mice injected with cyclophosphamide (CTX) and busulfan (BUS), and control mice. [Figure 16B] FIG. 16b is a graph comparing the expression of miR-4516 in the uterus of premature ovarian failure model mice injected with cyclophosphamide (CTX) and busulfan (BUS), and control mice. [Figure 17] FIG. 17 is a schematic diagram showing miR-4516 and its binding site in the 3′-UTR of STAT3, a target gene of miR-4516.

[0030] [Figure 18A]FIG. 18a is a schematic diagram showing the expression levels of STAT3, BAX, and p53 in the ovaries of premature ovarian failure model mice injected with cyclophosphamide (CTX) and busulfan (BUS), and control mice. [Figure 18B] FIG. 18b is a graph comparing the expression levels of STAT3 in FIG. 18a. [Figure 18C] FIG. 18c is a graph comparing the expression levels of BAX in FIG. 18a. [Figure 18D] FIG. 18d is a graph comparing the expression levels of p53 in FIG. 18a. [Figure 19] FIG. 19 is a schematic diagram showing the mechanism of action of miR-4516 in the ovary. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] Description of the Preferred Embodiments The present invention will be described in detail below. The present invention provides a composition for diagnosing premature ovarian failure and a composition for diagnosing premature ovarian failure caused by Turner syndrome, comprising an agent capable of measuring the expression level of one or more miRNAs selected from the group consisting of miR-4516, miR-29a-3p, and miR-30b-5p.

[0032] If the expression of miR-4516 is increased, it can be diagnosed as premature ovarian failure. If the expression of miR-29a-3p is decreased, it can be diagnosed as premature ovarian failure not caused by Turner syndrome, and if the expression of miR-30b-5p is decreased, it can also be diagnosed as premature ovarian failure not caused by Turner syndrome. On the other hand, if the expression of miR-29a-3p is increased, it can be diagnosed as premature ovarian failure caused by Turner syndrome, and if the expression of miR-30b-5p is increased, it can also be diagnosed as premature ovarian failure caused by Turner syndrome.

[0033] If the expression of miR-4516 is increased and the expression of miR-29a-3p is decreased, it can be diagnosed as premature ovarian failure not caused by Turner syndrome, and if the expression of miR-4516 is increased and the expression of miR-30b-5p is decreased, it can also be diagnosed as premature ovarian failure not caused by Turner syndrome. On the other hand, if the expression of miR-4516 and miR-29a-3p is increased, or if the expression of miR-4516 and miR-30b-5p is increased, it can be diagnosed as premature ovarian failure caused by Turner syndrome.

[0034] If the expression of miR-4516 is increased and the expression of miR-29a-3p and miR-30b-5p is decreased, it can be diagnosed as premature ovarian failure not caused by Turner syndrome, whereas if the expression of miR-4516, miR-29a-3p, and miR-30b-5p are all increased, it can be diagnosed as premature ovarian failure caused by Turner syndrome.

[0035] The agent may be a nucleic acid that contains a polynucleotide that is identical or complementary to the nucleic acid sequence of the miRNA.

[0036] Complementary polynucleotide means that the antisense oligonucleotide is sufficiently complementary to selectively hybridize to the miRNA target under a given hybridization or annealing condition, preferably under physiological conditions, and includes both substantially complementary and completely complementary polynucleotides. Substantially complementary means that it is not completely complementary, but is sufficiently complementary to produce a sufficient effect by binding to the target sequence.

[0037] The nucleic acid may be a primer or a probe.Specifically, it includes polynucleotides, oligonucleotides, DNA, RNA, and their analogs and derivatives, such as peptide nucleic acid (PNA) or mixtures thereof.In addition, the nucleic acid may be single-stranded or double-stranded, and may code for molecules including polypeptides, mRNA, microRNA, or siRNA.

[0038] A primer or probe may be labeled at its end or internally with a fluorescent substance, a chemiluminescent substance, or a radioisotope. The miRNA may be RNA obtained from exosomes isolated from a biological sample.

[0039] The biological sample may be cell culture supernatant, whole blood, serum, plasma, ascites, cerebrospinal fluid, bone marrow aspirate, bronchoalveolar lavage fluid, urine, vaginal fluid, mucus, saliva, sputum, or a lysate purified from a biological sample. Preferably, the biological sample is urine or a lysate purified from a biological sample.

[0040] The present invention also provides a kit for diagnosing premature ovarian failure, comprising the diagnostic composition. In addition, the present invention provides a kit for diagnosing premature ovarian failure caused by Turner syndrome, comprising the diagnostic composition.

[0041] The kit may further include a sample required for the diagnosis of premature ovarian failure. The kit may include a polymerase, a buffer, a nucleic acid, a coenzyme, a fluorescent substance, or a combination thereof for the detection of a nucleic acid. The polymerase may preferably be Taq polymerase.

[0042] The present invention also provides a method for diagnosing premature ovarian failure comprising the steps of: 1) obtaining a biological sample from an individual, isolating exosomes from the sample, and isolating RNA from the exosomes; and 2) measuring the expression level of one or more miRNAs selected from the group consisting of miR-4516, miR-29a-3p, and miR-30b-5p in the isolated RNA, and comparing the expression level with the expression level of the gene in a normal control group.

[0043] If the expression level of miR-4516 is increased compared to normal control group, or if the expression level of miR-29a-3p or miR-30b-5p is decreased compared to normal control group, the individual can be determined to be at high risk of premature ovarian failure.In addition, if the expression level of miR-29a-3p or miR-30b-5p is increased compared to normal control group, the subject can be determined to have premature ovarian failure caused by Turner syndrome.

[0044] The individual may be a mammal, and may be a human, dog, cat, rat, mouse, hamster, rabbit, horse, sheep, cow, goat or pig. The individual may preferably be a human.

[0045] The term "diagnosis" refers to determining the disease and can include the name, condition, stage, etiology, etc. of the disease for premature ovarian failure.

[0046] The exosomes may be spherical with an average diameter of 50-60 nm, preferably with an average diameter of about 57 nm.

[0047] The expression levels of miRNAs can be measured by electrophoresis, microarrays, Northern blotting, PCR (polymerase chain reaction), reverse transcription PCR (RT-PCR), real-time PCR (qRT-PCR), or a combination thereof.

[0048] In addition, the present invention provides a method for screening a therapeutic agent for premature ovarian failure, comprising the steps of: 1) treating a cell having a base sequence encoding one or more miRNAs selected from the group consisting of miR-4516, miR-29a-3p, and miR-30b-5p in vitro with a test substance; and 2) Analyzing the expression of miRNA in the isolated cells treated with the test substance and comparing the miRNA expression pattern with the miRNA expression pattern in isolated control cells not treated with the test substance.

[0049] If the expression level of miR-4516 in cells treated with the test substance is decreased compared to the expression level in control cells not treated with the test substance; or if the expression level of miR-29a-3p or miR-30b-5p in cells treated with the test substance is increased compared to the expression level in control cells not treated with the test substance, the test substance may be a candidate for the treatment of premature ovarian failure.

[0050] In addition, if the expression level of miR-4516 in cells treated with the test substance is decreased compared to the expression level in control cells not treated with the test substance; or if the expression level of miR-29a-3p or miR-30b-5p in cells treated with the test substance is decreased compared to the expression level in control cells not treated with the test substance, the test substance may be a candidate for the treatment of premature ovarian failure due to Turner syndrome.

[0051] Throughout the specification, "premature ovarian failure" refers to both premature ovarian failure caused by Turner syndrome and premature ovarian failure not caused by Turner syndrome, and "patients with premature ovarian failure" refers to both patients with premature ovarian failure caused by Turner syndrome and premature ovarian failure not caused by Turner syndrome.

[0052] "Premature ovarian failure due to Turner syndrome" specifically refers to premature ovarian failure resulting from Turner syndrome, and "a patient group with premature ovarian failure due to Turner syndrome" specifically refers to a group of patients with premature ovarian failure due to Turner syndrome.

[0053] Patients with premature ovarian failure are those with chronic oligo- or anovulation who have had no menses for more than 6 months before age 40 years and a serum follicle-stimulating hormone (FSH) greater than 40 mIU / mL on two occasions measured more than one month apart.

[0054] Specifically, this can be determined by measuring changes in follicle stimulating hormone (FSH) and estradiol levels.

[0055] Turner syndrome refers to a chromosomal condition that affects female development. Turner syndrome is a condition in which one normal X chromosome is present and the other sex chromosome is absent or structurally altered, encompassing cases of chromosomal mosaicism with the Turner syndrome karyotype (45,X). Turner syndrome is characterized by early loss of ovarian function and may present with short stature, disorders of sex development, heart disease, otitis media, venous vasculitis, lymphedema, kidney anomalies, hypothyroidism, obesity, diabetes, hypertension, osteoporosis, and chronic colitis.

[0056] Some cases of premature ovarian failure are due to Turner syndrome. Although women with Turner syndrome do not necessarily develop premature ovarian failure, they appear to be at high risk for premature ovarian failure. It is therefore necessary to distinguish whether the cause of premature ovarian failure is due to Turner syndrome.

[0057] "miR" or "microRNA" of the present invention refers to 21-23 non-coding RNAs that post-transcriptionally regulate gene expression by promoting the degradation of target RNAs or inhibiting their translation. The mature sequence of miRNAs used herein can be obtained from the miRNA database (http: / / www.mirbase.org). Generally, microRNAs are transcribed into precursors of about 70-80 nt (nucleotides) in length with hairpin structures called pre-miRNAs, and then cleaved by the RNAse III enzyme Dicer to generate the mature form. MicroRNAs form ribonucleocomplexes called miRNPs, which cleave target genes or inhibit translation through complementary binding to target sites. More than 30% of human miRNAs exist in clusters and are transcribed as single precursors, followed by a cleavage process to form the final mature miRNA.

[0058] In a specific experimental example of the present invention, patients with premature ovarian failure were divided into three groups: a group of patients with premature ovarian failure not caused by Turner syndrome, a group of patients with premature ovarian failure caused by Turner syndrome, and a normal control group. Urine was collected from the experimental group and the control group, and exosomes were isolated. The isolated exosomes were confirmed to have a spherical shape with an average diameter of 57.022 nm (Figure 1), and the exosomes could be identified by confirming the expression of exosome markers CD9, CD63, and TSG101 (Figure 2). In addition, it was confirmed that the isolated exosomes exhibited a typical exosome distribution pattern, and the size of the exosomes was about 50-300 nm (Figure 3).

[0059] Libraries were made with RNA from normal controls and premature ovarian failure patients (including both Turner syndrome and non-Turner syndrome premature ovarian failure patients), and genes with increased or decreased expression were identified (Figure 4). By identifying the biological functions of the above genes, it was confirmed that the differentially expressed miRNAs are associated with oocyte growth and maturation (Figure 5).

[0060] Among the above miRNAs, 12 miRNAs were selected and their expression was confirmed in the normal control group and the group of patients with premature ovarian failure not due to Turner syndrome (Figure 6 and Figure 7). Among the above miRNAs, the miRNAs that showed similar differences between the normal control group and the group of patients with premature ovarian failure not due to Turner syndrome were miR-16-5p, miR-20a-5p, miR-29a-3p, miR-30b-5p, miR-99b-3p, miR-151a-5p, miR-200b-3p, miR-423-3p, miR-941, miR-4516, miR-4492, and miR-7847-3p. In addition, the expression of 12 miRNAs was confirmed in the validation cohort of patients with premature ovarian failure due to Turner syndrome and the normal control group (Figure 6). Among the above miRNAs, the miRNAs that showed similar differences in sequencing between normal controls and patients with premature ovarian failure due to Turner syndrome were miR-20a-5p and miR-200b-3p (Figure 7).

[0061] When the expression levels of the above miRNAs were examined in each patient group, miR-4516 was expressed significantly higher in all groups of patients with premature ovarian failure than in the normal control group (Figure 8).

[0062] The expression levels of miR-29a-3p and miR-30b-5p were increased in patients with premature ovarian failure due to Turner syndrome compared with normal controls, but were decreased in patients with premature ovarian failure without Turner syndrome (Figures 9 and 10).

[0063] In the mouse model of premature ovarian failure injected with CTX and BUS, the size of the ovaries was decreased (Figs. 11a and 11b), the number of follicles was decreased (Figs. 12a and 12b), and the proportion of atretic follicles was increased (Fig. 12c). In addition, the reactivity of Sirius red staining, which indicates collagen accumulation, was increased, indicating the progression of tissue fibrosis (Fig. 13). In addition, TUNEL analysis showed increased apoptosis (Figs. 14a and 14b), and Ki67 staining showed a decrease in the number of Ki67-stained cells (Fig. 15).

[0064] In addition, it was confirmed that the expression of miR-4516 was increased in the ovaries of the premature ovarian failure mouse model, but not in the uterus (Fig. 16a, 16b). Through the analysis of miRTarBase2020, STAT3 was selected as the target gene of miR-4516, and two binding sites of miR-4516 were identified in the STAT3 3'-UTR (Fig. 17). It was confirmed that the expression of STAT3 was decreased in the ovaries of the premature ovarian failure mouse model (Fig. 18a-18d). Based on the above results, it is speculated that miR-4516 promotes oocyte death, follicle formation, and progenitor cell activity through regulating the expression of STAT3 (Fig. 19).

[0065] The present invention will now be described in detail with reference to the following experimental examples. However, the following experimental examples are merely intended to illustrate the present invention, and the contents of the present invention are not limited thereto.

[0066] Meanwhile, the embodiments of the present invention can be modified into various other forms, and the scope of the present invention is not limited to the embodiments described below. It is fully understood by those skilled in the art who have average knowledge in this field that the embodiments of the present invention are given in order to more precisely describe the present invention.

[0067] Experimental example 1: miRNA sequencing and biomarker screening <1-1> Selection of sequencing cohort for mRNA sequencing Healthy subjects without premature ovarian failure were selected as the control group, and a group of patients with premature ovarian failure not due to Turner syndrome and a group of patients with premature ovarian failure due to Turner syndrome were selected as the experimental groups.

[0068] According to the diagnostic method for premature ovarian failure, chronically oligo- or anovulatory women with serum follicle-stimulating hormone (FSH) >40 mIU / mL measured twice with an interval of more than one month were considered as patients with premature ovarian failure, while women with regular menstrual cycles and normal ovulation were selected as the control group.

[0069] For each group, seven patients with premature ovarian failure not caused by Turner syndrome, seven patients with premature ovarian failure caused by Turner syndrome, and five healthy subjects were selected and analyzed for age, height, weight, BMI, etc. (see Tables 1 and 2).

[0070] [Table 1]

[0071] In the group of patients with premature ovarian failure not due to Turner syndrome, total cholesterol was slightly higher and triglycerides were approximately 1.9 times higher than in the normal control group. In addition, it was found that HDL cholesterol (HDL-C) was slightly higher, prolactin was also slightly elevated, follicle-stimulating hormone (FSH) was extremely high, and on the other hand, estradiol was secreted only about 17% compared to the normal control group. Patients with premature ovarian failure due to Turner syndrome tend to be shorter than normal controls, with no significant difference in weight. They also have a high BMI in the overweight range. This is consistent with the developmental characteristics of Turner syndrome, which is characterized by slow growth with a final height in the range of 140 cm, and a tendency to gain weight due to insufficient burning of calories compared to the amount of food ingested, leading to obesity or diabetes.

[0072] The liver indices AST and ALT were also measured higher than in the normal control group. Total cholesterol levels were high, and HDL cholesterol levels were also high. Triglycerides were higher than in the normal control group, but lower than in patients with premature ovarian failure not due to Turner's syndrome, and thyroid-stimulating hormone was secreted in the highest amounts of the three groups. In cases of Turner's syndrome, hypothyroidism occurs frequently, and therefore thyroid-stimulating hormone levels are high.

[0073] Prolactin was lower in patients with premature ovarian failure due to Turner syndrome than in normal controls, but not in patients with premature ovarian failure not due to Turner syndrome. In patients with Turner syndrome, follicle-stimulating hormone was secreted more than in normal controls, but less than in patients with premature ovarian failure, and estradiol was secreted less than in patients with premature ovarian failure. This is a characteristic of Turner syndrome, which involves insufficient secretion of female hormones and often causes symptoms such as osteoporosis.

[0074] [Table 2]

[0075] Patients with premature ovarian failure not due to Turner syndrome began menarche at about age 13 years, had amenorrhea for at least 3 months, and did not have a mutation in the FMR-1 (fragile X messenger ribonucleoprotein 1) gene, the most common single gene mutation associated with premature ovarian failure. Patient 2 had a karyotype of 46,XX and 47,XXX, but this was not considered to be the direct cause of premature ovarian failure, so she was classified as a patient with premature ovarian failure not due to Turner syndrome. Patients with premature ovarian failure due to Turner syndrome showed primary amenorrhea and did not have a mutation in the FMR-1 gene. Some of these patients, such as patients 3, 6, and 7, had a Turner syndrome karyotype with a typical chromosomal abnormality of 45,X, while others, such as patients 2 and 4, had a Turner syndrome karyotype with structural chromosomal abnormalities such as deletions. Patients 1 and 5 showed mosaicism for the structural chromosomal abnormalities of 46,X,i and 45,X.

[0076] <1-2> Isolation of exosomes Urine was collected from subjects in the experimental and control groups selected in Experimental Example 1. The collected urine was first centrifuged at 2000g for 10 minutes to remove cells and sediments, and then centrifuged at 13,500g for 20 minutes. It was then filtered through a 0.22 μm filter and concentrated again using centrifugation (2000g, 30 minutes).

[0077] Exosome precipitation solution (Exoquick-TC) was mixed at a ratio of 1:4 and incubated overnight at 4° C. The next day, the incubated solution was centrifuged at 2000 g for 30 minutes to isolate the exosome pellet. The isolated pellet was resuspended, combined with beads for 15 minutes, and then centrifuged at 8000 g for 5 minutes to obtain the supernatant containing exosomes.

[0078] <1-3> Confirmation of exosomes The supernatant containing exosomes was lysed using lysis buffer (Cat. No. 9303, Cell Signaling Technology) and then microcentrifuged at 13,000 g for 10 min at 4°C. The supernatant of the centrifugation solution was collected and boiled in sodium dodecyl sulfate (SDS) buffer for 3 min, and then proteins were separated by SDS-PAGE. After electrophoresis, the gel was transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA) and the membrane was blocked in phosphate-buffered saline Tween (PBST) containing 5% (w / v) skim milk for 30 min at room temperature, and then incubated with primary antibodies in PBST containing 5% (w / v) skim milk overnight at 4°C. Exosomes were detected using secondary antibodies conjugated to horseradish peroxidase using Immobilon Western chemiluminescent HRP substrate (Millipore).

[0079] The detected exosomes were observed under an electron microscope. Specifically, it was placed on a copper grid coated with thin carbon foil (Cat. No. 01340, Ted Pella, Inc., USA) and stained with 2% (w / v) uranyl acetate (UrAc) for 1 min. Then, electron micrographs were taken using a bio-high voltage EM system (JEM-1400 Plus at 120 kV and JEM-1000BEF at 1000 kV; JEOL Ltd., Japan).

[0080] As a result, spherical exosomes with an average diameter of 57.022 nm were observed (Figure 1), and the expression of CD9, CD63, and TSG101 (Tumor Susceptibility 101), which are known as exosome markers, was confirmed (Figure 2).

[0081] Exosome size was measured by nanoparticle tracking analysis (NTA) using a particle analyzer (Litesizer 500, Anton Paar), and the results confirmed that exosomes showed a typical exosome distribution pattern, and most exosomes were distributed in the size range of 50 to 300 nm in diameter (Figure 3).

[0082] <1-4> Isolation of miRNA from exosomes MiRNAs were extracted from isolated exosomes using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA).

[0083] Specifically, exosomes were lysed by treatment with 0.5 ml of TRIzol reagent and incubated at room temperature for 5 minutes, followed by adding 0.1 ml of chloroform and mixing by vigorous shaking for 15 seconds. The mixture was incubated at room temperature for 2-3 minutes, centrifuged at 12,000 g at 4°C for 10 minutes, and then the supernatant was transferred to a new tube. RNA was precipitated by adding an equal volume of isopropanol (approximately 0.25 ml) to the tube and incubating at room temperature for 10 minutes. The RNA pellet was confirmed by centrifugation at 12,000 g at 4°C for 10 minutes, resuspended in 0.5 ml of 75% ethanol, centrifuged at 7500 g at 4°C for 5 minutes, and then the supernatant was removed and the RNA pellet was dried for 5 minutes. The RNA pellet was then dissolved and resuspended in 0.05 ml of water containing DEPC (diethylpyrocarbonate), and placed in a 55°C water bath for 10 minutes.

[0084] <1-5> mRNA sequencing Library construction was performed using the NEB Next Multiplex Small RNA Library Prep kit (New England BioLabs, Inc., USA) with RNA from normal controls and patients with premature ovarian failure (including both patients with premature ovarian failure due to Turner syndrome and patients with premature ovarian failure not due to Turner syndrome).

[0085] Specifically, 1 μg of total RNA from each sample was used for library construction to ligate adapters, and then cDNA was synthesized using reverse transcriptase with adapter-specific primers. The libraries were amplified by PCR, and the libraries were purified using the QIAquick PCR purification kit (Quaigen, Inc., German) and AMPure XP beads (Beckmancoulter, Inc., USA).

[0086] The yield and size distribution of the small RNA libraries were analyzed with an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., USA) for high-sensitivity DNA analysis, and high-throughput sequencing was performed on a NextSeq500 system (Illumina, San Diego, CA, USA) using single-end 75 sequencing.

[0087] <1-6> Analysis of changes in miRNA expression levels As a result of miRNA sequencing, 33 miRNAs with different expression levels were selected (fold change 1.3, normalization 2, significance 0.05) (Table 3).

[0088] [Table 3]

[0089] Hierarchical clustering analysis was performed by plotting the distribution of selected miRNAs as a heat map. As a result, the expression characteristics of miRNA genes that differed between the group of patients with premature ovarian failure and the normal control group were confirmed (Figure 4). Among them, the expression level of miR-4516 was confirmed to be significantly increased in the group of patients with premature ovarian failure. To confirm the biological effects of the target genes of the selected differentially expressed genes (DEGs), their distribution was investigated by gene category. The results showed that most of the selected miRNAs were involved in the embryonic development of the uterus, follicular development, and antral follicle growth, all of which processes are closely related to the development of POI (Figure 5). For the cases of the other categories, they were not considered to be included in the category because they lacked correlation with premature ovarian failure or because they were recently discovered mRNAs, and therefore no data on correlation was secured.

[0090] In other words, altered expression levels have been observed in genes involved in uterine embryo development, follicular development, and antral follicle growth, leading to premature ovarian failure due to reduced embryo and follicular development and growth within the uterus.

[0091] Experimental Example 2: Validation and analysis of selected miRNAs <2-1> Selection of validation cohort for miRNA validation Healthy subjects without premature ovarian failure were selected as the control group, and a group of patients with premature ovarian failure not due to Turner syndrome and a group of patients with premature ovarian failure due to Turner syndrome were selected as the experimental groups.

[0092] According to the diagnostic method for premature ovarian failure, chronically oligo- or anovulatory women with serum follicle-stimulating hormone (FSH) >40 mIU / mL measured twice with an interval of more than one month were considered as patients with premature ovarian failure, while women with regular menstrual cycles and normal ovulation were selected as the control group.

[0093] From June 2020 to July 2021, 15 patients with premature ovarian failure, 11 patients with premature ovarian failure due to Turner syndrome, and 20 healthy subjects were selected from each group and analyzed for age, height, weight, BMI, etc. The results are shown in the tables below (Tables 1 and 2).

[0094] [Table 5] [Table 6]

[0095] Patients with premature ovarian failure began menarche at approximately 13 or 14 years of age, and patients 1 and 14 did not have menarche, as did patients with premature ovarian failure due to Turner syndrome. These patients had amenorrhea for at least 6 months and did not have FMR-1 gene mutations analyzed as one of the causes of premature ovarian failure. Among them, patient 8 had karyotypes of 46,XX and 47,XXX, but this was not considered to be the direct cause of premature ovarian failure, so the patient was classified as a patient with premature ovarian failure not due to Turner syndrome. Patients with premature ovarian failure due to Turner syndrome showed primary amenorrhea and did not have mutations in the FMR-1 gene. Some of these patients, such as patients 1, 4, and 7, had the typical Turner syndrome karyotype with 45,X chromosome number abnormality, while others, such as patients 2, 6, and 10, had Turner syndrome karyotype with chromosomal structural abnormalities such as deletions. Patients 3, 5, 8, 9 and 11 showed mosaicism for the structural chromosomal abnormalities 46,X,i and 45,X.

[0096] <2-2> Isolation of exosomes Urine was collected from subjects in the experimental and control groups selected in Experimental Example 2. The collected urine was first centrifuged at 2000g for 10 minutes to remove cells and sediments, and then centrifuged at 13,500g for 20 minutes. It was then filtered through a 0.22 μm filter and concentrated again using centrifugation (2000g, 30 minutes).

[0097] Exosome precipitation solution (Exoquick-TC) was mixed at a ratio of 1:4 and incubated overnight at 4° C. The next day, the incubated solution was centrifuged at 2000 g for 30 minutes to isolate the exosome pellet. The isolated pellet was resuspended, combined with beads for 15 minutes, and then centrifuged at 8000 g for 5 minutes to obtain the supernatant containing exosomes.

[0098] <2-3> Isolation of miRNA from exosomes MiRNAs were extracted from isolated exosomes using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA).

[0099] Specifically, exosomes were lysed by treatment with 0.5 ml of TRIzol reagent and incubated at room temperature for 5 min, followed by adding 0.1 ml of chloroform and mixing by vigorous shaking for 15 s. The mixture was incubated at room temperature for 2-3 min, centrifuged at 12,000 g for 10 min at 4° C., and then the supernatant was transferred to a new tube. RNA was precipitated by adding an equal volume of isopropanol (approximately 0.25 ml) to the tube and incubated at room temperature for 10 min. The RNA pellet was confirmed by centrifugation at 12,000 g for 10 min at 4° C., resuspended in 0.5 ml of 75% ethanol, centrifuged at 7500 g for 5 min at 4° C., and then the supernatant was removed and the RNA pellet was dried for 5 min.

[0100] The RNA pellet was then dissolved and resuspended in 0.05 ml of water containing DEPC and placed in a 55° C. water bath for 10 minutes.

[0101] <2-4> miRNA PCR verification and analysis The relative expression of the miRNAs isolated in <Experimental Example 2-3> was analyzed using the miRCURY LNA SYBR Green PCR kit (QIAGEN, Hilden, Germany) and miRCURY LNA miRNA PCR assay primers for the selected miRNAs (QIAGEN, Hilden, Germany).

[0102] PCR was performed using a CFX384 C1000 thermal cycler (Bio-Rad, Hercules, CA, USA) with an initial heat activation at 95°C for 2 min, followed by 40 cycles of 95°C for 10 s and 56°C for 60 s, with PCR cycle conditions for melting curve analysis. The sequences of the miRNAs used here and information on the primers used for PCR of each miRNA are shown in Table 7.

[0103] [Table 7]

[0104] From the heatmaps and significance plots, 12 miRNAs were selected that appeared to be significantly differentially expressed in the group of patients with premature ovarian failure not due to Turner syndrome or in the group of patients with premature ovarian failure due to Turner syndrome, or equally expressed in each of the two patient groups (Table 8).

[0105] [Table 8]

[0106] Comparison of the relative expression levels of selected miRNAs between the sequencing cohort and the validation cohort is shown in Tables 9 and 10 and Figures 6 and 7. Table 9 shows the expression changes in the miRNA sequencing results of the sequencing cohort and the qRT-PCR validation results of the validation cohort in the group of premature ovarian failure patients not due to Turner syndrome and the normal control group. Of the 12 miRNAs selected for validation analysis, 9 miRNAs (miR-16-5p, miR-29a-3p, miR-30b-5p, miR-99b-3p, miR-151a-5p, miR-423-3p, miR-941, miR-4516, and miR-7847-3p) showed the same expression pattern in the validation cohort of patients with premature ovarian failure not due to Turner syndrome and in the miRNA sequencing cohort of patients with premature ovarian failure not due to Turner syndrome (Table 9 and Figure 6).

[0107] [Table 9] *Fold changes are expressed as percentages relative to normal controls. In addition, the expression of miR-4516, miR-20a-5p, and miR-200b-3p showed the same expression patterns in the miRNA sequencing cohort and validation cohort of patients with premature ovarian failure due to Turner syndrome (Table 10 and Figure 7).

[0108] [Table 10] *Fold change is expressed as a ratio to the normal control group.

[0109] As a result, miR-4516 was significantly more highly expressed in both the non-Turner syndrome premature ovarian failure and Turner syndrome premature ovarian failure groups compared with the normal control group (Figure 8).

[0110] On the other hand, the expression of miR-29a-3p and miR-30b-5p was confirmed to be decreased in the group of patients with premature ovarian failure not caused by Turner syndrome and increased in the group of patients with premature ovarian failure caused by Turner syndrome (Figures 9 and 10).

[0111] Since their expression differed between the groups of patients with premature ovarian failure and Turner syndrome, they can be used as biomarkers to distinguish between patients with premature ovarian failure and Turner syndrome.

[0112] Therefore, if miR-4516 is increased and miR-29a-3p and miR-30b-5p are decreased, the patient can be determined to have premature ovarian failure not due to Turner syndrome, and if miR-4516 is increased and miR-29a-3p and miR-30b-5p are also increased, the patient can be determined to have premature ovarian failure due to Turner syndrome.

[0113] Experimental Example 3: Confirmation of increased expression of miR-4516 in a mouse model of premature ovarian failure <3-1> Establishment of a mouse model of premature ovarian failure to identify miRNAs Eight-week-old female specific pathogen-free (SPF) grade C57B6L / N mice were obtained from Orient Bio Inc. (Seongnam, Korea). Mice were maintained under controlled temperature (20–22°C) and light (12 / 12-h light / dark cycle) with free access to water and food. Animals were randomly divided into two groups: control (n=7) and CTX+BUS-POI (n=7), and three independent experiments were performed. To induce premature ovarian failure, mice were intraperitoneally injected with 120 mg / kg CTX (cyclophosphamide) and 12 mg / kg BUS (busulfan) three times a week for 2 weeks, and saline was injected as the control group. All experimental procedures and maintenance and care in this study were performed in accordance with the Animal Care and Use Center of the Lee Gil-Yeo Cancer and Diabetes Institute, Gachon University, and the Laboratory Animal Care and Use Committee (IACUC) (Accession Number: LCDI-2021-0121). CTX (C0768) and BUS (B2635) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0114] <3-2> Hematoxylin and eosin (H&E) and Sirius red staining of ovaries in a mouse model of premature ovarian failure Ovarian tissues were fixed overnight in 4% paraformaldehyde, dehydrated, embedded in paraffin, and cut into 5 μm-thick sections. The fifth section was dewaxed, rehydrated, and stained with hematoxylin and eosin (H&E) or Sirius red for microscopic observation. H&E staining stains the nucleus with hematoxylin and the cytoplasm with eosin. Sirius red staining was performed to confirm collagen fibers in the ovarian tissue. H&E and Sirius red stained images were randomly captured using a Motic Easyscan Digital Slide digital slide scanner (Motic Hong Kong Limited, Hong Kong, China).

[0115] <3-3> TUNEL assay and Ki67 immunohistochemistry in ovaries of a mouse model of premature ovarian failure Apoptosis in ovarian tissues was confirmed using the DeadEnd™ Fluorometric TUNEL System (G3250, Promega, Fitchburg, WI, USA) according to the manufacturer's instructions. After preparing mounting medium for fluorescence with DAPI (4',6-diamidino-2-phenylindole, Vector Lab), slides were examined using an Olympus CKX3-Houn microscope (Olympus, Tokyo, Japan). To complete Ki67 immunohistochemistry, sections were incubated in 0.01 M citrate buffer (pH 6.0), deparaffinized and rehydrated, and then antigen retrieval was performed by applying intense microwave irradiation for 30 min. Endogenous peroxidase activity was inhibited with 3% H2O2 for 10 min, and nonspecific binding was blocked with 10% normal goat serum for 1 h. Sections were treated with Ki-67 primary antibody (1:200; ab16667 Abcam, UK) for 24 hours at 4°C. Diaminobenzidine (DAB) was used to visualize the antigen. Finally, slides were dehydrated, counterstained with hematoxylin, and mounted. Ki67 immunohistochemistry images were randomly captured using a Motic Easyscan digital slide scanner (Motic Hong Kong Limited, Hong Kong, China).

[0116] <3-4> Isolation of miRNA from mouse ovaries and uterus Total RNA was extracted from ovaries and uterus using TRIzol reagent (15596026, Thermo Fisher Scientific) according to the manufacturer's instructions. Specifically, tissues were pulverized and lysed by treatment with 0.5 ml of TRIzol reagent and incubated at room temperature for 5 min, followed by addition of 0.1 ml of chloroform and mixing by vigorous shaking for 15 s. The mixture was incubated at room temperature for 2-3 min, centrifuged at 12,000 g for 10 min at 4°C, and the supernatant was transferred to a new tube. RNA was precipitated by adding an equal volume of isopropanol (approximately 0.25 ml) and incubating at room temperature for 10 min. The mixture was centrifuged at 12,000 g for 10 min at 4°C to confirm the RNA pellet, resuspended in 0.5 ml of 75% ethanol, and centrifuged at 7500 g for 5 min at 4°C. The supernatant was removed and the RNA pellet was dried for 5 min. The RNA pellet was then dissolved, resuspended in 0.05 ml of water containing DEPC (diethylpyrocarbonate), and incubated in a 55° C. water bath for 10 minutes.

[0117] <3-5> miRNA PCR verification and analysis The relative expression of the miRNAs isolated in <Experimental Examples 3-4> was analyzed using the miRCURY LNA SYBR Green PCR Kit (QIAGEN, Hilden, Germany) and the miRCURY LNA miRNA PCR Assay Primers for selected miRNAs (QIAGEN, Hilden, Germany). Using the miRCURY LNA RT Kit (339340, QIAGEN, Maryland, Germany), cDNA was synthesized from 5 ng of total RNA using 10x miRCURY RT Enzyme Mix (QIAGEN, Germany) containing UniSp6 RNA spike-in (QIAGEN, Germany) in a reaction volume of 20 μL. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using a CFX384 C1000 Thermal Cycler (Bio-Rad, Hercules, CA, USA) with 2x miRCURY SYBR Green Master Mix (QIAGEN). Expression levels were analyzed as relative expression to UniSp6 (Kinoshita et al., 2017). The miRCURY LNA miRNA SYBR Green PCR primers used in this study are shown in Table 7.

[0118] PCR was performed using a CFX384 C1000 thermal cycler (Bio-Rad, Hercules, CA, USA) with an initial heat activation at 95°C for 2 min, followed by 40 cycles of 95°C for 10 s and 56°C for 60 s, with PCR cycle conditions for melting curve analysis.

[0119] <3-6> Protein expression analysis in a mouse model of premature ovarian failure Proteins in mouse ovaries were extracted using T-PER tissue protein extraction reagent supplemented with protease (Roche, Mannheim, Germany) and phosphatase inhibitors (Roche). Ovarian tissue (30 mg) was homogenized in extraction buffer for 1-2 min at 30 Hz using a TissueLyser II (Qiagen, Hilden, Germany). The lysate was centrifuged at 13,000xg for 10 min at 4°C to remove debris. The supernatant was mixed with sodium dodecyl sulfate sample buffer and boiled for 5 min. Proteins were electrophoresed on an SDS-polyacrylamide gel and then transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membrane was incubated with primary antibodies according to the manufacturer's instructions. HRP (horseradish peroxidase)-conjugated secondary antibodies were detected using Immobilon Western chemiluminescent HRP substrate (Millipore). Western blot band intensities were quantified by densitometry using ImageJ software.

[0120] To evaluate miR-4516 as a biomarker for premature ovarian failure, we established a mouse model of chemotherapy-induced premature ovarian failure by injecting mice with CTX and BUS. No obvious deaths or toxicity were observed in the premature ovarian failure mouse model, but the size of the ovaries was smaller than that in saline-injected control mice (Figs. 11a and 11b). Compared with saline-injected control mice, premature ovarian failure mice had a significant decrease in follicle number and an increase in the ratio of atretic follicles / total follicles (Figs. 12a-12c). There was also an increase in Sirius red staining, indicative of collagen deposition, suggesting the progression of tissue fibrosis (Fig. 13).

[0121] In addition, TUNEL positive cells were significantly increased in the premature ovarian failure group (Fig. 14a and 14b), while Ki67 positive cells were decreased (Fig. 15). These results confirmed that the premature ovarian failure mouse model was successfully established by chemotherapy. Next, the inventors confirmed the expression of miR-4516 in the ovary of chemotherapy-induced premature ovarian failure mouse model. As shown in Fig. 16a and Fig. 16b, the expression of miR-4516 was significantly increased in the ovary of the premature ovarian failure mouse model compared with the control group, but not in the uterus. These results indicate that the increased expression of miR-4516 is associated with pathological changes in the ovary.

[0122] To interpret the pathological relevance of miR-4516 overexpression, we analyzed the targets of miR-4516 using miRTarBase2020, an experimentally validated miRNA-target interaction database (Huang et al., 2020). This analysis method confirmed that STAT3 is a target of miR-4516 with experimental evidence (Figure 17). The 3'-UTR region of STAT3 contains two putative miR-4516 binding sites (Chowdhari and Saini, 2014) (Figure 17).

[0123] Consistent with this predicted interaction, STAT3 protein expression was significantly decreased in the ovaries of chemotherapy-induced premature ovarian failure mouse models, along with increased expression of apoptotic markers Bax and p53 (Figures 18a-18d). These results suggest that increased miR-4516, along with decreased STAT3 expression, is closely associated with POI pathology. Regulation of STAT3 expression by miR-4516 is thought to be involved in cell death, oocyte maturation, and activation of progenitor cells (Figure 19).

Claims

1. A composition for diagnosing premature ovarian failure (POI), comprising an agent capable of measuring the expression level of one or more miRNAs selected from the group consisting of miR-4516, miR-29a-3p, and miR-30b-5p, wherein the miRNAs are derived from an isolated biological sample selected from the group consisting of ovarian cell culture supernatant, urine, and vaginal fluid.

2. A composition for diagnosing premature ovarian failure (POI) caused by Turner syndrome, comprising an agent capable of measuring the expression level of one or more miRNAs selected from the group consisting of miR-4516, miR-29a-3p, and miR-30b-5p, wherein the miRNAs are derived from an isolated biological sample selected from the group consisting of ovarian cell culture supernatant, urine, and vaginal fluid.

3. 3. The diagnostic composition of claim 1 or 2, wherein the agent is a nucleic acid containing a polynucleotide identical or complementary to a nucleic acid sequence of a miRNA.

4. The diagnostic composition of claim 3, wherein the nucleic acid is a primer or a probe.

5. The diagnostic composition of claim 1 or 2, wherein the miRNA is obtained from exosomes isolated from the biological sample.

6. A kit for diagnosing premature ovarian failure comprising the diagnostic composition of claim 1.

7. A kit for diagnosing premature ovarian failure caused by Turner syndrome, comprising the diagnostic composition of claim 2.

8. Steps below: 1) obtaining an isolated biological sample from an individual, isolating exosomes from the sample, and isolating RNA from the exosomes, wherein the isolated biological sample is derived from ovarian cell culture supernatant, urine, or vaginal fluid; and 2) measuring the expression level of one or more miRNAs selected from the group consisting of miR-4516, miR-29a-3p, and miR-30b-5p in the isolated RNA, and comparing the expression level with the expression level of the gene in a normal control group.

13. A method for providing information for diagnosing premature ovarian failure, comprising:

9. The method for providing information for diagnosing premature ovarian failure described in claim 8, wherein an individual is determined to be at high risk of premature ovarian failure when the expression level of miR-4516 is increased compared to a normal control group, or when the expression level of miR-29a-3p or miR-30b-5p is decreased compared to a normal control group.

10. The method for providing information for diagnosing premature ovarian failure according to claim 8, wherein when the expression level of miR-29a-3p or miR-30b-5p is increased compared to a normal control group, the individual is determined to have premature ovarian failure caused by Turner syndrome.

11. Steps below: 1) treating a cell having a base sequence encoding one or more miRNAs selected from the group consisting of miR-4516, miR-29a-3p, and miR-30b-5p in vitro with a test substance, wherein the miRNA is derived from an isolated biological sample selected from the group consisting of ovarian cell culture supernatant, urine, and vaginal fluid; and 2) analyzing the expression of miRNAs in isolated cells treated with the test substance and comparing the miRNA expression pattern to the miRNA expression pattern in isolated control cells not treated with the test substance. A method for screening a therapeutic agent for premature ovarian failure, comprising:

12. Steps below: 1) treating a cell having a base sequence encoding one or more miRNAs selected from the group consisting of miR-4516, miR-29a-3p, and miR-30b-5p in vitro with a test substance, wherein the miRNA is derived from an isolated biological sample selected from the group consisting of ovarian cell culture supernatant, urine, and vaginal fluid; and 2) analyzing the expression of miRNAs in isolated cells treated with the test substance and comparing the miRNA expression pattern to the miRNA expression pattern in isolated control cells not treated with the test substance. A method for screening a therapeutic agent for premature ovarian failure caused by Turner syndrome, comprising:

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