KR20220115152A - Biomarker composition for diagnosing idiopathic nephrotic syndrome and use thereof - Google Patents

Abstract

The present invention relates to a micro RNA biomarker for diagnosis of idiopathic nephrotic syndrome and a method for providing information for diagnosis using the biomarker. More particularly, as a result of analyzing microRNA contained in exosomes isolated from blood of a normal group, idiopathic nephrotic syndrome and membranous glomerulonephritis, the expression of microRNA-335 and microRNA-17 was significantly increased or decreased in idiopathic nephrotic syndrome, compared to the normal group and membranous glomerulonephritis. By providing the microRNA as a biomarker for diagnosing idiopathic nephrotic syndrome, the microRNA can be usefully used in a method and a diagnostic kit for providing information for diagnosing idiopathic nephrotic syndrome.

Description

Biomarker composition for diagnosing idiopathic nephrotic syndrome and use thereof

The present invention relates to a microRNA biomarker for diagnosing idiopathic nephrotic syndrome and a method for providing information for diagnosis using the biomarker.

Primary nephrotic syndrome is defined as severe edema, massive proteinuria, and hypoalbuminemia of the blood. The causative disease of primary nephrotic syndrome is glomerulitis in which the glomerulus of the kidney is inflamed, and there are more than 10 types. Membranous glomerulonephritis (MGN) and idiopathic glomerulonephritis nephrotic syndrome (INS) accounts for the majority. Primary nephrotic syndrome is characterized by a variety of treatment methods and treatment prognosis for each disease. MGN generally does not respond to steroid treatment alone, and high-dose steroids and other immunosuppressive agents such as cyclophosphamide are required for resolution. There are also refractory MGNs that do not respond to treatment despite these treatments.

In nephrotic syndrome, an invasive kidney biopsy that collects kidney tissue using a gun needle is essential for an accurate diagnosis of the causative disease. In addition, invasive biopsy is a very dangerous test when taking antiplatelet agents and anticoagulation drugs for the recurrence of heart disease and cerebrovascular disease. Therefore, it is very important to develop a non-invasive biomarker that aids in the diagnosis of a disease along with biopsy, an invasive diagnostic method, or in cases where invasive biopsy is impossible.

On the other hand, microRNA (miRNA) is a non-coding small RNA that exists in the genome of both animals and plants and is known to be involved in all processes of living things. is becoming Most biomarkers currently used in clinical practice are proteins circulating in body fluids, but unfortunately, the development of new protein-based biomarkers faces considerable technical difficulties due to the low affinity of the target protein in the sample. Development can be an alternative that can overcome the limitations of protein biomarkers.

Republic of Korea Patent Publication No. 10-2019-0139075 (published on December 17, 2019)

The present invention provides a biomarker composition for effectively diagnosing idiopathic nephrotic syndrome in a non-invasive method, and a kit for diagnosing idiopathic nephrotic syndrome using the biomarker composition and an information providing method for diagnosing idiopathic nephrotic syndrome.

The present invention provides a biomarker composition for diagnosing idiopathic nephrotic syndrome comprising one or more microRNAs selected from the group consisting of miR-335 and miR-17.

The present invention provides an idiopathic nephrotic syndrome diagnostic kit comprising an agent for measuring the expression level of one or more microRNAs selected from the group consisting of miR-335 and miR-17.

In addition, the present invention comprises the steps of isolating a sample from the subject; and

It provides an information providing method for diagnosing idiopathic nephrotic syndrome, comprising measuring the expression level of one or more microRNAs selected from the group consisting of miR-335 and miR-17 in the isolated sample.

According to the present invention, as a result of analyzing the microRNA contained in the exosomes isolated from the blood of the normal group, idiopathic nephrotic syndrome and membranous glomerulonephritis, the expression significantly increased or By identifying the reduced microRNA-335 (microRNA-335) and microRNA-17 (microRNA-17), the microRNA is provided as a biomarker for diagnosing idiopathic nephrotic syndrome. A method of providing information for diagnosing idiopathic nephrotic syndrome and It can be usefully used in a diagnostic kit.

1 is a result of confirming the expression level of microRNAs in exosomes isolated from the serum of normal group (HV) and idiopathic nephrotic syndrome (INS) subjects. It is a heat map result obtained by performing hierarchical clustering according to the expression level of microRNAs with reduced or increased expression by more than 2 times (p < 0.05), and FIG. 1B is a scatter plot result according to the expression level of the microRNAs detected above. .
Figure 2 is the result of confirming the expression level of microRNAs in exosomes isolated from the serum of subjects with idiopathic nephrotic syndrome (INS) and membranous glomerulonephritis (MGN). (p < 0.05) is a heat map result obtained by performing hierarchical clustering according to the expression level of the microRNAs with decreased or increased expression, and FIG. 2B is a scatter plot result according to the expression level of the microRNAs detected above.
3 is a result of confirming the expression level of microRNAs in exosomes isolated from the serum of normal group (HV) and membranous glomerulonephritis (MGN) subjects. p < 0.05) is a heat map result obtained by performing hierarchical clustering according to the expression level of the microRNAs with decreased or increased expression, and FIG. 3B is a scatter plot result according to the expression level of the microRNAs detected above.
4a is a Venn diagram expressing microRNAs with significantly increased expression from the results of FIGS. 1, 2 and 3, and confirming microRNAs with increased expression only in idiopathic nephrotic syndrome compared to the normal group and membranous glomerulonephritis group (10 ), and FIG. 4b is a Venn diagram expressing microRNAs with significantly reduced expression from the results of FIGS. 1, 2, and 3, confirming the microRNAs with reduced expression in idiopathic nephrotic syndrome compared to the normal group and membranous glomerulonephritis group. (4).
5 is a result confirming the difference in the expression level of miR-335-5p or miR-17-5p between the normal group (normal) and idiopathic nephrotic syndrome (INS).

Hereinafter, the present invention will be described in more detail.

While the present inventors were researching a method for effectively diagnosing idiopathic nephrotic syndrome by a non-invasive method, microRNA specific for idiopathic nephrotic syndrome increased or decreased in serum-derived exosomes of idiopathic nephrotic syndrome patients compared to the normal group and membranous glomerulonephritis. By confirming, the present invention was completed.

The present invention may provide a biomarker composition for diagnosing idiopathic nephrotic syndrome comprising one or more microRNAs selected from the group consisting of miR-335 and miR-17.

The miR-335 may consist of the nucleotide sequence shown in SEQ ID NO: 1, and more specifically, may be hsa-miR-335-5p.

The miR-17-5p may consist of the nucleotide sequence shown in SEQ ID NO: 2, and more specifically, may be hsa-miR-17-5p.

The miR-335 may have increased expression compared to the normal group, and the miR-17 may have decreased expression compared to the normal group.

The present invention can provide an idiopathic nephrotic syndrome diagnostic kit comprising an agent for measuring the expression level of one or more microRNAs selected from the group consisting of miR-335 and miR-17.

The agent may be selected from the group consisting of primers, probes, antibodies, peptides and aptamers.

As used herein, the term “primer” refers to an oligonucleotide synthesized for use in diagnosis, DNA sequencing, etc. as a short gene sequence serving as a starting point of DNA synthesis. The primers can be synthesized and used with a length of typically 15 to 30 base pairs, but may vary depending on the purpose of use, and may be modified by methylation, capping, etc. by a known method.

As used herein, the term “probe” refers to a nucleic acid capable of specifically binding to mRNA having a length of several bases to several hundred bases produced through enzymatic chemical separation and purification or synthesis. The presence or absence of mRNA can be checked by labeling radioactive isotopes or enzymes, and can be designed and modified by a known method.

The diagnostic kit of the present invention consists of one or more other component compositions, solutions or devices suitable for the assay method.

For example, the kit of the present invention contains genomic DNA derived from a sample to be analyzed, a primer set specific for the marker gene of the present invention, an appropriate amount of a DNA polymerase, a dNTP mixture, a PCR buffer, and water to perform PCR. It may be a kit comprising The PCR buffer may contain KCl, Tris-HCl and MgCl ₂ . In addition, components necessary for performing electrophoresis that can confirm whether or not the PCR product is amplified may be additionally included in the kit of the present invention.

In addition, the kit of the present invention may be a kit including essential elements necessary for performing RT-PCR. In addition to each primer pair specific for a marker gene, the RT-PCR kit includes a test tube or other suitable container, reaction buffer, deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC -Water (DEPC-water), sterile water, etc. may be included. In addition, a primer pair specific for a gene used as a quantitative control may be included.

In addition, the present invention comprises the steps of isolating a sample from the subject; and measuring the expression level of one or more microRNAs selected from the group consisting of miR-335 and miR-17 in the isolated sample.

The information providing method may further include the step of confirming the increased level of proteinuria from the sample isolated from the subject.

The sample may be selected from the group consisting of tissue, cells, whole blood, blood, saliva, sputum, cerebrospinal fluid and urine, and more preferably may be blood or blood-derived exosomes, but is not limited thereto.

In the information providing method, the expression of miR-335 may be increased compared to the normal group, and when the expression of miR-17 is decreased compared to the normal group, idiopathic nephrotic syndrome can be diagnosed.

The expression level of the microRNA is 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), It may be measured by one or more methods selected from the group consisting of RNase protection assay (RPA), microarray, and northern blotting.

As used herein, the term “diagnosis” in a broad sense refers to judging the actual condition of a patient's disease in all aspects. The content of the judgment is the disease name, etiology, disease type, severity, detailed mode of the disease, and the presence or absence of complications. Diagnosis in the present invention is to determine whether or not the onset of idiopathic nephrotic syndrome and the level of progression.

Hereinafter, to help the understanding of the present invention, examples will be described in detail. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

<Experimental example>

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

1. Recruitment of analysis subjects

To discover idiopathic nephrotic syndrome diagnostic biomarkers for early diagnosis of idiopathic nephrotic syndrome and prevention of idiopathic nephrotic syndrome-related complications, 20 patients in the normal group (HV), 19 patients in the membranous glomerulonephritis group (MGN), and idiopathic nephrotic syndrome Twenty-one subjects with nephrotic syndrome were recruited and received serum from subjects in each of the above groups.

2. RNA Isolation and Sequencing from Serum

Previously, exosomes were isolated from serum samples collected from subjects in each group using Exoquick (SBI, USA), and total RNA was extracted from the isolated exosomes using Qiagen miRNeasy kit, and the extracted total RNA quality was measured using an Agilent RNA Bioanalyzer according to the manufacturer's instructions.

Thereafter, a library was prepared using the Takara SMARTer smRNA for illumina kit (Microgen) using the isolated RNA, and then the microRNA was detected by performing next-generation sequencing (NGS) with Hiseq 2500.

<Example 1> Discovering microRNA (microRNA) for diagnosis of idiopathic nephrotic syndrome

1. Identification of microRNAs showing differences in expression levels in normal group vs. idiopathic nephrotic syndrome

First, in order to identify microRNAs (miRNAs) with different expression levels in the blood-derived exosomes of 20 normal group and 21 idiopathic nephrotic syndrome patients recruited as in the previous experimental example, serum collected from each subject as in the experimental example. Exosomes were isolated from and NGS analysis was performed on the extracted total RNA. As a result of the analysis, 228 miRNAs were detected, and the expression levels of the detected miRNAs were compared.

As a result, as shown in FIG. 1a , among 44 miRNAs, 33 miRNAs with increased expression more than twice (p < 0.05) in idiopathic nephrotic syndrome compared to the normal group (HV) and 11 miRNAs with increased expression were confirmed. In addition, a scatter plot result according to the expression level of the miRNAs showing the significant expression change was confirmed as shown in FIG. 1B.

2. Idiopathic Nephrotic Syndrome vs. Analysis of microRNAs with differences in the expression level of membranous glomerulonephritis

In order to identify miRNAs having different expression levels in the blood-derived exosomes of 21 patients with idiopathic nephrotic syndrome and 19 patients with membranous glomerulonephritis, NGS analysis was performed on total RNA in the exosomes obtained in the same manner as above. As a result of the analysis, 228 miRNAs were detected, and the expression levels of the detected miRNAs were compared.

As a result, as shown in FIG. 2a , out of 11 miRNAs, 3 miRNAs with increased expression more than doubled (p < 0.05) in membranous glomerulonephritis compared to idiopathic nephritis, and 8 miRNAs with decreased expression were confirmed. In addition, a scatter plot result according to the expression level of the miRNA showing the significant expression change was confirmed as shown in FIG. 2b.

3. Normal control vs. Identification of microRNAs with differences in the expression level of membranous glomerulonephritis

In order to identify miRNAs having different expression levels in the blood-derived exosomes of 20 normal group and 19 membranous glomerulonephritis, NGS analysis was performed on total RNA in exosomes obtained in the same manner as above. As a result of the analysis, 228 miRNAs were detected, and the expression levels of the detected miRNAs were compared.

As a result, as shown in FIG. 3a , 31 miRNAs with increased expression and 27 miRNAs with decreased expression were identified in membranous glomerulonephritis in membranous glomerulonephritis, compared to the normal group (HV), among 58 miRNAs as shown in FIG. 3A . In addition, a scatter plot result according to the expression level of the miRNAs showing the significant expression change was confirmed as shown in FIG. 3b.

4. Identification of microRNA for diagnosis of idiopathic nephrotic syndrome

In order to discover miRNAs capable of specifically diagnosing idiopathic nephrotic syndrome from the previously confirmed results, miRNAs confirmed to have significantly increased expression as a result of the analysis are shown in a Venn diagram as shown in FIG. 4 .

As a result, 10 miRNAs with significantly increased expression in idiopathic nephrotic syndrome compared to the normal group and membranous glomerulonephritis and 4 miRNAs with reduced expression compared to the normal group and membranous glomerulonephritis were identified, and among the miRNAs, the most hsa-miR-335-5p and hsa-miR-17-5p, which show significant difference in expression level, were finally selected.

As shown in Table 1, it was confirmed that the final selected miRNAs showed a significant difference of 2.05 times and 2.04 times, respectively, in idiopathic nephrotic syndrome compared to normal persons, and as shown in FIG. 5 , hsa-miR-335-5p and hsa-miR-17- It was confirmed that 5p represents a significant difference in expression between the normal group and idiopathic nephrotic syndrome.

Idiopathic Nephrotic Syndrome/Normal
fold change hsa-miR-335-5p 2.05 *** hsa-miR-17-5p -2.04 ***

From the above results, the two types of miRNAs, whose expression is significantly increased or decreased only in idiopathic nephrotic syndrome compared to normal membranous glomerulonephritis, can be usefully used as biomarkers that can specifically diagnose idiopathic nephrotic syndrome. Confirmed.

<Example 2> Serum index confirmation

Changes in the level of various indicators were confirmed in the serum of 20 patients in the normal group (HV), 19 patients in the membranous glomerulonephritis group (MGN), and 21 patients in idiopathic nephrotic syndrome collected in the previous experimental example.

Variables study subjects P value
(MGN vs Idiopathic NS) HVs (n = 20) MGN (n = 19) Idiopathic NS
(n = 21) AGE 51.9±12.4 54.4±13.7 61.0±16.2 0.797 SEX (Male) 11 (55%) 11 (57.9%) 12 (57.1%) 0.962 WBC 5364.6±588.8 7490.7±1466.7 ^* 6985.5±1914.1 0.96 Hb 13.9±1.3 13.2±2.0 13.7±1.7 0.398 PLT (K) 219.9±48.1 303.0±100.2 ^* 289.3±70.3 ^** 0.209 BUN 12.9±3.2 14.1±5.5 21.3±10.4 0.009 Cr 0.9±0.2 0.9±0.2 1.3±0.8 ^** 0.04 eGFR 88.2±15.7 81.5±23.2 70.1±38.2 0.360 Baseline proteinuria (g/day) 82.3±49.3 7046.3±4423.2 ^* 9023.6±4062.6 ^** 0.220 AST 19.5±7.0 26.4±10.1 ^* 31.1±19.5 ^** 0.892 ALT 18.9±9.1 19.7±8.2 27.9±22.1 0.188 total protein 7.2±0.3 4.8±0.7 ^* 5.1±1.2 ^** 0.366 albumin 4.6±0.2 2.4±0.6 ^* 2.5±1.1 ^** 0.775 CRP 0.1±0.2 0.3±0.5 ^* 0.2±0.2 ^** 0.668 Total cholesterol 192.9±28.5 300.3±94.1 ^* 340.3±116.0 ^** 0.445 Triglycerides 108.7±48.4 332.2±214.9 ^* 454.1±350.9 ^** 0.871 HDL 60.8±9.3 53.1±19.0 58.1±21.4 0.552 LDL 123.8±30.1 180.8±98.7 201.9±84.3 ^** 0.913

* HVs vs MGN P value < 0.05; ** HVs vs Idiopathic NS P value < 0.05

HVs-Health volunteers, MGN-Membranous glomerulonephritis, NS-nephrotic syndrome. Hb-hemoglobin, PLT-platelet, BUN-blood urea nitrogen, Cr-creatinine, eGFR-estimated glomerular filtration rate, HDL-high density lipoprotein, LDL-low density lipoprotein

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> Research Cooperation Foundation of Yeungnam University <120> Biomarker composition for diagnosing idiopathic nephrotic syndrome and use thereof <130> ADP-2020-0616 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 23 <212> RNA <213> Unknown <220> <223> hsa-miR-335-5p <400> 1 ucaagagcaa uaacgaaaaa ugu 23 <210> 2 <211> 23 <212> RNA <213> Unknown <220> <223> hsa-miR-17-5p <400> 2 caaagugcuu acagugcagg uag 23

Claims (11)

A biomarker composition for diagnosing idiopathic nephrotic syndrome comprising one or more microRNAs selected from the group consisting of miR-335 (microRNA-335) and miR-17 (microRNA-17). The biomarker composition according to claim 1, wherein the miR-335 comprises the nucleotide sequence represented by SEQ ID NO: 1. The biomarker composition according to claim 1, wherein the miR-17 comprises the nucleotide sequence represented by SEQ ID NO: 2. The biomarker composition according to claim 1, wherein the miR-335 has increased expression compared to the normal group. The biomarker composition according to claim 1, wherein the miR-17 expression is reduced compared to the normal group. A diagnostic kit for idiopathic nephrotic syndrome comprising an agent for measuring the expression level of one or more microRNAs selected from the group consisting of miR-335 and miR-17. The diagnostic kit for idiopathic nephrotic syndrome according to claim 6, wherein the agent is selected from the group consisting of primers, probes, antibodies, peptides and aptamers. isolating the sample from the subject; and
An information providing method for diagnosing idiopathic nephrotic syndrome, comprising measuring the expression level of one or more microRNAs selected from the group consisting of miR-335 and miR-17 in the isolated sample. The method of providing information for diagnosing idiopathic nephrotic syndrome according to claim 8, wherein the method of providing information further comprises the step of confirming an increased level of proteinuria from a sample isolated from an individual. The method of claim 8, wherein the sample is selected from the group consisting of tissue, cell, whole blood, blood, saliva, sputum, cerebrospinal fluid, and urine. The method according to claim 8, wherein the expression level of the microRNA is 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 (microarray) and Northern blotting (northern blotting) characterized by measuring by one or more methods selected from the group consisting of idiopathic nephrotic syndrome diagnosis HOW TO PROVIDE INFORMATION.

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