KR102178919B1 - Biomarker microRNAs for diagnosing diabetic nephropathy and use thereof - Google Patents

Abstract

The present invention relates to a biomarker for diabetic nephropathy and its use, and more specifically, microRNA-642a (miR-642a), microRNA-1246 (miR-1246), microRNA-1255b (miR-1255b) , And a marker composition for diabetic nephropathy diagnosis comprising at least one selected from the group consisting of microRNA-5010 (miR-5010), a composition and kit for diabetic nephropathy diagnosis comprising an agent for measuring the expression level of the miRNA, the It relates to a method of diagnosing diabetic nephropathy using a marker. The microRNAs specific for the presence or absence of renal complications according to the present invention may be usefully used as biomarkers for diagnosing diabetic nephropathy and predicting progression.

Description

Biomarker microRNAs for diagnosing diabetic nephropathy and use thereof {Biomarker microRNAs for diagnosing diabetic nephropathy and use thereof}

The present invention relates to a biomarker for diabetic nephropathy and its use, and more specifically, microRNA-642a (miR-642a), microRNA-1246 (miR-1246), microRNA-1255b (miR-1255b) , And a marker composition for diabetic nephropathy diagnosis comprising at least one selected from the group consisting of microRNA-5010 (miR-5010), a diabetic nephropathy diagnostic composition and a diagnostic kit comprising an agent for measuring the expression level of the miRNA, It relates to a method for diagnosing diabetic nephropathy using the above marker.

Diabetes is a type of metabolic disease such as insufficient insulin secretion or inability to function normally.It is characterized by high blood sugar, which increases the concentration of glucose in the blood, and causes various symptoms and signs due to high blood sugar, and glucose is excreted from the urine. . Type 1 diabetes, also called'child diabetes', is caused by an absolute or relative lack of insulin production due to the destruction of beta cells in the pancreas by an autoimmune mechanism, whereas type 2 diabetes is induced by insulin resistance and obesity, It is known to be caused by various factors such as decrease in insulin receptor, decrease in insulin secretion, and genetic factors.

According to a 2016 presentation by the Korean Diabetes Association, the prevalence of diabetes among adults over 30 years of age in Korea recorded an all-time high of 13.7% as of 2014, and the risk of accompanying diseases of diabetes was also at a serious level. They reached 48.6% and 58.9%, and hypertension and hypercholesterolemia were also associated with 54.7% and 31.6%, respectively. As such, diabetes can lead to very serious complications such as cardiovascular disease, retinopathy, kidney failure, and peripheral neuropathy as well as its own risk.

Diabetic nephropathy, one of the complications of diabetes, is a disease in which the glomerulus inside the kidney is continuously damaged due to high blood sugar, and kidney function decreases. Diabetes slowly progresses kidney disease over 10 to 15 years. If not treated well in the early stages of diabetes, kidney function may be impaired, and in severe cases, end-stage renal failure requiring dialysis and transplantation can result. The etiology of diabetic nephropathy has not been accurately identified, but a combination of factors such as intrarenal hemodynamic alterations, glycosylation of glomerular proteins, and accumulation of sorbitol are related to the etiology of diabetic nephropathy. It is believed to be there.

Currently, renin-angiotensin system (RAS) inhibitors are mainly used to prevent diabetic nephropathy. RAS inhibitors not only have hemodynamic effects, but also exhibit excellent effects in protecting kidney function by inhibiting the production of cytokines, chemokines, and growth factors that induce chronic inflammatory reactions or fibrosis. However, the treatment effect is not meeting expectations because the patient's individual genetic tendencies are different, and there are side effects from long-term treatment and resistance to the treatment. In addition, despite intensive research on the cause of nephropathy, diagnosis, and treatment for the past several years, a definite therapeutic agent that can effectively treat nephropathy has not yet been developed. Therefore, it can be said that diabetic nephropathy is detected and treated in an early stage as the most effective method.

Conventional biomarkers for diagnosing diabetic nephropathy are limited. Diabetic nephropathy is diagnosed regularly and repeatedly through clinical symptoms and the patient's medical history. For example, patients with long-term diabetic history have nephrotic-range proteinuria and retinopathy. If there is no other obvious cause of kidney disease, diabetic nephropathy is diagnosed.

Microproteinuria (24 hours urinary albumin 30-300 mg/d or albumin excretion rate 20-200 ug/min) is the most widely used biomarker to predict the progression of diabetic nephropathy early and predict vascular complications other than nephropathy. . According to existing studies, microproteinuria is a risk factor for predicting a decrease in glomerular filtration rate and an independent risk factor for chronic renal failure, and therefore, measurement of microproteinuria is recommended in the current diabetes diagnosis and treatment guidelines. However, recent studies have shown that the frequency of spontaneous loss of microproteinuria is high in diabetic patients (Diabetes Care. 2001 Sep;24(9):1560-6.), and renal function is reduced without microproteinuria (glomerular filtration rate <60 ml/min/). 1.73m ² ) Due to the high frequency of diabetic patients, it is reported that there is a limit to early diagnosis of diabetic nephropathy with only microproteinuria and to judge it as a progression indicator.

Accordingly, the present inventors analyzed miRNAs present in exosomes derived from serum of a subject in order to discover biomarkers that can effectively diagnose diabetic nephropathy, and the expression was significantly increased in diabetic patients with renal complications compared to the normal control group. The present invention was completed by discovering 4 types of miRNAs.

In order to solve the conventional problems as described above, the present inventors collect serum from subjects corresponding to the normal group, the diabetic group without renal complications, and the diabetic group with renal complications, and obtain exosome RNA from them. As a result of comparative analysis of the expression level of exosome microRNA through NGS analysis after isolation, we found 4 types of microRNAs whose expression level significantly increased only in the diabetic group with renal complications compared to the normal group. The present invention has been completed.

Accordingly, the present invention is selected from the group consisting of microRNA-642a (miR-642a), microRNA-1246 (miR-1246), microRNA-1255b (miR-1255b), and microRNA-5010 (miR-5010). It is an object of the present invention to provide a marker composition for diabetic nephropathy diagnosis comprising at least one of which is to be.

In addition, the present invention is selected from the group consisting of microRNA-642a (miR-642a), microRNA-1246 (miR-1246), microRNA-1255b (miR-1255b), and microRNA-5010 (miR-5010) Another object of the present invention is to provide a diabetic nephropathy diagnostic composition and a diabetic nephropathy diagnostic kit comprising the composition, including an agent for measuring one or more expression levels.

In addition, the present invention provides microRNA-642a (miR-642a), microRNA-1246 (miR-1246), microRNA-1255b (miR-1255b), and microRNA-5010 (miR- Another object of the present invention is to provide a method of providing information for diabetic nephropathy diagnosis, including measuring the expression level of one or more selected from the group consisting of 5010).

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention is microRNA-642a (miR-642a), microRNA-1246 (miR-1246), microRNA-1255b (miR-1255b), and microRNA-5010 It provides a marker composition for diabetic nephropathy diagnosis comprising at least one selected from the group consisting of (miR-5010).

In addition, the present invention is selected from the group consisting of microRNA-642a (miR-642a), microRNA-1246 (miR-1246), microRNA-1255b (miR-1255b), and microRNA-5010 (miR-5010) It provides a composition for diabetic nephropathy diagnosis comprising an agent for measuring one or more expression levels.

In addition, the present invention provides a diabetic nephropathy diagnostic kit comprising the diabetic nephropathy diagnostic composition.

As an embodiment of the present invention, the miR-642a may be composed of the nucleotide sequence of SEQ ID NO: 1.

In another embodiment of the present invention, the miR-1246 may be composed of the nucleotide sequence of SEQ ID NO: 2.

In another embodiment of the present invention, the miR-1255b may be composed of the nucleotide sequence of SEQ ID NO: 3.

In another embodiment of the present invention, the miR-5010 may be made of the nucleotide sequence of SEQ ID NO: 4.

In another embodiment of the present invention, the agent for measuring the expression level may be a sense and antisense primer or probe that complementarily binds to the microRNA.

In addition, the present invention provides microRNA-642a (miR-642a), microRNA-1246 (miR-1246), microRNA-1255b (miR-1255b), and microRNA-5010 (miR- 5010) provides a method for providing information for diabetic nephropathy diagnosis comprising the step of measuring the expression level of one or more selected from the group consisting of.

In one embodiment of the present invention, the biological sample may be blood or serum-derived exosomes.

In another embodiment of the present invention, the expression level 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, and northern blotting.It may be measured by one or more methods selected from the group consisting of.

The present inventors analyzed the microRNA contained in the exosomes isolated from the blood of the normal group, the diabetic group without renal complications, and the diabetic group with renal complications. As a result, the expression was significant only in the diabetic group with renal complication compared to the normal group. MicroRNAs specific for the presence or absence of nephropathy, miR-642a, miR-1246, miR-1255b, and miR-5010, which were increased significantly. Accordingly, the discovered microRNAs may be usefully used as biomarkers for diagnosing diabetic nephropathy and predicting progression.

1 is a result of comparative analysis of the expression levels of microRNAs in exosomes isolated from serum of normal group (HV) and diabetic group (Nephro-X) subjects without renal complication. FIG. 1A shows renal complications compared to the normal group. Heat map results of hierarchical clustering according to the expression level of microRNAs with significantly reduced or increased expression level of 2 times or more (p <0.05) in the diabetic group without, Figure 1b is the result of the expression level of the microRNAs detected above. This is the result of a scatter plot.
2 is a result of comparative analysis of the expression levels of microRNAs in exosomes isolated from serum of normal group (HV) and diabetic group (Nephro-O) subjects with renal complication. FIG. 2A shows renal complications compared to the normal group. Heat map results of hierarchical clustering according to the expression level of microRNAs with significantly decreased or increased expression levels of 2 times or more (p <0.05) in the diabetic group, and FIG. 2B is the result of the hierarchical clustering according to the expression level of the microRNAs detected above. This is the result of a scatter plot.
3 is a result of comparative analysis of the expression levels of microRNAs in exosomes isolated from the serum of subjects of diabetic group without nephropathy (Nephro-X) and diabetic group with nephropathy (Nephro-O), FIG. 3a is a kidney A heat map result of hierarchical clustering according to the expression level of microRNAs with significantly decreased or increased expression level of microRNAs that was significantly decreased or increased by 2 times or more (p <0.05) in the diabetic group with renal complication compared to the non-complication diabetic group, and FIG. 3B is It is a scatter plot result according to the expression level of the microRNAs detected above.
Figure 4 is a multidimensional scaling method (MDS) for the normal group (HV), the diabetic group without renal complications (Nephro-X), and the diabetic group (Nephro-O) without renal complications based on the results of FIGS. 1 to 3 above. This is the result of the analysis.
5 is a result of deriving only microRNAs with significantly increased expression only in the diabetic group with renal complications compared to the normal group by expressing only microRNAs with significantly increased expression from the results of FIGS. 1 to 3.
6 shows hsa-miR-642a-3p, hsa-miR-1246, hsa-miR-1255b-5p and hsa-miR-5010-5p between the normal group (HV) and the diabetic group (Nephro-O) with kidney complications. This is the result of comparing and measuring the expression level of.

As a result of intensive research to discover biomarkers for diabetic diagnosis, the present inventors have shown that microRNA-642a (miR-642a), microRNA-1246 (miR-1246), micro The present invention was completed by discovering RNA-1255b (miR-1255b) and microRNA-5010 (miR-5010).

Accordingly, the present invention is selected from the group consisting of microRNA-642a (miR-642a), microRNA-1246 (miR-1246), microRNA-1255b (miR-1255b), and microRNA-5010 (miR-5010). It provides a marker composition for diabetic nephropathy diagnosis, including one or more.

In addition, the present invention is selected from the group consisting of microRNA-642a (miR-642a), microRNA-1246 (miR-1246), microRNA-1255b (miR-1255b), and microRNA-5010 (miR-5010) It provides a diabetic nephropathy diagnostic composition and a diabetic nephropathy diagnostic kit comprising the diagnostic composition, including an agent for measuring one or more expression levels.

Accordingly, the miR-642a, more preferably hsa-miR-642a-3p, which is a biomarker for diabetic nephropathy discovered in the present invention, may be made of a nucleotide sequence represented by SEQ ID NO: 1 (NCBI, MIMAT0020924). In this case, the nucleotide sequence represented by SEQ ID NO: 1 and 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more may include a nucleotide sequence having sequence homology. .

In addition, the miR-1246, more preferably hsa-miR-1246, which is a biomarker for diabetic nephropathy discovered in the present invention, may be composed of a nucleotide sequence represented by SEQ ID NO: 2 (NCBI, MIMAT0005898). In this case, the nucleotide sequence represented by SEQ ID NO: 2 and 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more may include a nucleotide sequence having sequence homology. .

In addition, the miR-1255b, more preferably hsa-miR-1255b-5p, which is a biomarker for diabetic nephropathy discovered in the present invention, may be composed of a nucleotide sequence represented by SEQ ID NO: 3 (NCBI, MIMAT0005945). At this time, the nucleotide sequence represented by SEQ ID NO: 3 and 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more may include a nucleotide sequence having sequence homology. .

In addition, the miR-5010, more preferably hsa-miR-5010-5p, which is a biomarker for diabetic nephropathy discovered in the present invention, may be made of a nucleotide sequence represented by SEQ ID NO: 4 (NCBI, MIMAT0021043). In this case, the nucleotide sequence represented by SEQ ID NO: 4 and 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more may include a nucleotide sequence having sequence homology. .

The term "diagnosis" used in the present invention means in a broad sense to judge the condition of a patient's disease in all aspects. The content of the judgment is the name of the disease, etiology, disease type, severity, detailed mode of the bed, and the presence or absence of complications. In the present invention, the diagnosis is to determine whether the onset of diabetic complications and the level of progression, and more preferably, the diabetic complication may be diabetic nephropathy.

In the present invention, the agent for measuring the expression level of miR-642a, miR-1246, miR-1255b and/or miR-5010 may be a sense and antisense primer or probe that complementarily binds to each of the microRNAs. However, it is not limited thereto.

The term “primer” used in the present invention refers to a short gene sequence that serves as a starting point for DNA synthesis, and refers to an oligonucleotide synthesized for use in diagnosis or DNA sequencing. The primers are usually synthesized and used in a length of 15 to 30 base pairs, but may vary depending on the purpose of use, and may be modified by methylation or capping by a known method.

The term "probe" as used herein refers to a nucleic acid capable of specifically binding to an mRNA having a length of several to several hundreds of bases produced through enzymatic chemical separation or purification or synthesis. The presence or absence of mRNA can be confirmed by labeling radioactive isotopes or enzymes, and it can be designed and modified by a known method.

The diagnostic kit of the present invention is composed of one or more different component compositions, solutions, or devices suitable for analysis methods.

For example, in order to perform PCR, the kit of the present invention uses 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 DNA polymerase, dNTP mixture, PCR buffer solution, and water. It may be a kit including. The PCR buffer solution may contain KCl, Tris-HCl and MgCl ₂ . In addition, constituents necessary for electrophoresis that can confirm whether a 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. RT-PCR kits include test tubes or other suitable containers, reaction buffers, deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC, in addition to each primer pair specific for the marker gene. -May include DEPC-water, sterilized water, etc. In addition, a primer pair specific to a gene used as a quantitative control may be included.

The present inventors discovered and identified miR-642a, miR-1246, miR-1255b and miR-5010 as biomarkers for diabetic nephropathy diagnosis through specific examples.

In one embodiment of the present invention, subjects corresponding to the normal group, the diabetic group without renal complications, and the diabetic group with renal complications are recruited and provided with their serum to separate exosomes from the serum to express miRNA in exosomes. The levels were compared and analyzed (see Example 1).

In another embodiment of the present invention, microRNAs with differences in the normal group and the diabetic group without renal complications, the diabetic group without renal complications, the diabetic group without renal complications, and the diabetic group with renal complications were analyzed, respectively. And, as a result of analyzing the microRNAs expression level result by a multidimensional scale method, it was confirmed that a pattern was distinguished between the normal group and the diabetic group in two dimensions, and there was no significant difference with respect to the presence or absence of renal complications. In addition, microRNAs showing a significant increase in expression in each of the comparative analysis results were shown and compared with a Venn diagram. Specifically, in 40 miRNAs whose expression was increased in the diabetic group with renal complications compared to the normal group, kidneys compared to the normal group In the non-complication diabetic group, 8 types except for the common 32 miRNAs with increased expression were selected, and furthermore, among the 8 miRNAs, one type with increased expression in the diabetic group with renal complication compared to the diabetic group without renal complication was selected. The 4 miRNAs showing the most significant difference in expression were finally selected among the 7 types excepted. The hsa-miR-642a-3p, hsa-miR-1246, hsa-miR-1255b-5p and hsa-miR-5010-5p selected above actually showed a significant difference in expression between the normal group and the diabetic group with renal complications. It confirmed what was shown (Examples 2-1 to 2-4).

Accordingly, as another aspect of the present invention, the present invention provides microRNA-642a (miR-642a), microRNA-1246 (miR-1246), microRNA-1255b (miR-1255b), and microRNA in a biological sample derived from a subject. It provides a method of providing information for diabetic nephropathy diagnosis comprising the step of measuring the expression level of at least one selected from the group consisting of RNA-5010 (miR-5010).

The biological sample derived from the subject may include tissue, cells, whole blood, blood, saliva, sputum, cerebrospinal fluid, urine, and the like, and more preferably, blood or serum-derived exosomes, but is not limited thereto.

In the present invention, if the expression level of miR-642a, miR-1246, miR-1255b and/or miR-5010 in exosomes isolated from a sample derived from a subject according to the above method is higher than that of a normal person, it can be determined as diabetic nephropathy. have.

The expression level of the microRNAs is determined by conventional methods known in the art: polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (Real-time PCR), and RNase protection assay (RNase). Protection assay; RPA), microarray (microarray), or may be measured by one or more methods selected from the group consisting of northern blotting (northern blotting), but is not limited thereto.

Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. Experiment preparation and experiment method

1-1. Recruitment of targets for analysis

In order to discover biomarkers capable of early diagnosis of diabetic nephropathy, which is a kidney complication caused by diabetes, the present inventors have 18 normal groups (HV), 33 diabetic patients without kidney complications (Nephro-X), and 23 diabetic patients with nephropathy (Nephro-O) were recruited, and serum samples were collected from them to find biomarkers representing differences between groups. To this end, serum was provided from subjects in each group.

1-2. RNA isolation and sequencing from serum

Exoquick (SBI, USA) was used to isolate exosomes for serum samples collected from subjects of each group in Example 1-1, and total RNA from the isolated exosomes using Qiagen miRNeasy kit Was extracted, and the quality of the extracted total RNA was measured according to the manufacturer's instructions using an Agilent RNA Bioanalyzer.

Thereafter, using the isolated RNA, a library was prepared using the Takara SMARTer smRNA for illumina kit from Macrogen, and then next-generation sequencing (NGS) was performed with Hiseq 2500 to detect microRNA.

Example 2. Discovery of microRNA for diabetic nephropathy diagnosis

2-1. Normal vs. Analysis of microRNAs with differences in expression levels in diabetic groups without kidney complications

First, in order to identify microRNAs (miRNAs) having a difference in expression level in the blood-derived exosomes of 18 normal groups and 33 diabetic groups without renal complications, the methods of Examples 1-1 and 1-2 According to the following, exosomes were isolated from serum collected from each subject, and NGS analysis was performed on the extracted total RNA. As a result of the analysis, 107 miRNAs were detected, and further, the expression levels of the detected miRNAs were compared.

As a result, as shown in FIG. 1A, 62 miRNAs with reduced expression more than twice (p <0.05) in the diabetic group (Nephro-X) without renal complications (Nephro-X) among 107 miRNAs were expressed, compared to the normal group (HV). There were 45 of these increasing miRNAs. In addition, a scatter plot result according to the expression level of miRNAs showing the significant expression change is shown in FIG. 1B.

2-2. Normal vs. Analysis of microRNAs with differences in expression levels in diabetic groups with kidney complications

In addition to the above results, in order to identify miRNAs having a difference in expression level in the blood-derived exosomes in the 18 normal group and 23 diabetic groups with kidney complications, NGS analysis was performed. As a result of the analysis, 95 miRNAs were detected, and further, the expression levels of the detected miRNAs were compared.

As a result, as shown in Fig. 2a, 55 miRNAs whose expression is reduced by more than two times (p <0.05) in the diabetic group (Nephro-O) with renal complications compared to the normal group (HV) among 95 miRNAs were expressed. There were 40 increasing miRNAs. In addition, a scatter plot result according to the expression level of miRNAs showing the significant expression change is shown in FIG. 2B.

2-3. Diabetic group without kidney complication vs. Analysis of miRNAs with differences in expression levels in diabetic groups with kidney complications

In addition, in order to identify miRNAs having a difference in expression level in the blood-derived exosomes of 33 diabetic groups without renal complications and 23 diabetic groups with renal complications, the present inventors have obtained total RNA in exosomes obtained by the same method as above. NGS analysis was carried out. As a result of the analysis, 12 miRNAs were detected, and further, the expression levels of the detected miRNAs were compared.

As a result, as shown in Fig. 3a, the expression of 12 miRNAs decreased by more than twice (p <0.05) in the diabetic group (Nephro-O) with renal complication compared to the diabetic group (Nephro-X) without renal complication. There were 9 miRNAs and 3 miRNAs with increasing expression. In addition, a scatter plot result according to the expression level of miRNAs showing the significant expression change is shown in FIG. 3B.

2-4. Normal vs. Diabetic group without kidney complication vs. Analysis of miRNAs with differences in expression levels between diabetic groups with kidney complications

Furthermore, for the normal group, the diabetic group without renal complications, and the diabetic group with renal complications, the amount of change for individual samples was derived through the multidimensional scale (MDS), and this was visualized and analyzed. As a result, as shown in FIG. 4, the two-dimensional spatial positions of the normal group and the diabetic group were clearly divided into two groups, but the difference according to the presence or absence of renal complications was not largely divided. However, if miRNAs that differ depending on the presence or absence of renal complications are discovered, they can be used as useful biomarkers for diagnosing renal complications, so the present inventors based on the analysis results obtained through the results of Examples 2-1 to 2-3. Thus, an additional comparative analysis was conducted to discover miRNA for diagnosing renal complications.

To this end, miRNAs confirmed to be significantly increased in expression as a result of each analysis are shown in the Venn diagram of FIG. 5. As a result, 40 miRNAs (C/A) with significantly increased expression in the diabetic group (Diabetes-Nephro-O) with kidney complications compared to the normal group (HV), and the diabetic group without kidney complications ( Diabetes-Nephro-X) compared to the normal group by comparing 45 miRNAs (B/A) with significantly increased expression and excluding 32 miRNAs (C/A & B/A) that are common. Eight miRNAs with increased expression were obtained only in the diabetic group. Thereafter, the result of comparing the results of 3 miRNAs (C/B) with significantly increased expression in the diabetic group with renal complication compared to the diabetic group without renal complication in Example 2-3 was compared with the obtained 8 miRNAs, common One type of miRNA with increased expression was identified. Accordingly, 7 kinds of miRNAs except for the one common miRNA were obtained from the 8 miRNAs, and 4 kinds of miRNAs showing the most significant difference in expression level were finally selected. The selected four miRNAs are shown in Table 1 below, and it was confirmed that they showed significant differences of 2.51, 2.02, 2.08, and 2.54 times, respectively, in the group with renal complications compared to the normal group.

Diabetes-renal complication/normal group (C/A)
fold change hsa-miR-642a-3p 2.51 * hsa-miR-1246 2.02 ** hsa-miR-1255b-5p 2.08 ** hsa-miR-5010-5p 2.54 **

In addition, as shown in Figure 6, the four miRNAs discovered above between the normal group (HV) and the diabetic group (Nephro-O) with kidney complications, that is, hsa-miR-642a-3p, hsa-miR-1246, It was confirmed that the expression of hsa-miR-1255b-5p and hsa-miR-5010-5p showed a significant difference.

Therefore, the above results show that 4 kinds of miRNAs, whose expression is significantly increased only in the diabetic group with renal complications compared to the normal person, can be usefully used as a biomarker capable of specifically diagnosing diabetic nephropathy.

The above-described description of the present invention is for illustration purposes only, and those of ordinary skill in the art to which the present invention pertains can understand that it is possible to easily transform it into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not limiting.

<110> Soonchunhyang University Industry Academy Cooperation Foundation <120> Biomarker microRNAs for diagnosing diabetic nephropathy and use thereof <130> PD18-145 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> RNA <213> hsa-miR-642a-3p <400> 1 agacacauuu ggagagggaa cc 22 <210> 2 <211> 19 <212> RNA <213> hsa-miR-1246 <400> 2 aauggauuuu uggagcagg 19 <210> 3 <211> 22 <212> RNA <213> hsa-miR-1255b-5p <400> 3 cggaugagca aagaaagugg uu 22 <210> 4 <211> 22 <212> RNA <213> hsa-miR-5010-5p <400> 4 agggggaugg cagagcaaaa uu 22

Claims (19)

A marker composition for diabetic nephropathy diagnosis comprising microRNA-1246 (miR-1246).
The method of claim 1,
The marker composition is in the group consisting of microRNA-642a-3p (miR-642a-3p), microRNA-1255b-5p (miR-1255b-5p), and microRNA-5010-5p (miR-5010-5p). Characterized in that it further comprises one or more selected, the marker composition.
The method of claim 1,
The miR-1246 is characterized in that consisting of the nucleotide sequence of SEQ ID NO: 2, marker composition.
The method of claim 2,
The miR-642a-3p, miR-1255b-5p, and miR-5010-5p are characterized in that consisting of the nucleotide sequences of SEQ ID NOs: 1, 3 and 4, respectively, a marker composition.
A composition for diabetic nephropathy diagnosis comprising an agent for measuring the expression level of microRNA-1246 (miR-1246).
The method of claim 5,
The diagnostic composition is in the group consisting of microRNA-642a-3p (miR-642a-3p), microRNA-1255b-5p (miR-1255b-5p), and microRNA-5010-5p (miR-5010-5p). A composition for diagnosing diabetic nephropathy, characterized in that it further comprises an agent for measuring one or more selected expression levels.
The method of claim 5,
The miR-1246 is characterized in that consisting of the nucleotide sequence of SEQ ID NO: 2, diabetic nephropathy diagnostic composition.
The method of claim 6,
The miR-642a-3p, miR-1255b-5p, and miR-5010-5p are each composed of the nucleotide sequence of SEQ ID NO: 1, 3 and 4, diabetic nephropathy diagnostic composition.
The method of claim 5,
The agent for measuring the expression level is a sense and antisense primer, or a probe that complementarily binds to the microRNA, a composition for diabetic nephropathy diagnosis.
A kit for diabetic nephropathy diagnosis comprising the composition of any one of claims 5 to 9.
A method for providing information for diabetic nephropathy, comprising measuring the expression level of microRNA-1246 (miR-1246) in blood or serum-derived exosomes derived from a subject.
The method of claim 11,
The information providing method is a group consisting of microRNA-642a-3p (miR-642a-3p), microRNA-1255b-5p (miR-1255b-5p), and microRNA-5010-5p (miR-5010-5p). The method of providing information, characterized in that it further comprises the step of measuring one or more expression levels selected from.
The method of claim 11,
The miR-1246 is characterized in that consisting of the nucleotide sequence of SEQ ID NO: 2, information providing method.
The method of claim 12,
The miR-642a-3p, miR-1255b-5p, and miR-5010-5p are characterized in that consisting of nucleotide sequences of SEQ ID NOs: 1, 3 and 4, respectively.
The method of claim 11,
The expression level is 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. (RNase protection assay; RPA), microarray (microarray), and characterized in that measured by one or more methods selected from the group consisting of northern blotting (northern blotting), information providing method. delete delete delete delete

Images