KR102165841B1 - Biomarker microRNA let-7b or microRNA-664a for diagnosing diabetes and use thereof - Google Patents

Abstract

The present invention relates to a biomarker for diagnosing diabetes and its use, and more specifically, a marker composition comprising let-7b or microRNA-664a, a microRNA capable of diagnosing diabetes, the let-7b or microRNA- It relates to a composition and kit for diagnosing diabetes, including an agent for measuring the expression level of 664a, and a method for diagnosing diabetes using the marker. Accordingly, it is possible to diagnose diabetes early using the microRNAs according to the present invention, and it will be useful as a biomarker for diagnosing diabetes, predicting prognosis, and preventing related complications.

Description

Biomarker microRNA let-7b or microRNA-664a for diagnosing diabetes and use thereof {Biomarker microRNA let-7b or microRNA-664a for diagnosing diabetes and use thereof}

The present invention relates to a biomarker for diagnosing diabetes and its use, and more specifically, a marker composition for diagnosing diabetes containing let-7b or microRNA-664a (miR-664a), the let-7b or miR- It relates to a composition and kit for diagnosing diabetes, including an agent for measuring the expression level of 664a, and a method for diagnosing diabetes using the 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. Type 2 diabetes accounts for more than 90% of all diabetes, and recently, it is rapidly increasing with the increase of obesity and lifestyle habits of low activity in modern people. In particular, obesity has a positive correlation with type 2 diabetes. It can increase diabetes by 90 times.

According to a 2016 presentation by the Korean Diabetes Association, as of 2014, the prevalence of diabetes among adults over 30 years old in Korea reached a record high of 13.7%, and in the elderly over 65 years old, the prevalence rate reached over 30%, and fasting, pre-diabetes The prevalence of blood sugar disorders was also 24.8%, indicating that 1 out of 4 adults over 30 years of age was an ultra-high risk group waiting for diabetes. In addition, the risk of concomitant diseases of diabetes was also serious, and the prevalence of obesity and abdominal obesity in diabetic patients reached 48.6% and 58.9%, and hypertension and hypercholesterolemia were also associated with 54.7% and 31.6%, respectively. As such, diabetes can cause not only its own risk, but also very serious complications such as cardiovascular disease, retinopathy, kidney failure, and peripheral neuropathy, so it is essential to accurately diagnose and manage early.

Diabetes is diagnosed through blood tests such as urine glucose measurement, fasting blood glucose measurement, glucose tolerance test by oral or intravenous injection, and glycated hemoglobin measurement. There are Nilander's method, Benedict's method, and Testappe method for measuring urine glucose. When diabetes is mild, urinary sugar appears only after meals, and if there is a decrease in the diabetic discharge threshold of the kidney, there is also kidney diabetes, where urine sugar appears even without diabetes. Diabetes cannot be diagnosed only with the proof of. Fasting blood glucose measurement is diagnosed as diabetes if the fasting capillary blood glucose value before breakfast is 140 mg/dℓ or more, but mild cases may be within the normal range. Therefore, there is a need for a more accurate way to diagnose diabetes.

On the other hand, microRNA (miRNA) is a non-coding small RNA that exists in the genomes of both animals and plants, and is known to be involved in all processes of living organisms, and studies have recently been conducted to use these miRNAs as biomarkers for disease diagnosis. Has become. Currently, most biomarkers 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 to overcome the limitations of protein biomarkers. Recently, several miRNAs have an important effect on the development and function of beta cells and are involved in the development of diabetes, and reports have been published that these diabetes-related miRNAs are related to insulin resistance in target tissues such as fat, muscle, and hepatocytes (J. Endocrinol 222:R1-R10, 2014). Accordingly, analyzing changes in circulating miRNAs in diabetic patients is important for diagnosing and monitoring diabetes.

Accordingly, the present inventors analyzed miRNAs present in serum-derived exosomes to discover miRNAs biomarkers that can effectively diagnose diabetes, and identified two types of miRNAs whose expression is significantly increased in the diabetic patient group compared to the normal control group. By doing this, the present invention was completed.

In order to solve the conventional problems as described above, the present inventors collect serum from subjects corresponding to the normal group, obesity group, and obesity-diabetes group, and isolate exosome RNA from this, and then through NGS analysis. As a result of comparative analysis of the expression level of exosome microRNAs, two types of microRNAs were found whose expression levels were significantly increased only in the obese-diabetic group, not in the obese group compared to the normal subjects, and the present invention was completed based on this.

Accordingly, an object of the present invention is to provide a marker composition for diagnosing diabetes, including microRNA (microRNA) let-7b or microRNA-664a (microRNA-664a).

In addition, the present invention provides a composition for diagnosing diabetes, including an agent for measuring the expression level of microRNA (microRNA) let-7b or microRNA-664a (microRNA-664a), and a diabetes diagnosis kit comprising the composition. For a different purpose.

In addition, the present invention provides a method for diagnosing diabetes, comprising measuring the expression level of microRNA let-7b or microRNA-664a in a biological sample derived from a subject. It has another purpose.

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 provides a marker composition for diagnosing diabetes, including microRNA (microRNA) let-7b or microRNA-664a (microRNA-664a).

In addition, the present invention provides a composition for diagnosing diabetes, including an agent measuring the expression level of microRNA (microRNA) let-7b or microRNA-664a (microRNA-664a; miR-664a).

In addition, the present invention provides a diagnostic kit for diabetes comprising the composition for diagnosing diabetes.

As an embodiment of the present invention, let-7b may be composed of the nucleotide sequence of SEQ ID NO: 1.

In another embodiment of the present invention, the miR-664a may be composed of the nucleotide sequence of SEQ ID NO: 2.

In another embodiment of the present invention, the diabetes may be type 2 diabetes.

In another embodiment of the present invention, the agent for measuring the expression of let-7b or miR-664a may be a sense and antisense primer or probe that complementarily binds to the microRNA.

In addition, the present invention includes measuring the expression level of microRNA (microRNA) let-7b or microRNA-664a (microRNA-664a; miR-664a) in a biological sample derived from a subject, information for diabetic diagnosis Provide a method of delivery.

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 of let-7b or miR-664a is next generation sequencing (NGS), polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR). , Real-time PCR, RNase protection assay (RPA), microarray, and northern blotting through one or more methods selected from the group consisting of It can be measured.

The present inventors analyzed the microRNA contained in the exosomes isolated from the blood of the normal group, obesity group, and obesity-diabetic group, and as a result, the expression was significantly increased only in the obese-diabetic group compared to the normal group, and the expression was not increased in the obese group. Diabetes-specific microRNAs that do not have let-7b and miR-664a were identified. Accordingly, diabetes can be diagnosed early using the identified microRNAs, and may be usefully used as a biomarker for diagnosing diabetes, predicting prognosis, and preventing related complications.

1 is a result of comparative analysis of the expression level of microRNAs in exosomes isolated from serum of subjects in the normal group (HV) and obese group (Obese), and FIG. 1A is significantly more than twice (p) in the obese group compared to the normal group. <0.05) It is a heat map result of performing hierarchical clustering according to the expression level of microRNAs with decreased or increased expression, and FIG. 1B is a result of a scatter plot according to the expression level of the microRNAs detected above.
2 is a result of comparative analysis of the expression levels of microRNAs in exosomes isolated from serum of subjects in the normal group (HV) and obesity-diabetes group, and FIG. 2A is compared to the normal group in the obesity-diabetes group. A heat map result of hierarchical clustering according to the expression level of microRNAs with significantly decreased or increased expression of 2 times or more (p <0.05), and FIG. 2B is a scatter plot according to the expression level of the microRNAs detected above. ) Is the result.
3 is a result of comparative analysis of the expression levels of microRNAs in exosomes isolated from serum of obese and obese-diabetes subjects. It is a heat map result of performing hierarchical clustering according to the expression level of microRNAs with decreased or increased expression of 2 times or more (p <0.05), and FIG. 3B is a scatter plot result according to the expression level of the microRNAs detected above. to be.
FIG. 4 is a result of multidimensional scaling (MDS) analysis for the normal group (HV), the obese group (Obese), and the obesity-diabetes group (Ob-diabetes) based on the results of FIGS. 1 to 3.
5 is a result of deriving only microRNAs with increased expression only in the diabetic group compared to the normal group by expressing only microRNAs with significantly increased expression in a Venn diagram from the results of FIGS. 1 to 3.
6 is a result of measuring and comparing the expression levels of let-7b-5p and miR-664a-5p between the normal and obesity-diabetes group.

The present inventors of the present invention by finding a microRNA (microRNA) let-7b or microRNA-664a (microRNA-664a) as a biomarker that can be used for diabetes diagnosis as a result of intensive research to discover a biomarker for diabetes diagnosis. Was completed.

Accordingly, the present invention provides a marker composition for diagnosing diabetes including microRNA (microRNA) let-7b or microRNA-664a (microRNA-664a).

In addition, the present invention provides a diabetes diagnostic composition comprising an agent for measuring the expression level of microRNA (microRNA) let-7b or microRNA-664a (microRNA-664a), and a diabetes diagnostic kit comprising the diagnostic composition.

The present inventors discovered let-7b and microRNA-664a (miR-664a) as biomarkers for diagnosing diabetes through Examples.

In one embodiment of the present invention, subjects corresponding to the normal group, obesity group, and obesity-diabetes group were recruited, their serum was provided, and the exosomes were separated from the serum to compare and analyze the expression level of miRNA in the exosomes ( See example 1).

In another embodiment of the present invention, microRNAs that differ in the normal group and obesity group, the normal group and obesity-diabetes group, the obesity group and the obesity-diabetes group, respectively, were analyzed, and the results of analysis by a multidimensional scale method using the microRNA expression level result It was confirmed that the normal group, the obese group, and the obesity-diabetic group showed a sequential pattern on the 2D. In addition, microRNAs showing a significant increase in expression in the above comparative analysis results were shown and compared with a Venn diagram. Specifically, in 24 miRNAs with increased expression in the obesity-diabetes group compared to the normal group, expression in the obese group compared to the normal group 13 types excluding the increased common 11 miRNAs were selected, and furthermore, 2 types showing the most significant difference in expression among 9 types except 4 types with increased expression in the obesity-diabetes group compared to the obesity group among the 13 miRNAs. The miRNAs were finally selected. It was confirmed that the hsa-let-7b-5p and hsa-miR-664a-5p selected above actually showed a significant difference in expression between the normal group and the obesity-diabetic group (Examples 2-1 to 2-4).

Accordingly, the let-7b, more preferably hsa-let-7b-5p, which is a biomarker for diagnosing diabetes discovered in the present invention may be composed of a nucleotide sequence (NCBI, MIMAT0000063) represented by SEQ ID NO: 1. At this time, 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-664a, more preferably hsa-miR-664a-5p, which is a biomarker for diagnosing diabetes discovered in the present invention may be composed of a nucleotide sequence represented by SEQ ID NO: 2 (NCBI, MIMAT 0005948). 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.

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 the onset of diabetes and the level of progression, and more preferably, the diabetes may be type 2 diabetes.

In the present invention, the agent for measuring the expression of let-7b or miR-664a may be a sense and antisense primer or probe that complementarily binds to the microRNA, but 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.

As another aspect of the present invention, the present invention provides a method of providing information for diagnosing diabetes, comprising measuring the expression level of microRNA let-7b or miR-664a in a biological sample derived from a subject. .

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 let-7b or miR-664a in exosomes isolated from a sample derived from a subject according to the above method is higher than that of a normal person, diabetes may be determined.

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 diagnose diabetes early and discover biomarkers for diagnosing diabetes to prevent the onset of diabetes-related complications, the present inventors have 18 normal groups (HV, BMI less than 25) and obese groups without diabetes (obese, BMI 30- Severe obesity over 35) and 13 subjects in the obese-diabetes group (Obese-diabetes, BMI 30-35 or higher + fasting blood glucose 126 or higher) were recruited, and serum samples were collected from them. Thus, we tried 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. Diabetes diagnosis microRNA discovery

2-1. Normal vs. MicroRNA analysis with differences in expression levels in obese groups

First, in order to identify microRNAs (miRNAs) having a difference in expression levels in the blood-derived exosomes of 18 normal groups and 16 obese groups without diabetes, according to the methods of Examples 1-1 and 1-2 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, 54 miRNAs were detected, and further, the expression levels of the detected miRNAs were compared.

As a result, among the 54 miRNAs, as shown in FIG. 1A, 28 miRNAs decreased expression (p <0.05) in the obese group (Obese) compared to the normal group (HV), and 26 miRNAs increased expression. appear. 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. MicroRNA analysis with differences in expression levels in obesity-diabetes group

In addition to the above results, the present inventors identified miRNAs having a difference in expression level in the blood-derived exosomes of the 18 normal group and 13 obese-diabetic groups with obesity and diabetes, in exosomes obtained by the same method as above. NGS analysis was performed for total RNA. As a result of the analysis, 53 miRNAs were detected, and further, the expression levels of the detected miRNAs were compared.

As a result, as shown in Fig. 2a, among 53 miRNAs, 29 miRNAs with a decrease in expression more than twice (p <0.05) in the obesity-diabetic group (Ob-Diabetes) compared to the normal group (HV) were 29, and the expression increased. There were 24 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. Obese vs. Analysis of miRNAs with differences in expression levels in obesity-diabetes group

In addition, the inventors of the present invention in order to identify the miRNA having a difference in expression level in the blood-derived exosomes of the obese group without diabetes and the obese and obese-diabetic group of 13 people with diabetes, total in exosomes obtained by the same method as above NGS analysis was performed for RNA. As a result of the analysis, 25 miRNAs were detected, and further, the expression levels of the detected miRNAs were compared.

As a result, as shown in FIG. 3A, 11 miRNAs whose expression is decreased by more than two times (p <0.05) in the obese-diabetic group (Ob-Diabetes) compared to the obese group (Obese) were 11, and the expression increased. There were 14 miRNAs. 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. Obese vs. Analysis of miRNAs with differences in expression levels in obesity-diabetes group

Furthermore, for the normal group, obesity group, and obesity-diabetes group, the amount of change for individual samples was derived through the multidimensional scale method (MDS), and this was visualized and analyzed. As a result, as shown in FIG. 4, the two-dimensional spatial locations that appear in the normal group and the obese group were clearly divided into two groups, but in the case of the obese group and the obese-diabetic group, there were many overlapping parts of the spatial location. appear. However, it was confirmed that the normal group, the obese group, and the obese-diabetic group showed sequential patterns.

Thus, in order to discover miRNAs capable of specifically diagnosing diabetes based on the analysis results obtained through the results of Examples 2-1 to 2-3, it was confirmed that the expression of each analysis result was significantly increased. The miRNAs are shown in the Venn diagram of FIG. 5. As a result of the analysis, 24 miRNAs (C/A) with significantly increased expression in the obese-diabetic group compared to the normal group and 26 miRNAs (B/A) with a significantly increased expression in the obese group compared to the normal group were compared. By excluding the 11 miRNAs (C/A & B/A) present, 13 miRNAs with increased expression were obtained only in the obese-diabetic group compared to the normal group. Thereafter, compared to the obese group in Example 2-3, the results of 14 miRNAs (C/B) with significantly increased expression in the obese-diabetic group were compared with the obtained 13 miRNAs, and as a result, 4 types with increased expression in common Of miRNAs were identified. Accordingly, 9 types of the 13 miRNAs excluding the 4 types of miRNAs in common were obtained, and two types of miRNAs showing the most significant difference in expression level were finally selected. The selected two miRNAs are shown in Table 1 below, and it was confirmed that the obesity-diabetic group showed a significant difference of 2.18 and 2.23 times, respectively, compared to the normal person.

Obesity-diabetes/normal group (C/A) fold change hsa-let-7b-5p 2.18 *** hsa-miR-664a-5p 2.23 **

In addition, it was confirmed that the expression of hsa-let-7b-5p and hsa-miR-664a-5p between the normal group and the obesity-diabetic group (Ob-Diabetes) showed a significant difference as shown in FIG. .

Therefore, the above results show that two types of miRNAs whose expression is significantly increased only in the obesity-diabetic group compared to the normal person can be effectively used as a biomarker capable of specifically diagnosing diabetes.

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 microRNA let-7b or microRNA-664a for diagnosing diabetes and use thereof <130> PD18-132 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> RNA <213> hsa-let-7b-5p <400> 1 ugagguagua gguugugugg uu 22 <210> 2 <211> 24 <212> RNA <213> hsa-miR-664a-5p <400> 2 acuggcuagg gaaaaugauu ggau 24

Claims (16)

delete delete delete delete A composition for diagnosing type 2 diabetes, comprising an agent for measuring the expression level of microRNA-664a-5p (microRNA-664a-5p).
The method of claim 5,
The diagnostic composition further comprises an agent for measuring the expression level of microRNA (microRNA) let-7b-5p, a composition for diagnosing type 2 diabetes.
The method of claim 5,
The microRNA-664a-5p is a composition for diagnosing type 2 diabetes, characterized in that consisting of the nucleotide sequence of SEQ ID NO: 2.
The method of claim 6,
The let-7b-5p is a composition for diagnosing type 2 diabetes, characterized in that consisting of the nucleotide sequence of SEQ ID NO: 1.
delete The method of claim 5 or 6,
The agent for measuring the expression level of the microRNA is a sense and antisense primer, or a probe that complementarily binds to the microRNA, a composition for diagnosing type 2 diabetes.
A kit for diagnosing type 2 diabetes, comprising the composition of claim 5 or 6.
A method for providing information for diagnosing type 2 diabetes, comprising measuring the expression level of microRNA-664a-5p (microRNA-664a-5p) in a biological sample derived from a subject.
The method of claim 12,
The information providing method further comprises the step of measuring the expression level of microRNA (microRNA) let-7b-5p. A method of providing information for diagnosing type 2 diabetes.
The method of claim 12 or 13,
The method for providing information for diagnosing type 2 diabetes, characterized in that the biological sample is an exosome derived from blood or serum.
The method of claim 12 or 13,
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 (RNase protection assay; RPA), microarray (microarray), characterized in that measured by one or more methods selected from the group consisting of northern blotting (northern blotting), for diagnosing type 2 diabetes How to provide information. delete

Images