KR20200002241A - Biomarker microRNA-26b or microRNA-4449 for diagnosing obesity and use thereof - Google Patents

Abstract

The present invention relates to a biomarker for obesity diagnosis and usages thereof, and more specifically, to a marker composition comprising micro RNA-26b or micro RNA-4449 which can diagnose obesity; a composition and a kit for diagnosing obesity which comprise agents for measuring expression levels of the micro RNA-26b or micro RNA-4449; and a method for diagnosing obesity by using the marker. The expressions of micro RNAs according to the present invention are significantly increased in a boundary group compared with a normal control group even in a normal group, and obesity can be diagnosed in an early stage by using the micro RNAs. In addition, the present invention can be useful as a biomarker for targeting and developing materials for preventing or treating obesity and relevant complications.

Description

Biomarker microRNA-26b or microRNA-4449 for diagnosing obesity and use approximately}

The present invention relates to a biomarker and its use for diagnosing obesity, more specifically a marker composition comprising a microRNA-26b or microRNA-4449 capable of diagnosing obesity, the microRNA-26b or microRNA-4449 It relates to a composition and kit for diagnosing obesity, comprising an agent for measuring the expression level of, and to a method for diagnosing obesity using the marker.

Obesity is caused by excessive fat tissue in the body and caused by various causes such as mental and social factors, heredity, disease, drugs, etc. Especially in modern times, the prevalence of obesity due to excessive calorie intake or high fat diet is increasing. . According to the World Health Organization (WHO) in 2010, the world's overweight adult population is estimated to be 1.6 billion people worldwide and 400 million people who are obese, with 2.6 million people reported dying from obesity and overweight each year. In Korea, according to the report by the Ministry of Health and Welfare and the Centers for Disease Control and Prevention, the adult obesity rate is continuously increasing, and as of 2010, it was about 30.8% of the total population (36.3% for men and 24.8% for women). As such, the obese population is continuously increasing worldwide, and the burden of medical expenses is also increasing.

Obesity is more serious because it can cause a variety of complications if persisted. Specifically, obesity is known to increase the risk of developing hypertension, hyperlipidemia, metabolic syndromes such as diabetes, fatty liver, joint abnormalities, and cancer.As published by the World Health Organization in 2010, hypertension in people who are obese compared to people of normal weight , Diabetes mellitus, dyslipidemia is reported to be more than twice the risk (hypertension 2.5 times, diabetes 2 times, hypercholesterolemia 2.3 times, hypertriglyceridemia 2.4 times). In addition to the above diseases, excess body fat in women may lead to infertility in severe sex hormone balance, which is known to increase the risk of endometrial and breast cancer. In addition, since obesity can cause not only physical diseases but also mental illnesses such as social isolation, alienation, lack of confidence, and depression, the need for the prevention and treatment of obesity is very important. Therefore, early diagnosis and management of obesity is very important to prevent obesity and related complications.

Obesity can be measured by height and weight and diagnosed by measuring the thickness of skin wrinkles using a caliper. As a method of quantifying the degree of obesity, the modified broca's method and the body mass index (BMI) are used. Body mass index is the weight divided by the square of height. In the West, more than 30, considering the differences between races in Korea, diagnosed as over 25. However, since these diagnostic methods rely only on limited physical characteristics, it is difficult to accurately diagnose obesity using these measures alone. Thus, the present inventors completed the present invention by discovering the obesity-specific biomarker that the expression is specifically increased as the degree of obesity increases for the early diagnosis of obesity.

In order to solve the conventional problems as described above, the present inventors collected the serum of subjects corresponding to the normal group and the obese group and isolate the exosome RNA from the exosome RNA from the expression level of the exosome microRNA through NGS analysis As a result of the comparative analysis, as the obesity level was found to increase the expression level significantly increased microRNAs, the present invention was completed based on this.

Accordingly, an object of the present invention is to provide a marker composition for diagnosing obesity, including microRNA-26b (microRNA-26b; miR-26b) or microRNA-4449 (microRNA-4449; miR-4449).

In addition, the present invention comprises a composition for diagnosing obesity and the composition comprising an agent for measuring the expression level of microRNA-26b (microRNA-26b; miR-26b) or microRNA-4449 (microRNA-4449; miR-4449) Another object is to provide a diagnostic kit for obesity.

The present invention also includes measuring the expression level of microRNA-26b (microRNA-26b; miR-26b) or microRNA-4449 (microRNA-4449; miR-4449) in a biological sample derived from a subject. Another object is to provide a method for providing information for diagnosing obesity.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is 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 comprises a microRNA-26b (microRNA-26b; miR-26b) or microRNA-4449 (microRNA-4449; miR-4449), diagnostic marker composition for obesity To provide.

The present invention also provides a composition for diagnosing obesity, comprising an agent for measuring the expression level of microRNA-26b (microRNA-26b; miR-26b) or microRNA-4449 (microRNA-4449; miR-4449).

The present invention also provides a kit for diagnosing obesity comprising the composition for diagnosing obesity.

In one embodiment of the invention, the microRNA-26b may be composed of the nucleotide sequence of SEQ ID NO: 1.

In another embodiment of the present invention, the microRNA-4449 may be composed of the nucleotide sequence of SEQ ID NO: 2.

In another embodiment of the invention, the agent for measuring the expression of the microRNA-26b or microRNA-4449 may be a sense and antisense primer, or probe that complementarily binds to the microRNA.

The present invention also includes measuring the expression level of microRNA-26b (microRNA-26b; miR-26b) or microRNA-4449 (microRNA-4449; miR-4449) in a biological sample derived from a subject. It provides information providing method for diagnosing obesity.

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

In another embodiment of the present invention, the expression level of the microRNA-26b or microRNA-4449 is next generation sequencing (NGS), polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR) Through one or more methods selected from the group consisting of real-time PCR, RNase protection assay (RPA), microarray, and northern blotting. Can be measured.

The present inventors analyzed microRNAs contained in exosomes isolated from blood of normal and obese groups, and identified obesity-specific microRNA-26b and microRNA-4449 that significantly increased expression in obese groups compared to normal groups. The identified microRNAs have significantly increased expression in the borderline group of normal and obesity in the normal group compared to the normal control group. The microRNAs can be used to diagnose obesity early and prevent or treat obesity and related complications. It may be usefully used as a target and a biomarker for development.

Figure 1a is a result of analyzing the expression level of microRNAs in exosomes isolated from the serum of normal and obese group subjects, Figure 1b is a result of performing a multi-dimensional scaling (MDS) analysis based on the results of Figure 1a.
FIG. 2 illustrates age, height, weight, and weight of subjects included in the normal control group included in the normal group identified in FIG. 1B and the middle and normal groups of obesity. And body mass index (BMI).
Figure 3a is a result of analyzing the expression level of microRNAs in exosomes isolated from the serum of the normal control group and obese group subjects, except for the border group, Figure 3b is based on the results of Figure 3a multidimensional scaling (MDS) analysis The result is.
4 shows the expression of hsa-miR-26b-3p (FIG. 4A) and hsa-miR-4449 (FIG. 4B) between normal and obese, or between normal and middle. This is a result of measuring the level.

The present inventors completed the present invention by finding microRNA-26b or microRNA-4449 as a biomarker that can be used for diagnosing obesity as a result of earnestly researching to find a genetic marker for the diagnosis of obesity.

Accordingly, the present invention provides a marker composition for diagnosing obesity, including microRNA-26b (microRNA-26b; miR-26b) or microRNA-4449 (microRNA-4449; miR-4449).

In addition, the present invention comprises an agent for measuring the expression level of microRNA-26b (microRNA-26b; miR-26b) or microRNA-4449 (microRNA-4449; miR-4449), a diagnostic composition for obesity and the diagnostic composition It provides a diagnostic kit for obesity.

The inventors have discovered the microRNA-26b and microRNA-4449 as a biomarker for the diagnosis of obesity through the examples.

In one embodiment of the present invention, subjects corresponding to the normal group and the obese group of BMI 25 or more were recruited and received their serum to separate the exosomes from the serum to compare and analyze the expression level of microRNA in the exosomes (Example 1 Reference).

In another embodiment of the present invention, as a result of analyzing the microRNA having a difference in the normal group and the obese group, it was confirmed that there is a significant difference in the expression level in 51 microRNA, and also by the multidimensional scaling method using the microRNA expression level results As a result of the analysis, it was observed that the border group appeared in the middle of the normal group and the obese group in two dimensions. As a result of analyzing the information of the subjects corresponding to the normal control group and the border group in the normal group, there was a significant difference in age, height, and weight, and it was confirmed that a significant difference appeared based on the body mass index (BMI) 23 (Example 2). -1).

In another embodiment of the present invention, it was confirmed that there is a difference in the level of 72 microRNA in the serum-derived exosomes of the normal control group and obese group subjects except the border group (see Example 2-2). Furthermore, by analyzing the results, we finally identified hsa-miR-26b-3p or hsa-miR-4449 as microRNAs with significantly increased expression in the borderline group and higher expression in the obese group than the normal control group. (See Example 2-3).

In the present invention, the microRNA-26b (miR-26b), more preferably hsa-miR-26b-3p may be composed of the nucleotide sequence of SEQ ID NO: 1.

In the present invention, the microRNA-4449 (miR-4449), more preferably hsa-miR-4449 may be composed of the nucleotide sequence of SEQ ID NO: 2.

As used herein, the term “diagnosis” in the broad sense means to determine the actual condition of the patient's illness in all aspects. The contents of the judgment include the name of the disease, the etiology, the type of disease, the severity, the details of the condition, and the presence or absence of complications. Diagnosis in the present invention is to determine whether the onset of obesity and progression level.

In the present invention, the agent for measuring expression of miR-26b or miR-4449 may be, but is not limited to, a sense and antisense primer or probe that binds to the microRNA complementarily.

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

As used herein, the term “probe” refers to a nucleic acid capable of specifically binding to mRNA of several to several hundred bases in length, which has been produced through enzymatic chemical separation or purification. The presence of mRNA can be confirmed by labeling radioisotopes or enzymes, and can be designed and modified by known methods.

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

For example, the kit of the present invention may be used to perform 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, 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, the components necessary for performing electrophoresis to confirm amplification of PCR products may be further 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 appropriate containers, reaction buffers, deoxynucleotides (dNTPs), enzymes such as Taq-polymerases and reverse transcriptases, DNases, RNase inhibitors, DEPCs, as well as individual primer pairs specific for the marker gene. -May include DEPC-water, sterile water, and the like. It may also include primer pairs specific for the gene used as a quantitative control.

In another aspect of the invention, the invention measures the expression level of microRNA-26b (microRNA-26b; miR-26b) or microRNA-4449 (microRNA-4449; miR-4449) in a subject-derived biological sample. It provides a method for providing information for diagnosing obesity, comprising the steps.

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

In the present invention, when the expression level of miR-26b or miR-4449 in the exosomes isolated from the subject-derived sample according to the above method can be determined as obese.

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

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand 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 analysis target

The present inventors recruited subjects corresponding to the normal group and obese group (obese, BMI 25 or more), respectively, in order to discover obesity diagnostic biomarkers for early diagnosis of obesity and prevention of the development of obesity and related complications. Serum samples were taken to find biomarkers representing differences between groups. To this end, serum was given to 33 normal and 16 obese groups, respectively.

1-2. RNA isolation and sequencing from serum

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

Thereafter, using the isolated RNA, a library was prepared using the Takara SMARTer smRNA for illumina kit from Macrogen, followed by Next Generation Sequence Analysis (NGS) with Hiseq 2500 to detect microRNA.

Example 2. Excavation of miRNA for obesity diagnosis

2-1. Normal group (normal control and border group) vs. Analysis of miRNAs with different expression levels in obese groups

First, the present inventors, according to the method of Examples 1-1 and 1-2 in order to identify the miRNA having a difference in the expression level in the blood-derived exosomes of the 33 normal group and 16 obese group (BMI 25 or more) Exosomes were isolated from serum collected from each subject and the expression levels of miRNAs contained therein were compared.

As a result, as shown in Figure 1a it was confirmed through the heat map (Heatmap) that the difference in the expression level in 51 miRNA between the normal group and the obese group. In addition, as a result of deriving and visualizing changes of individual samples through multidimensional scaling (MDS) for the normal group and the obese group, as shown in FIG. It was confirmed that the spatial position of the dimensional space was not clearly divided into two groups, and the boundary group between the obese group and the normal group appeared. Therefore, the normal group was divided into a normal control group and a border group by referring to the results of FIGS. 1A and 1B.

Furthermore, as a result of analyzing the information on the subjects of the normal control group and the border group, as shown in FIG. 2, the age of the border group was significantly lower than that of the normal control group, and the height and weight were It was confirmed that it was significantly high. In comparison, BMI, which is a representative indicator of obesity, was higher in the border group on average, and when the BMI index was 23, almost 90% of the normal control group had less than 23. In the borderline group, more than half exceeded 23.

These results are in line with the addition of BMI 23 to 24.9 as a 'pre-obesity level' in the Korean Obesity Society's 2018 Obesity Guidelines for Obesity. .

2-2. Normal control vs. Analysis of miRNAs with different expression levels in obese groups

In addition to the above results, the present inventors, in order to identify miRNA having a difference in expression level in blood-derived exosomes between these groups in 18 subjects and 16 obese groups classified as normal controls except the border group, Example 1 According to the method of -1 and 1-2, the expression level of miRNA contained in serum-derived exosomes collected from each subject was compared.

As a result, it was confirmed by heat map analysis that the expression change in 72 miRNAs, as shown in Figure 3a. In addition, as a result of analyzing the analysis results by the multidimensional scaling method (MDS) as in the method of Example 2-1, as shown in FIG. 3b, the two-dimensional spatial position and the normal control group appearing in the obesity group different from the result of FIG. It is confirmed that the two-dimensional spatial position appearing at is clearly divided into two groups.

2-3. Discovery of hsa-miR-26b-3p and hsa-miR-4449 biomarkers for the diagnosis of obesity

From the results of the above example, the microRNA results of significantly changed expression level from the normal control group to the border group and the obese group were compared and analyzed. As a result, as shown in Table 1, it was confirmed that the expression of hsa-miR-26b-3p or hsa-miR-4449 was significantly increased as the obesity level was increased in normal individuals. In addition, as shown in FIGS. 4A and 4B, the hsa-miR-26b-3p and hsa-miR-4449 groups between the normal and obese groups, or between the normal and middle groups. It was confirmed that the expression showed a significant difference.

Therefore, these results show that hsa-miR-26b-3p or hsa-miR-4449 can be usefully used as a biomarker for the diagnosis of obesity.

Obesity-related miRNA Obese / normal Normal / middle hsa-miR-26b-3p 10.2 -3.18 hsa-miR-4449 9.70 -3.08

The description of the present invention set forth above is for illustrative purposes, and it will be understood by those skilled in the art that the present invention may be easily modified 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 exemplary in all respects and not restrictive.

<110> Soonchunhyang University Industry Academy Cooperation Foundation <120> Biomarker microRNA-26b or microRNA-4449 for diagnosing obesity          and use according <130> PD18-065 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> RNA <213> hsa-miR-26b-3p <400> 1 ccuguucucc auuacuuggc u 21 <210> 2 <211> 22 <212> RNA <213> hsa-miR-4449 <400> 2 cgucccgggg cugcgcgagg ca 22

Claims (13)

A marker composition for diagnosing obesity, comprising microRNA-26b (microRNA-26b; miR-26b) or microRNA-4449 (microRNA-4449; miR-4449).
The method of claim 1,
The microRNA-26b is characterized in that consisting of the nucleotide sequence of SEQ ID NO: 1, marker composition.
The method of claim 1,
The microRNA-4449 is a marker composition, characterized in that consisting of the nucleotide sequence of SEQ ID NO: 2.
A composition for diagnosing obesity, comprising an agent for measuring the expression level of microRNA-26b (microRNA-26b; miR-26b) or microRNA-4449 (microRNA-4449; miR-4449).
The method of claim 4, wherein
The microRNA-26b is characterized in that consisting of the nucleotide sequence of SEQ ID NO: 1, a diagnostic composition for obesity.
The method of claim 4, wherein
The microRNA-4449 is characterized in that consisting of the nucleotide sequence of SEQ ID NO: 2, obesity diagnostic composition.
The method of claim 4, wherein
The agent for measuring the expression level of the microRNA-26b or microRNA-4449 is characterized in that the sense and antisense primers, or probes that complementarily bind to the microRNA, obesity diagnostic composition.
Claim 4 diagnostic kit, comprising the composition of obesity.
Information for diagnosing obesity, comprising measuring the expression level of microRNA-26b (microRNA-26b; miR-26b) or microRNA-4449 (microRNA-4449; miR-4449) in a biological sample from a subject How to Provide.
The method of claim 9,
The microRNA-26b is an information providing method, characterized in that consisting of the nucleotide sequence of SEQ ID NO: 1.
The method of claim 9,
The microRNA-4449 is characterized in that consisting of the nucleotide sequence of SEQ ID NO: 2, information providing method.
The method of claim 9,
The biological sample is characterized in that the blood or serum-derived exosomes, information providing method.
The method of claim 9,
The expression level of the microRNA-26b or microRNA-4449 is next generation sequencing (NGS), polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real time polymerase chain reaction (Real) information measured by one or more methods selected from the group consisting of: -time PCR), RNase protection assay (RPA), microarray, and northern blotting. How to Provide.

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