KR20200021334A - Method for Monitoring Metabolic Obesity Disease Using Feces - Google Patents

Abstract

The present invention relates to a method for monitoring metabolic obesity using feces, and more particularly, to a method for monitoring metabolic obesity using feces that can be easily obtained without using blood. According to the present invention, since metabolic obesity is diagnosed using feces that are easy to collect without using blood, a large amount of samples can be easily obtained from a subject who has difficulties in having his or her blood drawn, and it is easy to predict the possibility of developing metabolic obesity.

Description

Method for Monitoring Metabolic Obesity Disease Using Feces}

The present invention relates to a method for monitoring metabolic obesity disease using feces, and more particularly, to a method for monitoring metabolic obesity disease from feces that can be easily obtained without using blood.

In recent years, obesity patients have increased due to improved eating habits and easier lifestyles. Most of the obesity is the energy consumed and the remaining portion is converted to fat, which accumulates in various parts of the body, especially the subcutaneous tissue and abdominal cavity.

Obesity prevents and prevents early treatment because much more fat accumulates in the body than the amount of fat required for the functioning of adipose tissue, which impairs normal biochemical and physiological functions and causes various diseases, especially diabetes, hypertension and coronary artery disease. Much effort must be made.

In particular, obesity induced by a high-fat diet (HFD) is the most important risk factor for the development of diabetes mellitus, arteriosclerosis, chronic liver disease, coronary heart disease, fatty liver and hypertension, so their prevention and treatment Should be considered as an extension of a healthy lifestyle.

Recently, we have been aware of the causes of obesity and are trying to understand and respond to the causes of obesity, but despite such efforts, obesity is increasing rapidly.

Most of the metabolic obesity has been diagnosed using blood. For example, Korean Patent Publication Nos. 10-1759786, 10-1556403, 10-1256928, and the like have provided a method for diagnosing obesity from a blood sample.

However, the method of collecting and testing blood is limited to about 1 mL or less, and some patients may have difficulty collecting blood. Therefore, the method of diagnosing and monitoring metabolic obesity disease may be more efficient. Development is required.

MicroRNAs (miRNAs) are small single-stranded RNAs of 21-23 nucleotides in size that are not directly involved in protein translation, and their physiology has been studied since their initial discovery in 1993. It has been found to regulate the translation process.

MiRNA can be used as one of the means of treating diseases including cancer because it can regulate the expression of genes associated with various diseases such as cancer. In addition, the miRNA is also used for early diagnosis because the type and amount of miRNA detected in the tissues are different depending on the type and progression of the disease.

The inventors of the present invention have found that miRNAs can be extracted from fecal samples and utilized as biochemical biomarkers of metabolic obesity.

An object of the present invention for solving this problem is to provide a method for providing information for diagnosing metabolic obesity from feces without collecting blood.

It is also an object of the present invention to provide a method for monitoring metabolic obesity from feces.

To achieve the above object, the present invention provides a method of providing information regarding the diagnosis of metabolic obesity, comprising the following steps:

(a) measuring the expression level of miR-129-5p from the fecal of the subject;

(b) comparing the expression level measured in step (a) with a reference value; And

(c) providing information regarding the diagnosis of metabolic obesity from the comparison of step (b).

In the present invention, the information on the diagnosis of metabolic obesity may be information for diagnosing the occurrence of metabolic obesity, predicting the risk of development, or determining the progress after the onset.

In the present invention, the reference value may be miR-129-5p expression level in a healthy subject.

The step (c) may be characterized in that it corresponds to metabolic obesity if the expression level of miR-129-5p is lower than the reference value.

The present invention also provides a method for monitoring metabolic obesity comprising the following steps:

(a) measuring the expression level of miR-129-5p from the fecal of the subject;

(b) comparing the expression level measured in step (a) with a subject's previous expression level; And

(c) monitoring the status of metabolic obesity from the comparison of step (b).

In the present invention, step (c) determines that the metabolic obesity is alleviated when the expression level of miR-129-5p is higher than the previous expression level, and that the metabolic obesity is deteriorated when it is lower than the previous expression level. It may be characterized by.

According to the present invention, since metabolic obesity is diagnosed using feces that are easy to sample without using blood, a large amount of samples can be easily obtained from a blood difficult subject and the possibility of easily inventing a metabolic obesity disease It can be predicted.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

The present invention relates to a method of monitoring metabolic obesity from a fecal in a subject.

In the present invention, the term "expression level" refers to, for example, the measured level of expression of a nucleic acid of interest. Expression levels can also be assessed with respect to expression in different tissues, patients versus healthy controls, and the like.

In the present invention, the term "gene" is a set of nucleic acid fragments containing the information needed to produce a functional RNA product in a controlled manner.

In the present invention, the term “gene product” is a biological molecule, eg miRNA or translated protein, produced through the transcription or expression of a gene.

In the present invention, the term “miRNA” is a short naturally occurring RNA molecule and has the common meaning understood by those skilled in the art.

In the present invention, the term "array" refers to an arrangement of addressable positions on a device, for example a chip device. The number of locations can range from several to at least hundreds or thousands. Each position represents an independent reaction site. Arrays include, but are not limited to, nucleic acid arrays, protein arrays, and antibody arrays. "Nucleic acid array" refers to an array containing a larger portion of nucleic acid probes, such as oligonucleotides, polynucleotides or genes. The nucleic acids on the array are preferably single stranded. "Microarray" refers to an array of biochips or biological chips, ie areas having a density of discrete regions with an immobilized probe of at least about 100 / cm ² .

The present invention provides a method of providing information regarding the diagnosis of metabolic obesity, comprising the following steps:

(a) measuring the expression level of miR-129-5p from the fecal of the subject;

(b) comparing the expression level measured in step (a) with a reference value; And

(c) providing information regarding the diagnosis of metabolic obesity from the comparison of step (b).

Step (a) is a step of measuring the expression level of miR-129-5p from the fecal sample of the subject. The fecal sample can measure the expression level of miRNA by microarray analysis.

The present invention found that miR-129-5p can be utilized as a marker for diagnosing the invention and prognosis of metabolic obesity disease among miRNAs that can be expressed from feces.

Specifically, the subject who has alleviated the metabolic obesity disease may exhibit miR-129-5p expression level about twice that of the miR-129-5p expression level of the subject having metabolic obesity disease.

Therefore, in the step (b), it can be determined that the result of measuring the expression level of miR-129-5p corresponds to the metabolic obesity when it shows a lower level compared to the reference value.

The reference value refers to the miR-129-5p expression level of a healthy subject, may be a numerical value in the literature, or may be a miR-129-5p expression level measured when the subject is healthy.

In the step (c), the information on the diagnosis of metabolic obesity may be information for diagnosing the occurrence of metabolic obesity, predicting the risk of development, or determining the progress after the onset.

The present invention also provides a method for monitoring metabolic obesity comprising the following steps:

(a) measuring the expression level of miR-129-5p from the fecal of the subject;

(b) comparing the expression level measured in step (a) with a subject's previous expression level; And

(c) monitoring metabolic obesity from the comparison of step (b).

In the present invention, step (c) determines that the metabolic obesity is alleviated when the expression level of miR-129-5p is higher than the previous expression level, and that the metabolic obesity is deteriorated when it is lower than the previous expression level. It may be characterized by.

According to the present invention, it is possible for a patient with metabolic obesity to monitor the progress of metabolic obesity in a simple manner using feces without having to collect blood every time. The monitoring can determine the prognosis of metabolic obesity by measuring the expression level of miR-129-5p from feces and comparing it with previous expression levels.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only to illustrate the invention, it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as limited by these examples.

1. Materials and Methods

1.1 Animals and Diet

Four-week-old male C57BL / 6J mice were purchased from Orient Bio Co. and adapted to the laboratory environment for one week. The rats were divided into two groups of 10 rats, and then fed for free diet with high-fat diet (HFrD) or high-fat diet + 5wt% grape seed powder (ChrSd) shown in Table 1 for 8 weeks. Body weights were measured weekly and intake was monitored twice a week.

ingredient HFrD HFrD + 5% ChrSd Lard fat 225 225 Soybean oil 27 23 Cholesterol 0.8 0.8 Microcrystalline cellulose (MCC) 50 21.4 ChrSd 0 50 Casein 200 199 Corn starch 91.2 74.8 Fructose 355 355 L-Cystine 3 3 Choline bitartrate 3 3 Mineral mix 35 35 Vitamin mix 10 10 Total Diet (g) 1000 1000 Fat% 46.7 46.7 Protein% 16.7 16.7 Carbohydrate% 36.6 36.1

1.2 Sample Collection

The mice were anesthetized after starving for 12 hours. Blood samples were collected using the cardiac puncture method. Blood was centrifuged at 4,500 rpm for 10 minutes and then stored for fat analysis. After weighing, liver and epididymal fat cells were collected, fixed in formalin and frozen in liquid nitrogen for further analysis.

Fecal samples were taken from natural bowel movements.

1.3 Biochemical Test

Plasma lipoprotein cholesterol was analyzed using size exclusion chromatography.

In addition, using a biochemical automatic analyzer (7020, HITACHI) triglyceride (triglyceride; TG, mg / dL), total cholesterol (TC, mg / dL), glucose (glucose, mg / dL), insulin ( Insulin, ng / mL) was measured.

1.4 miRNA microarray expression analysis

Fecal total RNA was extracted using a fecal kit (Power Microbiome RNA Isolation Kit, MoBio).

Purity of total RNA extracted from blood and feces was analyzed using a spectrophotometer (ND-1000, NanoDrop), and integrity was analyzed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto).

Total RNA 745 ng was analyzed using Affymetrix Genechip miRNA 4.0 Array and Genechip Fluidics Station 450 (Affymetrix, Santa Clara).

2. Experimental Results

2.1 Effect of Grape Seed Flour on Body Weight, Organ Weight and Blood Cholesterol Levels

Table 2 shows the weight, dietary intake, adipose tissue weight, blood sugar, lipid, and cholesterol changes of rats fed high-fat diet (HFrd) and high-fat diet + 5% grape seed meal for 8 weeks.

HFrD HFrD + 5% ChrSD Body weight gain 12.4 ± 0.76 6.37 ± 0.49 Food intake 2.94 ± 0.33 2.82 ± 0.33 Liver weight 1.01 ± 0.03 0.96 ± 0.03 Adipose tissue weight 1.34 ± 0.09 0.49 ± 0.06 Blood glucose 203 ± 18.6 224 ± 20.6 Triglyceride 93.9 ± 8.88 74.3 ± 3.55 HDL 78.7 ± 2.88 71 ± 4.12 LDL 17 ± 0.69 14 ± 1.06 Total-Cholesterol 136 ± 5.61 115 ± 7.30

In Table 2, the rat group fed high fat diet with 5% by weight grape seed powder for 8 weeks showed 49% less body weight and 5% less liver weight than the group not ingesting grape seed powder. In addition, the 5% by weight grape seed powder also reduced the fat cell weight of the mice 63%, lipid and cholesterol levels were also reduced.

2.2 Fecal miRNA Microarray Expression Analysis

Expression of miRNA collected from feces of two groups of mice was analyzed using miRNA expression microarray analysis. Expression of miRNA was statistically analyzed to determine miRNAs that showed a statistically significant 1.5-fold difference in the 5% ChrSd diet (| FC | ≥1.5, fold change) compared to the control group. A total of 82 miRNAs were determined, as shown in Table 3 below.

2.3 Blood miRNA Microarray Expression Analysis

The miRNA microarray analysis was also compared to the miRNAs collected from blood.

Similarly, miRNA expression was analyzed to determine miRNAs that showed a statistically significant 1.5-fold difference in 5% ChrSd diet compared to controls. A total of 53 miRNAs were determined, as shown in Table 4 below.

2.4 Correlation Analysis between miRNA Expression and Metabolic Obesity Physiological Biomarkers

To determine whether miRNA results in feces and blood correlate with metabolic obesity and physiological biomarkers, biological biomarkers of expressed miRNA and seven metabolic obesity (weight, liver weight, fat cell weight, triglycerides, Correlation between total cholesterol and HDL) was analyzed.

As a result, 23 of the miRNAs extracted from feces correlated with metabolic obesity biomarkers (Tables 5 and 6), and 31 of miRNAs extracted from blood correlated with metabolic obesity markers (Tables 7 and Table). 8) confirmed ( p <0.05 Or p <0.01).

Among miRNAs correlated with metabolic obesity biomarkers, miRNAs detected in both feces and blood were confirmed to be miR-129-5p expressed in both feces and blood.

In the table above,

*: Significantly (p <0.05) different from HFrD

#: Significantly (p <0.01) different from HFrD

BW: body weight gain

HDL: high density lipoprotein

As described above in detail a specific part of the content of the present invention, for those skilled in the art, such a specific description is only a preferred embodiment, which is not limited by the scope of the present invention Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (6)

How to provide information for diagnosing metabolic obesity, including the following steps:
(a) measuring the expression level of miR-129-5p from the fecal of the subject;
(b) comparing the expression level measured in step (a) with a reference value; And
(c) providing information for diagnosing metabolic obesity from a comparison result of step (b).
The method of claim 1,
The information for diagnosing metabolic obesity is information for diagnosing the onset of metabolic obesity, predicting the risk of development, or determining the progress after the onset, providing information for diagnosing metabolic obesity.
The method of claim 1,
The reference value is miR-129-5p expression level of a healthy subject, characterized in that for providing information for the diagnosis of metabolic obesity.
The method of claim 1,
The step (c) is characterized in that if the expression level of miR-129-5p is lower than the reference value metabolic obesity, characterized in that for providing information for the diagnosis of metabolic obesity.
Metabolic obesity monitoring method comprising the following steps:
(a) measuring the expression level of miR-129-5p from the fecal of the subject;
(b) comparing the expression level measured in step (a) with a subject's previous expression level; And
(c) monitoring the status of metabolic obesity from the comparison of step (b).
The method of claim 1,
Step (c) is determined that the metabolic obesity is alleviated when the expression level of miR-129-5p is higher than the previous expression level, characterized in that the metabolic obesity is deteriorated when it is lower than the previous expression level, Method of monitoring metabolic obesity.