KR102133893B1 - Method for Monitoring Metabolic Obesity Disease Using Feces - Google Patents

Abstract

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

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, as the diet is improved and the lifestyle is convenient, obese patients are increasing. Most of obesity is a phenomenon in which energy consumed and the remaining portion is converted into fat and accumulated in various parts of the body, particularly subcutaneous tissue and abdominal cavity.

Obesity not only impairs normal biochemical and physiological functions by accumulating much more fat in the body than the amount of fat needed for the function of adipose tissue, but also causes various diseases, especially diabetes, hypertension, and coronary artery disease. It takes a lot of effort.

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

In recent years, we have been alert to obesity, understand the causes of obesity, and strive globally to respond to it, but despite such efforts, obesity is increasing rapidly.

Most of the diagnosis of metabolic obesity has been done with blood. For example, Korean Patent Registration Nos. 10-1759786, 10-1556403, and 10-1256928 have provided methods for diagnosing obesity from blood samples.

However, the method of collecting and testing blood is limited to about 1 mL or less of blood collection, and depending on the patient, it may be difficult to collect blood, so a more efficient method can diagnose and monitor metabolic obesity disease. Development is required.

On the other hand, micro-ALN (miRNA) is a small single-stranded RNA of 21-23 nucleotide size that is not directly involved in protein translation, and its physiological activity has been studied since it was first discovered in 1993. It was found to control the translation process.

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

The inventors of the present invention have completed the present invention by discovering that miRNA can be extracted from fecal samples and used as a biochemical biomarker of metabolic obesity.

An object of the present invention to solve this problem is to provide a method for providing information for diagnosing metabolic obesity from feces without taking 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 for providing information regarding the diagnosis of metabolic obesity, comprising the following steps:

(a) measuring the expression level of miR-129-5p from the feces 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 result of step (b).

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

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

Step (c) may be characterized in that it is determined to correspond to metabolic obesity when 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 feces of the subject;

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

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

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

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

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

The present invention relates to a method for monitoring metabolic obesity from feces of a subject.

In the present invention, the term "expression level" refers to the measured level of expression of the nucleic acid of interest, for example. Expression levels can also be assessed for 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 necessary to produce a functional RNA product in a controlled manner.

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

In the present invention, the term "miRNA" is a short naturally occurring RNA molecule and has the usual meaning understood by one of ordinary skill in the art.

In the present invention, the term "array" refers to an arrangement of addressable locations 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 a nucleic acid probe, such as an oligonucleotide, polynucleotide, or an array containing a larger portion of a gene. The nucleic acids on the array are preferably single stranded. “Microarray” refers to an array of biochips or biological chips, ie regions with a density of discontinuous regions with an immobilized probe of at least about 100/cm ² .

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

(a) measuring the expression level of miR-129-5p from the feces 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 result of step (b).

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

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

Specifically, in the case where the metabolic obesity disease is alleviated, the miR-129-5p expression level may be about twice the miR-129-5p expression level of the subject suffering from the metabolic obesity disease.

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

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

In step (c), the information related to the diagnosis of metabolic obesity may be information for diagnosing the onset of metabolic obesity, predicting the risk of developing the disease, 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 feces of the subject;

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

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

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

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

Example

Hereinafter, the present invention will be described in more detail through examples. It will be apparent to those skilled in the art that these examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

1. Substances and methods

1.1 Animals and diet

Four week old male C57BL/6J mice were purchased from Orient Bio and adapted to the laboratory environment for one week. After dividing the experimental rats into two groups of 10 animals, the high-fat diet (HFrD) or high-fat diet + 5wt% chardonnay grape seed (ChrSd) diet shown in Table 1 for 8 weeks was used as a free diet. Body weight was 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 Sampling

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

Fecal samples were taken from natural feces.

1.3 Biochemical testing

Plasma lipoprotein cholesterol was analyzed using size exclusion chromatography.

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

1.4 miRNA microarray expression analysis

Total RNA of feces was extracted using a fecal kit (Power Microbiome RNA Isolation Kit, MoBio).

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

2. Experimental results

2.1 Effects of Grapeseed Powder on Body Weight, Organ Weight and Blood Cholesterol Concentration

Table 2 shows changes in body weight, dietary intake, adipose tissue weight, blood sugar, lipids, and cholesterol in the rats who consumed the high-fat diet (HFrd) and the high-fat diet +5% grape seed powder diet 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 experimental rat group, which consumed the high-fat diet with 5% by weight grape seed powder for 8 weeks, showed 49% less weight and 5% less liver weight than the group without the grape seed powder. In addition, 5% by weight of grape seed powder, the fat cells of the mice were also reduced by 63%, and lipid and cholesterol levels were also reduced.

2.2 Fecal miRNA microarray expression analysis

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

2.3 Blood miRNA microarray expression analysis

The results of miRNA microarray analysis on miRNA collected from blood were also compared and analyzed.

Similarly, by analyzing the expression of miRNA, it was determined that the miRNA showed a statistically significant difference of 1.5 times or more in the 5% ChrSd diet compared to the control group. As shown in Table 4 below, a total of 53 miRNAs were determined.

2.4 Correlation analysis between miRNA expression and metabolic obesity physiological biomarkers

Biological biomarkers of expressed miRNA and 7 metabolic obesities (weight, liver weight, fat cell weight, triglyceride, Correlation between total cholesterol and HDL) was analyzed.

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

Among the miRNAs correlated with the metabolic obesity biomarker, it was confirmed that miR-129-5p was 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, specific parts of the present invention have been described in detail. For those skilled in the art, these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. It will be obvious. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (6)

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