KR20230130433A - Biomarker composition for diagnosing non-alcoholic fatty liver disease comprising miRNA204 - Google Patents

Abstract

The present invention relates to a biomarker composition for diagnosing non-alcoholic fatty liver disease containing miRNA204.

Description

Biomarker composition for diagnosing non-alcoholic fatty liver disease comprising miRNA204 {Biomarker composition for diagnosing non-alcoholic fatty liver disease comprising miRNA204}

The present invention relates to a biomarker composition for diagnosing non-alcoholic fatty liver disease containing miRNA204.

Fatty liver disease, also called fatty liver disease, is a disease that causes liver damage due to abnormal accumulation of fat (triglycerides, etc.) in liver cells. It is known that the initial condition of fatty liver disease is simple fatty liver, in which only fat is deposited in liver cells, and that the condition then progresses to steatohepatitis (including liver fibrosis), cirrhosis, and hepatocellular carcinoma. In general, causes of fat deposition in the liver include alcohol consumption, obesity, diabetes, abnormalities in lipid metabolism, drugs (steroids, tetracycline, etc.), Cushing's syndrome, poisoning (yellow phosphorus, etc.), and severe nutritional disorders. I can hear it.

Non-alcoholic fatty liver disease is a modern disease with a rapidly increasing mortality rate worldwide. Non-alcoholic fatty liver disease is characterized by no noticeable symptoms even when fat accumulates chronically, and if left untreated, it is an early stage disease that leads to liver cirrhosis and liver cancer. Therefore, the need for early diagnosis and detection is increasing for effective treatment. .

Among patients with non-alcoholic fatty liver disease, it is very important to distinguish between simple fatty liver disease and non-alcoholic steatohepatitis because the prognosis of the disease and the application of treatment therapy vary depending on whether it is simple fatty liver disease or non-alcoholic steatohepatitis. However, in the case of non-alcoholic steatohepatitis, most of the symptoms are absent or mild, so it is very difficult to detect non-alcoholic steatohepatitis. Generally, when liver function (ALT/AST) levels are elevated and fatty liver findings are seen on imaging, non-alcoholic steatohepatitis is diagnosed after excluding other diseases; however, liver function (ALT/AST) levels in non-alcoholic steatohepatitis Even if it increases, it appears fluctuating and does not exceed 4 times the upper limit of normal, making diagnosis difficult, and there are limitations in differentiating simple fatty liver from non-alcoholic steatohepatitis using imaging tools such as ultrasound, CT, and MRI. In addition, gamma-glutamyl transferase (GTP) or alkaline phosphatase may also be increased, but there is a problem in that the sensitivity and specificity are too low to diagnose non-alcoholic steatohepatitis with these test findings.

Liver biopsy is required to confirm the diagnosis of non-alcoholic steatohepatitis with these clinical characteristics, but there is still much controversy about when the biopsy should be performed. In general, it is recognized that a biopsy is necessary when liver function (ALT/AST) levels rise more than twice the normal level. However, there is a limit to performing biopsies on all patients with non-alcoholic steatohepatitis, which is gradually increasing in clinical practice, and there is an urgent need to develop tools or methods to non-invasively diagnose non-alcoholic steatohepatitis in the future.

Korean Patent No. 1570269 discloses mIR-451, a biomarker for diagnosing non-alcoholic steatohepatitis, Korean Patent No. 2242639 discloses miRNA4668-5p, a biomarker for diagnosing liver fibrosis, and Korean Patent Publication No. 20180105156. relates to non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) biomarkers and their uses, and discloses a method of determining whether an individual suffers from non-alcoholic fatty liver disease using various biomarker proteins.

However, there has been no report on the biomarker composition for diagnosing non-alcoholic fatty liver disease including miRNA204 of the present invention.

Korean Patent No. 1570269 Korean Patent No. 2242639 Korean Patent Publication No. 20180105156

The present invention was derived from the above-mentioned needs, and the present inventors completed the present invention by confirming the diagnostic effect of non-alcoholic fatty liver disease of a biomarker composition containing miRNA204 as an active ingredient.

In order to solve the above problems, the present invention provides a biomarker composition for diagnosing non-alcoholic fatty liver disease containing miRNA204.

In one example of the present invention, the miRNA204 is characterized in that it has SEQ ID NO: 1, and the expression of the miRNA204 is increased in non-alcoholic fatty liver.

Additionally, a composition for diagnosing non-alcoholic fatty liver disease comprising an agent capable of measuring the expression level of miRNA204 is provided.

In another example of the present invention, the miRNA204 is SEQ ID NO: 1, and the agent capable of measuring the expression level of the miRNA204 is a primer or probe capable of specifically binding to the miRNA204, and determines the expression level of the miRNA204. The agent that can be measured is a primer set consisting of SEQ ID NO: 2 and SEQ ID NO: 3.

The present invention provides a kit for diagnosing non-alcoholic fatty liver disease, including a composition for diagnosing non-alcoholic fatty liver disease including an agent capable of measuring the expression level of miRNA204.

In another example of the present invention, the kit is for RT-PCR, real-time RT-PCR, Northern blotting, RNA protection assay, or microarray chip.

The present invention provides a method of providing information for diagnosing non-alcoholic fatty liver disease, including the step of measuring the expression level of miRNA204 from an isolated sample.

In another example of the present invention, the separated sample is one or more selected from the group consisting of blood, serum, plasma, lymph fluid, ascites, urine, feces, and tissue biopsy.

The biomarker composition containing miRNA204 of the present invention as an active ingredient has a significant correlation with CD68, an indicator of fat accumulation and macrophage infiltration in liver tissue, and can be usefully used in the diagnosis of non-alcoholic fatty liver disease.

Figure 1 shows the change in body weight in mice fed a high-fat diet according to an embodiment of the present invention. #p<0.05 vs. WT-ND
Figure 2 shows a photograph of the abdomen in a high-fat diet mouse according to an embodiment of the present invention.
Figure 3 shows abdominal fat mass (Fat mass) in high-fat diet mice according to one embodiment of the present invention. *p<0.05 vs. WT-ND
Figure 4 shows liver tissue fat accumulation in high-fat diet mice according to an embodiment of the present invention.
Figure 5 shows changes in macrophage markers (brown) in high-fat diet mice according to one embodiment of the present invention.
Figure 6 shows the expression of miR204 in high-fat diet mice according to one embodiment of the present invention. *p<0.05 vs. WT-ND

Hereinafter, preferred embodiments of the present invention will be described in detail. In addition, in the following description, many specific details, such as specific components, are shown, but this is provided to facilitate a more general understanding of the present invention, and it is known in the art that the present invention can be practiced without these specific details. It will be self-evident to those who have the knowledge. Additionally, in describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description will be omitted.

The present invention provides a biomarker composition for diagnosing non-alcoholic fatty liver disease containing miRNA204.

In the present invention, the diagnostic biomarker composition is a substance that can diagnose non-alcoholic fatty liver disease by distinguishing it from normal liver tissue, and is a polypeptide or nucleic acid (e.g., a polypeptide or nucleic acid (e.g., : mRNA, etc.), organic biomolecules such as lipids, glycolipids, glycoproteins, or sugars (monosaccharides, disaccharides, oligosaccharides, etc.), preferably miRNA204, and more preferably, the miRNA204 is SEQ ID NO: 1, The expression of the miRNA204 is increased in non-alcoholic fatty liver disease, but is not limited thereto.

In the present invention, the “microRNA (miRNA)” is a small RNA encoded in the genome across several species, including primary microRNA (primary RNA), precursor microRNA (precursor miRNA), and mature microRNA (mature). The microRNA that is generated and functions as a (miRNA) and is selected from the group consisting of base sequences represented by SEQ ID NOs: 1 to 101 according to the present invention is a mature microRNA, but the present invention is not limited thereto.

Additionally, a composition for diagnosing non-alcoholic fatty liver disease comprising an agent capable of measuring the expression level of miRNA204 is provided.

In the present invention, “diagnosis” means confirming the presence or characteristics of a pathological condition. For the purposes of the present invention, the “diagnosis” may be confirming whether non-alcoholic fatty liver disease exists. Since changes in the expression of the microRNA according to the present invention occur in patients suffering from non-alcoholic fatty liver disease, it is possible to predict whether the individual has non-alcoholic fatty liver disease by checking the expression level of the microRNA of the present invention from the individual's sample. .

In another example of the present invention, the miRNA204 is SEQ ID NO: 1, and the agent capable of measuring the expression level of the miRNA204 is a primer or probe capable of specifically binding to the miRNA204, and determines the expression level of the miRNA204. The agent that can be measured is a primer set consisting of SEQ ID NO: 2 and SEQ ID NO: 3.

In the present invention, “primer” includes a continuous polynucleotide and/or a polynucleotide complementary thereto that specifically recognizes and amplifies RNA generated by gene expression or a polynucleotide derived therefrom.

In the present invention, “probe” includes a polynucleotide used to specifically detect RNA generated by gene expression or a polynucleotide derived therefrom and/or a polynucleotide complementary thereto.

Here, the complementary polynucleotide (complementary strand, reverse strand) refers to the full-length sequence of a polynucleotide consisting of a base sequence defined by the sequence number, or a base sequence where u is t in the base sequence, or a partial sequence thereof (here, for convenience, This refers to a polynucleotide in a base-complementary relationship based on base pairing relationships such as A:T(U) and G:C. However, such complementary strand is not limited to cases where it forms a completely complementary sequence with the nucleotide sequence of the target positive strand, and may have a complementary relationship to the extent that it can hybridize with the target positive strand under strict conditions.

The present invention provides a kit for diagnosing non-alcoholic fatty liver disease, including a composition for diagnosing non-alcoholic fatty liver disease including an agent capable of measuring the expression level of miRNA204.

In the present invention, the expression level of miRNA204 is measured using reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), realtime reverse transcription polymerase reaction (Realtime RT-PCR), and RNase protection assay (RPA). ; RNase protection assay), Northern blotting, DNA chip, etc. can be used, and preferably RT-PCR can be used to compare the expression of normal expression to determine the number of patients suffering from non-alcoholic fatty liver disease. Diagnosis can be made by confirming the increase or decrease in the expression of the microRNA in the patient.

The terms used in referring to the above expression levels may refer to differential expression or having a differential level or value compared to a “normal” expression level or value, and may refer to qualitative as well as quantitative differences in expression. Includes both differences.

The present invention provides a method of providing information for diagnosing non-alcoholic fatty liver disease, including the step of measuring the expression level of miRNA204 from an isolated sample.

In the present invention, the “isolated sample” includes blood and other liquid samples of biological origin, biopsy specimens, solid tissue samples such as tissue culture, or cells derived therefrom. More specifically, it may be selected from the group consisting of blood, serum, plasma, lymph, ascites, urine, feces, and tissue biopsy, and the sample may be preprocessed before use for detection. For example, it may include filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, etc. Additionally, nucleic acids can be separated from the sample and used for detection. Preferably, it refers to liver tissue, but is not limited thereto.

The advantages and features of the present invention and methods for achieving them will become clear with reference to the embodiments described in detail below. However, the present invention is not limited to the embodiments disclosed below and will be implemented in various different forms. The present embodiments are merely intended to ensure that the disclosure of the present invention is complete and that common knowledge in the technical field to which the present invention pertains is not limited. It is provided to fully inform those who have the scope of the invention, and the present invention is only defined by the scope of the claims.

Example 1. Establishment of non-alcoholic fatty liver mouse model

Ten 6-week-old wild-type mice were purchased from Orient Bio Co., Ltd. and bred according to the rules of the Preclinical Center at Chungnam National University Hospital. 1 kg of high fat diet (60% fat) feed was purchased from Duyeol Biotech.

The weight of the mice was recorded every 2 days while being fed regular feed and high-fat diet. After 12 weeks, each mouse was anesthetized using 25% urethane, heart blood was collected, abdominal fat was collected (fat mass measurement), liver tissue was obtained, and 1/2 of the liver was placed in 4% paraformaldehyde solution. It was fixed by immersion, and 1/2 was stored in liquid nitrogen.

Example 2. Detection of miR204 expression in liver tissue (qPCR)

The liver tissue stored in liquid nitrogen was taken out, treated with 2 mL of Trizol solution (Trizol reagent, Invitrogen, USA), dissolved, and ground through a homogenizer machine. 1 mL of supernatant was separated through centrifugation (12,000 g, 10 minutes) and transferred to a new tube. The solution was treated with 200 ㎕ of chloroform. The tube was shaken vigorously more than 10 times and stored at room temperature for 5 minutes. From the three-layer solution obtained by centrifugation (12,000 g, 15 minutes, 4°C), only the top transparent solution was transferred to a new tube. The solution was treated with 500 ㎕ of 100% isopropanol. Next, the RNA precipitate obtained by centrifugation (12,000 g, 10 minutes, 4°C) was washed with 1 mL of 75% ethanol. The RNA precipitate obtained by centrifugation (7,500 g, 5 minutes, 4°C) was dried at room temperature for 20-30 minutes. The precipitate was dissolved in 50 μl of RNase-free water. After quantifying the RNA concentration through nanodrop, the amount of RNA equivalent to 1 ㎍ was used to obtain cDNA of miRNA using the miScript II RT kit (Cat. #218161, Qiagen, Hilden, Germany). Afterwards, real-time quantitative PCR (realtime qPCR) was performed using the miScript SYBR Green PCR kit (Cat. #218073, Qiagen). The sequence of miR204-5-P (SEQ ID NO: 1) is 5'-UUC CCU UUG UCA UCC UAU GCC U-3', and the forward primer of miR204 (SEQ ID NO: 2, 5'-CGC TTC CCT TTG TCA TCC TA) -3') was used, and the reverse primer was mixed in equal amounts using the Universal primer (SEQ ID NO: 3, 5'-GCG AGC ACA GAA TTA ATA CGA C-3') in the SYBR green kit. RNA-U6 small nuclear 1 (RNU6) was used as a loading control. The forward primer (5'-GCA AAT TCG TGA AGC GTT CC-3') of RNU6 was ordered and used, and the reverse primer was mixed in equal amounts using the above universal primer.

Example 3. Histological analysis

- Hematoxylin & Eosin staining

After sufficiently fixing in 4% PFA solution for more than 16 hours, paraffin was infiltrated to create a block. Sections were made on slides with a thickness of 4 μm. After dissolving the paraffin by immersing it in a xylene solution for 15 minutes, the moisture density is gradually increased in the order of 100% ethanol -> 90% ethanol -> 80% ethanol -> 70% ethanol -> PBS, making it easy to stain. was created gradually. After staining in hematoxylin solution for 10 seconds, the cells were immersed in eosin solution for 10 seconds and dehydrated in the following order: PBS -> 70% ethanol -> 80% ethanol -> 90% ethanol -> 100% ethanol -> xylene. Afterwards, it was mounted and photographs were taken under a microscope.

- CD68 (macrophage) DAB staining

After sufficiently fixing in 4% PFA solution for more than 16 hours, paraffin was infiltrated to create a block. Sections were made on slides with a thickness of 4 μm. After dissolving the paraffin by immersing it in a xylene solution for 15 minutes, the moisture density is gradually increased in the order of 100% ethanol -> 90% ethanol -> 80% ethanol -> 70% ethanol -> PBS, making it easy to stain. was created gradually. The antigen was recovered again by placing it in 1 mM sodium citrate solution and boiling it at 100°C for 20 minutes. Afterwards, blocking was performed using 1% bovine serum albumin, and the primary antibody was diluted 1:500 with the same solution and reacted at 4°C for 16 hours. The next day, the secondary antibody was diluted 1:500 and reacted at room temperature for 1 hour, then stained with DAB solution, and the brown-stained area was observed as positive using an optical microscope.

<Test example: results>

Figure 1 shows the change in body weight in mice fed a high-fat diet according to an embodiment of the present invention. The body weight of high-fat diet mice increased statistically significantly from 8 weeks compared to the normal diet group.

Figures 2 and 3 show abdominal fat mass (Fat mass) in high-fat diet mice according to one embodiment of the present invention. As shown in the photo, 12 weeks after starting the high-fat diet, it was observed that the abdominal fat of the high-fat diet mice increased compared to the normal diet group, and the actual fat amount also increased statistically significantly.

Figure 4 shows liver tissue fat accumulation in high-fat diet mice according to an embodiment of the present invention. As shown in the photo, 12 weeks after starting the high-fat diet, fat accumulation in the liver tissue of the high-fat diet mice significantly increased compared to the normal diet group.

Figure 5 shows changes in macrophage markers (brown) in high-fat diet mice according to one embodiment of the present invention. As shown in the photo, 12 weeks after starting the high-fat diet, CD68, an indicator of macrophages, was significantly increased in the liver tissue of the high-fat diet mice compared to the normal diet group.

Figure 6 shows the expression of miR204 in high-fat diet mice according to one embodiment of the present invention. At 12 weeks after starting the high-fat diet, the level of miR204 in the liver tissue of the high-fat diet mice was statistically significantly increased compared to the normal diet group.

<110>CNU <120> Biomarker composition for diagnosing non-alcoholic fatty liver disease comprising miRNA204 <130>M22-0000 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> RNA <213> Artificial Sequence <220> <223>miR204-5-P <400> 1 uucccuuugu cauccuaugc cu 22 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223>miR204 forward primer <400> 2 cgcttccctt tgtcatccta 20 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223>miR204 reverse primer <400> 3 gcgagcacag aattaatacg ac 22

Claims (11)

Biomarker composition for diagnosing non-alcoholic fatty liver disease containing miRNA204 The biomarker composition for diagnosing non-alcoholic fatty liver disease according to claim 1, wherein the miRNA204 is SEQ ID NO: 1. The biomarker composition for diagnosing non-alcoholic fatty liver disease according to claim 1, wherein the expression of the miRNA204 is increased in non-alcoholic fatty liver disease. A composition for diagnosing non-alcoholic fatty liver disease comprising an agent capable of measuring the expression level of miRNA204 The composition for diagnosing non-alcoholic fatty liver disease according to claim 4, wherein the miRNA204 is SEQ ID NO: 1. The composition for diagnosing non-alcoholic fatty liver disease according to claim 4, wherein the agent capable of measuring the expression level of the miRNA204 is a primer or probe capable of specifically binding to the miRNA204. The composition for diagnosing non-alcoholic fatty liver disease according to claim 4, wherein the agent capable of measuring the expression level of miRNA204 is a primer set consisting of SEQ ID NO: 2 and SEQ ID NO: 3. A kit for diagnosing non-alcoholic fatty liver disease comprising the composition of any one of claims 4 to 7 The kit for diagnosing non-alcoholic fatty liver disease according to claim 8, wherein the kit is for RT-PCR, real-time RT-PCR, Northern blotting, RNA protection analysis, or microarray chip. Information provision method for diagnosing non-alcoholic fatty liver disease including measuring the expression level of miRNA204 from an isolated sample The method of claim 10, wherein the separated sample is one or more selected from the group consisting of blood, serum, plasma, lymph, ascites, urine, feces, and tissue biopsy.