KR20150044332A - Composition and kit for detecting tissue or organ damage in a subject, method for predicting or detecting tissue or organ damage in a subject and method for screening a therapeutic agent for tissue or organ damage - Google Patents

Abstract

In the present invention, provided are a composition and a kit for diagnosing damage to tissues or organs of subjects, a method for predicting or diagnosing damage to tissues or organs of subjects and a method for searching for drug for treating damage to tissues or organs of subjects.

Description

TECHNICAL FIELD The present invention relates to a composition and a kit for diagnosing tissue or organ damage of an individual, a method of predicting or diagnosing tissue or organ damage of an individual, and a method of searching for a therapeutic agent. organ damage in a subject and method for screening a therapeutic agent for tissue or organ damage}

Compositions and kits for diagnosing tissue or organ damage of an individual, methods of predicting or diagnosing tissue or organ damage of an individual, and methods of screening therapeutic agents for tissue or organ damage.

miRNAs bind to mRNA and directly regulate gene expression. miRNAs are also found in vertebrates such as primates, rodents, and fish, invertebrates such as insects, and viruses, and are well-preserved evolutionarily. Studies have shown that miRNA plays an important role in the developmental process, and its expression pattern is tissue specific. In humans, small intestinal epithelium and intestinal epithelial cells showed distinctive miRNA expression pattern, respectively. Another study reported the types and patterns of miRNAs expressed in the intestinal mucosa of rats. Transgenic mice were used to express miRNAs in a variety of gastrointestinal physiological systems, including epithelial cell differentiation, It is proven that it plays a role in the function. Analysis of miRNA expression patterns of endoderm-derived organs such as liver, plant, and pancreas has shown that a large number of miRNAs show tissue specificity and also correlate with tissue-specific protein expression patterns.

miRNAs are expressed by different physiological and pathological conditions in animals. To date, disease-related miRNA studies have been performed primarily on cancer. Recently, various miRNAs have been specifically expressed in various cancers and have been playing an important role in the carcinogenesis mechanism, and efforts to utilize them for early diagnosis and segmentation of cancer have been continued. Currently, some researchers have succeeded in developing methods that use miRNA expression patterns in lung cancer as diagnostic and prognostic markers. In addition to cancer, the expression patterns of miRNAs in the blood of patients with ulcerative colitis and Crohn's disease, known as inflammatory and autoimmune diseases, have been reported in gastrointestinal diseases, and differential diagnosis of intestinal diseases using microRNA patterns in peripheral blood has been reported The utility and possibility of this has been suggested.

One aspect provides a composition for diagnosing tissue or organ damage of an individual.

Another aspect provides a kit for the diagnosis of tissue or organ damage in an individual.

Another aspect provides a method of predicting or diagnosing tissue or organ damage in an individual.

Another aspect provides a method for screening a therapeutic agent for tissue or organ damage of an individual.

Ea-miR-101-2, eca-mir-29b, eca-mir-106b, eca-mir-450, eca-mir-101-2, -mir-196b, eca-mir-18a, and eca-mir-488. The composition for diagnosing a tissue or organ damage of an individual is provided.

The term "miRNA" can be referred to as microRNA or miRs. The length of the miRNA may be about 19 to 25, or about 21 to 23 nucleotides in length. Because miRNAs are transcribed from DNA but not translated into proteins, they are encoded by genes that contain non-coding RNAs. miRs are processed from a known primary transcript pri-miRNA into a short stem-loop structure called pre-miRNA and finally processed with the resulting single-stranded miRNA. The pre-miRNA can form a self-folding structure within the self-complementary region. The structure is then processed by the nuclease in the animal. A mature miRNA molecule is partially complementary to one or more mRNA molecules and can function to regulate transcription of the protein. Identified sequences of miRNAs are available at www.microRNA.org, www.mirbase.org, or www.mirz.unibas.ch/cgi/miRNA.cgi. ≪ RTI ID = 0.0 > and / or < / RTI > miRNAs are generally numbered according to a naming convention such as "mir- [number]". The number of miRNAs is assigned according to the sequence found for miRNA species that have already been identified. If the miRNA is found to be similar to a known miRNA of a different organism, the name is provided in the form of [organism identifier] - mir- [number], the name of the selector organism identifier. Mature microRNAs usually have the prefix "miR" attached, and gene or precursor miRNAs are prefixed with "mir". In the context of the present invention, any microRNA (miRNA or miR) with the prefix mir- * or miR- * should be understood to include both precursors and / or mature species unless explicitly stated otherwise. For more information on miR nomenclature, see www.mirbase.org or Ambros et al., A uniform system for microRNA annotation, RNA 9: 277-279 (2003).

The above eca-mir-365-2, eca-mir-190, eca-mir-29b, eca-mir-106b, -196b, eca-mir-18a, and eca-mir-488 may have the nucleotide sequences of SEQ ID NOS: 1 to 10, respectively.

The miRNAs shown in Table 1 are expressed specifically from organs and exhibit high expression levels and are present at low concentrations in the plasma of normal subjects. The miRNAs leak into the plasma in proportion to the degree of tissue damage.

miRNAs are stable and not degraded by external conditions such as nucleic acid degrading enzymes such as RNAse A, repetition of freezing and thawing, or pH changes, and the amount of miRNA expression can be measured repeatedly and consistently. In addition, miRNAs can be quantitated by amplification, so that miRNAs can be detected even in a trace amount in a sample, and the degree of miRNA expression level can be detected to a small extent.

The miRNA is derived from Equus caballus. The above eca-mir-365-2, eca-mir-190, eca-mir-29b, eca-mir-106b, -196b, eca-mir-18a, and eca-mir-488 has been found by the present inventors to be associated with tissue or organ damage of an individual. Thus, the level of expression of one or more miRNAs can be used to diagnose damage to a tissue or organ of an individual, such as damage to the colon, liver or muscle.

A composition for diagnosing colon damage of an individual comprising an agent for measuring the expression level of one or more miRNA selected from the group consisting of eca-mir-155, eca-mir-196b, eca-mir-18a and eca- .

Also provided is a composition for diagnosing hepatic injury of an individual comprising an agent for measuring the expression level of at least one miRNA selected from the group consisting of eca-mir-106b, eca-mir-450, and eca-mir-101-2 do.

Also provided is a composition for diagnosing muscle damage of an individual comprising an agent for measuring the expression level of one or more miRNAs selected from the group consisting of eca-mir-365-2, eca-mir-190, and eca-mir-29b do.

The Aceession Number of the mature sequence of eca-mir-155 is MIMAT0013182 and has the nucleotide sequence of SEQ ID NO: 1 and the stem-loop sequence has the Accession Number of MI0012927. The Aceession Number of the mature sequence of eca-mir-196b is MIMAT0012939 and has the nucleotide sequence of SEQ ID NO: 2, and the stem-loop sequence has the Accession Number of MI0012693. The Aceession Number of the mature sequence of eca-mir-18a is MIMAT0013085 and has the nucleotide sequence of SEQ ID NO: 3, and the stem-loop sequence has the Accession Number of MI0012832. The Aceession Number of the mature sequence of eca-mir-488 is MIMAT0012965 and has the nucleotide sequence of SEQ ID NO: 4, and the stem-loop sequence has the Accession Number of MI0012718.

The Aceession Number of the mature sequence of eca-mir-106b is MIMAT0013054 and has the nucleotide sequence of SEQ ID NO: 5, and the stem-loop sequence has the Accession Number of MI0012797. The Aceession Number of the mature sequence of eca-mir-450 is MIMAT0013219 and has the nucleotide sequence of SEQ ID NO: 6, and the stem-loop sequence has the Accession Number of MI0012965. The Aceession Number of the mature sequence of eca-mir-101-2 is MIMAT0012951 and has the nucleotide sequence of SEQ ID NO: 7, and the stem-loop sequence has the Accession Number of MI0012861.

The Aceession Number of the mature sequence of eca-mir-365-2 is MIMAT0013038 and has the nucleotide sequence of SEQ ID NO: 8, and the stem-loop sequence has the Accession Number of MI0012802. The Aceession Number of the mature sequence of eca-mir-190 is MIMAT0012896 and has the nucleotide sequence of SEQ ID NO: 9, and the stem-loop sequence has the Accession Number of MI0012652. The Aceession Number of the mature sequence of eca-mir-29b is MIMAT0012941 and has the nucleotide sequence of SEQ ID NO: 10, and the stem-loop sequence has the Accession Number of MI0012695.

The term "eca-mir-155", "eca-mir-196b", "eca-mir-18a", "eca- Mir-190 "," eca-mir-101b "," eca-mir-101b " , eca-mir-196b, eca-mir-18a, eca-mir-488, eca-mir-106b, eca-mir-450, -190 and eca-mir-29b, and functional variants thereof.

miRNA order eca-mir-155 UUAAU GCUAA UCGUG AUAGG GGU (SEQ ID NO: 1) eca-mir-196b UAGGU AGUUU CCUGU UGUUG GG (SEQ ID NO: 2) eca-mir-18a UAAGG UGCAU CUAGU GCAGA UAG (SEQ ID NO: 3) eca-miR-488 UUGAA AGGCU AUUUC UUGGU C (SEQ ID NO: 4) eca-mir-106b UAAAG UGCUG ACAGU GCAGAI (SEQ ID NO: 5) eca-mir-450 UUUUG CGAUG UGUUC CUAAU AU (SEQ ID NO: 6) eca-mir-101-2 UACAG UACUG UGAUA ACUGA A (SEQ ID NO: 7) eca-mir-365-2 UAAUG CCCCU AAAAA UCCUU AU (SEQ ID NO: 8) eca-mir-190 UGAUA UGUUU GAUAU AUUAG GU (SEQ ID NO: 9) eca-mir-29b UAGCA CCAUU UGAAA UCAGU GUU (SEQ ID NO: 10)

The composition for diagnosing leprosyphritis of the individual may comprise an agent for measuring the amount of the at least one miRNA. The agent may be a substance that specifically binds to the one or more miRNAs. The agent comprises a primer, probe, or antisense sequence thereof that specifically binds to the one or more miRNAs or a complementary sequence thereof; Or a combination thereof. The primer may be one which provides a polymerization initiation point in a polymerization reaction with a polymerase. The primer may be one used in a nucleic acid amplification reaction. The term "amplification" refers to increasing the number of copies of the target sequence or its complementary sequence. The nucleic acid amplification reaction can be performed by any method known in the art. Amplification of nucleic acids involves either a method requiring multiple cycles during amplification or a method performed at a single temperature. Examples of cycling techniques include those that require thermal cycling. Methods that require thermocycling include polymerase chain reaction (PCR). PCR is known in the art. PCR typically involves denaturing double stranded DNA to single stranded DNA by thermal degradation, annealing the primer to the single stranded DNA; And synthesizing a complementary strand from the primer. An isothermal amplification method is a method in which a single temperature or a major aspect of the amplification process is performed at a single temperature. In contrast to PCR, in which the reaction product is heated to allow additional primers to bind in order to separate the double strands, the isothermal method relies on a strand displacing polymerase to separate the double strands and rescopy the template do. Isothermal methods can be distinguished by methods that rely on substitution of primers to initiate reiterative template copying and methods that rely on sequential reuse or novel synthesis of single primer molecules. Methods that rely on primer substitution include helicase dependent amplification (HDA), exonuclease dependent amplification, recombinase polymerase amplification (RPA) and loop mediated amplification and loop mediated amplification (LAMP). Methods that rely on sequential reuse or novel synthesis of single primer molecules include those selected from the group consisting of strand displacement amplification (SDA) and nucleic acid based amplification (NASBA and TMA). The primers may be included in one, two, three, four, five, six or more sets according to the selected amplification method. The primer may be a PCR primer.

The miRNA expression level measurement can be a measure of the amount of miRNA as a process of identifying the presence and expression level of an miRNA of an individual to diagnose damage to a tissue or organ such as colon, liver and muscle. This can be done by isolating the miRNA directly or by using a primer or probe for the miRNA. Analysis methods include RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), Northern blotting ), Nucleic acid microarrays comprising DNA, or combinations thereof. RT-PCR is a method of analyzing RNA. It is a method of amplifying and analyzing cDNA obtained by reverse transcription of miRNA by PCR. After the RT-PCR, a pair of primers prepared specifically for the gene was used in the amplification step of the RT-PCR, and the band pattern and the band thickness were confirmed by the RT-PCR, thereby confirming the miRNA expression and the expression level of the gene And comparing it with a normal control, it is possible to judge the degree of damage or the damage of a tissue or an organ such as colon, liver and muscle of an individual.

As used herein, the term "primer" refers to a nucleic acid sequence that can form base pairs with a complementary template with a free 3 'hydroxyl group and serves as a starting point for template strand replication . Primers can initiate DNA synthesis in the presence of reagents for polymerization (i. E., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at appropriate buffer solutions and temperatures. For example, PCR amplification is carried out using a sense and antisense primer having 7 to 50 nucleotide sequences as a specific primer for the above-mentioned one or more miRNAs to measure the amount of production of a desired product, The presence or the degree of damage to the same tissue or organ can be confirmed. The PCR conditions, the lengths of the sense and antisense primers can be appropriately selected according to techniques known in the art. The primers may be used in an amount of 10 to 100, 15 to 100, 10 to 80, 10 to 50, 10 to 30, 15 to 80, 15 to 50, 15 to 30, 20 to 100, 20 to 80, 20 to 50, 30 nt. ≪ / RTI >

As used herein, the term "probe" refers to a target nucleic acid, for example, a nucleic acid fragment such as RNA or DNA capable of specifically binding to miRNA, and is labeled to confirm the presence, amount and amount of specific miRNA. . The probe may be prepared in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, or an RNA probe. For example, hybridization is carried out using a probe complementary to the miRNA, and the expression level of miRNA is measured through the degree of hybridization to determine whether or not a tissue or organ such as colon, liver, and muscle is damaged or not . Selection of suitable probes and hybridization conditions can be appropriately selected according to techniques known in the art. Wherein said probe is selected from the group consisting of 10 to 100, 15 to 100, 10 to 80, 10 to 50, 10 to 30, 15 to 80, 15 to 50, 15 to 30, 20 to 100, 20 to 80, 20 to 50, 30 nt. ≪ / RTI >

In addition, the primer or probe can be chemically synthesized using a phosphoramidite solid support synthesis method or other well-known methods. Such nucleic acid sequences may also be modified through a variety of methods known in the art. Examples of such modifications include, but are not limited to, methylation, capping, substitution with one or more of the natural nucleotide analogs, or modifications between nucleotides such as uncharged linkers (e.g., methylphosphonate, phosphotriester, phosphoramidate, carbamate Etc.) or charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.). The primer or probe may also be modified using a label that can provide a detectable signal directly or indirectly. Examples of such labels may include radioactive isotopes, fluorescent molecules, or biotin.

The expression level may be an expression level in a sample isolated from the subject. The sample may be blood, plasma, serum, urine, feces, saliva, tears, cerebrospinal fluid, cells, tissue or a combination thereof separated from an individual. The tissue may be colon, liver, or muscle. The entity may belong to Perissodactypa . The entity may be an Equus genus . Equus can include horses, donkeys, or zebras. The horse is referred to as equus ferus . The above equus ferus ) species is Equus Peruvian Scavalus ferus caballus ), Equus Perus ( Equus ferus ferus , or Equus ferus przewalskii ) subspecies. The subject may include horses, buffalo, rhinoceros, or camels.

Other aspects include eca-mir-155, eca-mir-196b, eca-mir-18a, eca-mir-488, eca-mir-106b, 29. A kit for the diagnosis of tissue or organ damage of an individual, comprising an agent for measuring an expression level of one or more miRNAs selected from the group consisting of -365-2, eca-mir-190, and eca-mir-29b.

For the preparation "," individual "and" for the diagnosis of tissue or organ damage "for the determination of the level of expression of one or more miRNA, as described in the description of the composition for the diagnosis of damage to tissues or organs such as colon, liver and muscle .

The kit can be, for example, a microarray for the diagnosis of damage to tissues or organs such as colon, liver and muscle, which can measure the expression level of miRNA. Microarrays for the diagnosis of damage to tissues or organs such as the colon, liver and muscle can be easily produced by those skilled in the art according to methods known in the art using the miRNA. The microarray may be one in which the above-described miRNA, a complementary sequence thereof, or a nucleic acid sequence corresponding thereto is attached to a substrate as a probe. The nucleic acid sequence may be selected from the group consisting of DNA, PNA (peptide nucleic acids), LNA (locked nucleic acids), combinations thereof, and analogs thereof.

For example, when the kit is used for measuring the expression level of the miRNA of the gene described above, it may be a kit containing essential elements necessary for conducting RT-PCR. In addition to the respective primers specific for the miRNA of the marker gene, the RT-PCR kit may also contain enzymes such as test tubes or other appropriate containers, reaction buffers, deoxyribonucleotides (dNTPs), Taq polymerase and reverse transcriptase, DNase, RNase Inhibitor, DEPC-water (dEPC-water), or sterile water. It may also contain a primer pair specific for the gene used as a quantitative control.

Other aspects include eca-mir-155, eca-mir-196b, eca-mir-18a, eca-mir-488, eca-mir-106b, Measuring the expression level of one or more miRNAs selected from the group consisting of eca-mir-36, eca-mir-365-2, eca-mir-190, and eca-mir-29b, Comparing the level of expression of one or more of the miRNAs measured in the control to the level of expression of one or more of the miRNAs.

The entity may belong to Perissodactyla . Equus (Equus) can be deceiving. Equus can include horses, donkeys, or zebras. The horse may be an equus ferus species. The above equus ferus ) species is the Equus ferus caballus , Equus Perus ferus ferus , or Equus Perus, ferus przewalskii ) subspecies. The subject may include horses, buffalo, rhinoceros, or camels.

The expression level may be an expression level of the miRNA. Thus, the measurement may be an amount of miRNA. The measurement of the amount of the miRNA is as described above.

The sample may be blood, plasma, serum, urine, feces, saliva, tears, cerebrospinal fluid, cells, tissue or a combination thereof separated from an individual. The tissue may be colon, liver, or muscle.

The method includes comparing the level of expression of the one or more miRNAs measured to the level of expression of one or more miRNAs measured in a control. The control group may be an individual whose tissue or organ is not damaged.

The method may further comprise determining that the tissue or organ of the individual is damaged if the expression level of the one or more miRNAs measured is higher than the expression level of one or more miRNAs measured in the control. The miRNA expression level of the gene is at least about 2 to 8 times, such as about 3 to 8 times, at least 4 to 8 times, at least 5 to 8 times, at least 6 to 8 times , Or 7 to 8 times or more, it can be determined that there is damage to the organ.

In addition, when the expression level of the one or more miRNAs measured is equal to or lower than the expression level of one or more miRNAs measured in the control, the subject may further comprise determining that the tissue or organ is not damaged.

Another aspect includes the steps of administering a candidate agent to a subject; Eca-mir-186, eca-mir-188, eca-mir-106b, eca-mir-450, eca-mir-101-2 , eca-mir-365-2, eca-mir-190, and eca-mir-29b; And comparing the level of expression of the one or more miRNAs with the level of expression of one or more miRNAs measured in the control.

The method comprises administering a candidate agent to a subject. The candidate substance may be any substance expected to be effective in the treatment of colon. The administration can be appropriately selected depending on the substance to be selected. The administration is administered directly to the subject by any means, for example, intravenously, intramuscularly, orally, transdermal, mucosal, intranasal, intratracheal or subcutaneous. The material may be administered systemically or locally, for example, to the colon, liver, or muscle.

Eca-mir-186, eca-mir-188, eca-mir-106b, eca-mir-450, 2, eca-mir-365-2, eca-mir-190, and eca-mir-29b "and" measuring the expression level of one or more miRNAs Quot; comparing the expression level of one or more miRNAs measured in the control group "is as described above.

The subject may be a tissue or an organ, for example a colon, a liver, and a damaged muscle.

The method further comprises determining the candidate agent as being effective in treating the tissue or organ damage of the individual if the level of expression of the one or more miRNAs measured is lower than the expression level of the one or more miRNAs measured in the control As shown in Fig.

According to a composition for diagnosing tissue or organ damage of an individual according to one aspect, it is possible to efficiently diagnose whether or not tissue of a subject is damaged or damaged. In addition, the composition can be stable without deterioration due to external factors such as sample processing and storage process.

According to a kit for diagnosing tissue or organ damage of an individual according to one aspect, it is possible to efficiently diagnose whether or not an individual has tissue or organ damage.

According to a method of predicting or diagnosing tissue or organ damage of an individual according to one aspect, tissue or organ damage of the individual can be efficiently predicted or diagnosed.

According to a method of searching for a therapeutic agent for tissue or organ damage of an individual according to one aspect, a therapeutic agent for tissue or organ damage of an individual can be effectively screened.

1 is a view showing a library making process.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  One. miRNA Colon, liver and Muscle  Diagnosis of the same tissue or organ damage

This example identifies miRNAs specifically expressed in the colon, liver and muscle of horses and confirms that the level of miRNA expression can be used to diagnose malignant tissue or organ damage such as colon, liver and muscle .

1. Plasma and tissue miRNA NGS  Analysis and tissue damage specific miRNA  Selection

(1) Plasma sampling

Plasma was collected from the jugular vein and placed in BD vacutainer EDTA-K2 10.8 mg (6 ml) and centrifuged at 3000 rpm for 10 minutes using a centrifuge.

(2) tissue sampling

Euthanasia (200 mg of mebezonium iodine 50 mg Tetracaine per ml, T-61 ^® , 50 ml, European Union) after sedation (Domosedan ^® , 10 mg / kg, Orion Co./Ketamine, After the autopsy was carried out, the tissues of the colon, liver and muscle of the horses were collected and stored at -70 ° C after quenching in liquid nitrogen.

Histopathologic examination was performed to determine the extent and severity of organ damage. For histopathological examination, the collected tissues were fixed in 10% formalin, embedded in paraffin according to a conventional method, cut into 4-6 μm and stained with hematoxylin and eosin. Colon, liver, and muscle, respectively. Collected colon, liver and muscles were generally flared.

(3) RNA  extraction

Plasma and 0.75 ml of Trizol per 50 mg of the three organ tissues of colon, liver and muscle were homogenized. The homogenized samples were incubated at room temperature for 5 minutes, 0.2 ml of chloroform per 0.75 ml of Trizol was added, and the mixture was shaken for 15 seconds. The mixture was incubated at RT for 3 minutes and centrifuged at 14000 rpm, 4 < 0 > C for 15 minutes. After centrifugation, approximately 500 μl of supernatant was obtained, mixed with isopropyl alcohol 1: 1, and incubated at RT for 10 minutes. After centrifugation at 14000 rpm at 4 ° C for 10 minutes, RNA pellet was confirmed and all supernatants were removed. After washing, 1 ml of 70% EtOH was added, followed by centrifugation at 8000 rpm and 4 ° C for 5 minutes. After removing the supernatant, the RNA pellet was completely dried with the tube lid open at RT. When the pellet is completely dried, elute it with 20 ~ 100 ul of HPLC water. Dnase I was then treated at RT for 15 min to extract pure RNA by completely removing the DNA.

(4) RNA  Concentration and quality analysis

Total RNA isolated from the three organ tissues of horses plasma and muscle, liver, and colon was analyzed using RNA standard and nano drop machine and bioanalyzer machine using RNA standard and quality.

Nucleic acid and protein have the maximum absorbance at 260 nm and 280 nm, respectively. The absorbance ratio (260/280) at 260 nm and 280 nm is a criterion for evaluating the purity of DNA and RNA. Ratio 1.8 is generally pure DNA and ratio 2.0 is pure RNA. When the 260/280 value of the extracted RNA was 1.6 - 2.0, it was judged that the purity was high and used for the subsequent analysis.

Total RNA was isolated from the plasma of the target horses, resulting in a high total RNA of 123 ng per ml of plasma.

(5) NGS  ( Next Generation Sequencing ) In the tissue miRNA  analysis

(5.1) stem loop RT - PCR Quantification using

Small RNAs of 18-32 nt size were isolated using Size-fractionation method. Adapters were attached to these small RNAs, and a size-fractionated cDNA library was constructed through a series of RT-PCR and cDNA cloning procedures. 1 is a view showing a library making process.

The extracted total RNA was loaded on polyacrylamide gel, separated by size through electrophoresis, and then a portion corresponding to small <40-nt RNA was cut out. The <40-nt fraction was washed out with a flashPAGE clean up kit (Life Technologies Corp.) and dissolved in 30 μl of elution buffer. It was then washed with about 80% ethanol. The pellet was resuspended in 50 ul of RNase-free TE. The quality of <40-nt RNA was then checked.

The reverse transcriptase reaction was performed as follows: The purified total RNA sample was mixed with an RT reaction premix containing 50 nM stem-loop RT primer, 1 × RT buffer, 0.25 mM dNTPs, reverse transcriptase and RNase inhibitor 7.5 uL reaction mixture was prepared. The prepared mixture was loaded on a 96-well plate and subjected to sequential reactions holding at about 16 ° C for about 30 minutes, at about 42 ° C for about 30 minutes, at about 85 ° C for about 5 minutes, and at about 4 ° C. The reverse transcription reaction was carried out using negative controls and no-template control and reverse transcriptase minus controls. In addition, the reaction was performed in duplicate or triplicate to reduce the error range.

Real-time qPCR was performed as follows: Approximately 18.7 uL of PCR primmix containing a forward primer, a reverse primer, and a hydrolysis probe was prepared. This premix was loaded onto a 96-well plate, and about 1.3 ul of the RT product was transferred. The real-time quantitative polymerization reaction was carried out in a triplicate manner at 40 ° C for about 10 minutes at about 95 ° C, followed by about 15 seconds at about 95 ° C and about 1 minute at about 60 ° C.

(5.2) NGS  ( Next Generation Sequencing ) Perform

Small RNA cDNA was loaded and NGS (Next Generation Sequencing) was performed by reading 50 bp single-end using HiSeq 2000 machine (Iluumina inc.). Subsequently, the results of the NGS obtained from the plasma of the horses were primarily subjected to the following data cleaning according to the following criteria:

And removed low quality read data. Thereafter, the lead data with the 5 'primer contaminant was removed. Thereafter, the lead data without the 3 &apos; primer was removed. Thereafter, lead data with Poly A was removed. Thereafter, lead data shorter than 18 nt was removed. After that, the lead data without the insert tag was removed.

As a result, 95%, and 94% or more of clean reads were obtained for NSG results obtained from plasma and organ samples, respectively. This indicates that total RNA isolation and size fractionation from horses' plasma and tissues has been well performed and that data reliability for standard bioinformatics analysis is high. After selecting only clean read data among the small RNAs identified through data cleaning, standard bioinformatics analysis shows the expression patterns of known microRNAs, new microRNA, rRNA, tRNA, snRNA, and snoRNA on small RNA Respectively. Standard bioinformatics analysis was performed using miRBase (www.mirbase.org), which is a data base of information on the nucleotide sequence and annotation of miRNAs published in papers.

A small RNA analysis of plasma and organ showed a total of about 250 known miRNAs, and about 55-460 new miRNA candidates were found for each sample. Original microRNAs derived from horse plasma and tissue miRNA sequences and miRNA NGS original data for a total of 15 samples, including the newly discovered miRNA sequences, were obtained from the US NCBI Sequence Read Archive (SRA) database www.ncbi.nlm.nih.gov/Traces/sra/) and received the Submission Numbers SRA056221 and SRA058805. The new miRNA species and nucleotide sequences found for each sample were reported in the results and original data were reported to the NCBI.

A more specific analysis of identified miRNAs has confirmed the bias of positional sequence. For example, microRNAs found in muscle showed more than 80% base pair consistency at positions other than "position 23". In addition, it showed more than 60% first base pair consistency in other parts except 19, 21, 25, 26 length miRNA.

3. Results

As a result of comparing the expression levels of miRNAs of organs such as horses, colon, liver, and muscle with those of whole blood plasma, miRNAs specifically expressed in organs such as colon, liver, and muscle were identified, 2 was expressed at high levels in the blood.

Table 2 shows miRNAs with high expression levels in plasma among miRNAs specifically expressed in organs such as colon, liver, and muscle. The expression level of the miRNA was about 8 times higher (3 Ct (threshold cycle)) than the expression level of miRNA in plasma isolated from normal human.

Types of Organs miRNA Colon ea-mir-155, eca-mir-196b, eca-mir-18a, eca-mir-488, eca-mir-7-2, ea-mir-138, eca-mir-342, eca-mir-1842, eca-mir-361, eca- 132, eca-mir-592, eca-mir-301b, eca-mir-153-2, eca-mir-184, Liver eca-mir-106b, eca-mir-101-2, eca-mir-450b Muscle eca-mir-365-2, eca-mir-190, eca-mir-29b,

From the above results, it was confirmed that the miRNAs shown in Table 2 leaks into the plasma specific miRNAs in each organs when the specific damage of the colon, liver, and muscles causes the expression level of the miRNA to increase in the plasma. Therefore, it was confirmed that the damage of the colon, liver, and muscle can be predicted or diagnosed by examining the degree of change of the miRNA expression level in the blood.

<110> SNU R & DB Foundation <120> Composition and kit for detecting tissue damage or organ damage in          subject, method for predicting or detecting tissue or organ          damage to a subject and method for screening a therapeutic agent          for tissue or organ damage <130> PN102419 <160> 10 <170> Kopatentin 2.0 <210> 1 <211> 23 <212> DNA <213> Equus caballus <400> 1 uuaaugcuaa ucgugauagg ggu 23 <210> 2 <211> 22 <212> DNA <213> Equus caballus <400> 2 uagguaguuu cguguuguug gg 22 <210> 3 <211> 23 <212> DNA <213> Equus caballus <400> 3 uaaggugcau cuagugcaga uag 23 <210> 4 <211> 21 <212> DNA <213> Equus caballus <400> 4 uugaaaggcu auuucuuggu c 21 <210> 5 <211> 21 <212> DNA <213> Equus caballus <400> 5 uaaagugcug acagugcaga u 21 <210> 6 <211> 22 <212> DNA <213> Equus caballus <400> 6 uuuugcgaug uguuccuaau au 22 <210> 7 <211> 21 <212> DNA <213> Equus caballus <400> 7 uacaguacug ugauaacuga a 21 <210> 8 <211> 22 <212> DNA <213> Equus caballus <400> 8 uaaugccccu aaaaauccuu au 22 <210> 9 <211> 22 <212> DNA <213> Equus caballus <400> 9 ugauauguuu gauauauuag gu 22 <210> 10 <211> 23 <212> DNA <213> Equus caballus <400> 10 uagcaccauu ugaaaucagu guu 23

Claims (11)

A composition for diagnosing colon damage of an individual comprising an agent for measuring the expression level of one or more miRNAs selected from the group consisting of eca-mir-155, eca-mir-196b, eca-mir- . The composition of claim 1, wherein the agent is a substance that specifically binds to the one or more miRNAs. The composition of claim 1, wherein the agent is a primer, a probe, or a combination thereof that specifically binds to the one or more miRNAs or a complementary sequence thereof. A kit for diagnosing a colon lesion of an individual, comprising an agent for measuring an expression level of one or more miRNAs selected from the group consisting of eca-mir-155, eca-mir-196b, eca-mir- . 5. The kit of claim 4, wherein the agent is a substance that specifically binds to the one or more miRNAs. 5. The kit of claim 4, wherein the agent is a primer, a probe, or a combination thereof that specifically binds to the one or more miRNAs or a complementary sequence thereof. Measuring the expression levels of one or more miRNAs selected from the group consisting of eca-mir-155, eca-mir-196b, eca-mir-18a, and eca-mir-
Comparing the level of expression of said one or more miRNAs with the level of expression of one or more miRNAs measured in a control. 8. The method of claim 7, further comprising determining that the colon of the individual is damaged if the level of expression of the one or more miRNAs measured is greater than the level of expression of one or more miRNAs measured in the control. 9. The method of claim 8, wherein said measuring step measures the amount of said one or more miRNAs. Administering the candidate substance to the individual;
Measuring the expression level of one or more miRNAs selected from the group consisting of eca-mir-155, eca-mir-196b, eca-mir-18a, and eca-mir-488 in samples isolated from said subject; And
Comparing the level of expression of said one or more miRNAs with the level of expression of one or more miRNAs measured in a control. 11. The method of claim 10, further comprising determining if the measured level of expression of one or more miRNAs is lower than the expression level of a miRNA measured in a control, determining the candidate agent to be effective in treating a diseased colon of the subject .

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