KR101988578B1 - A marker for the determination of Mibyeong - Google Patents

Abstract

The present invention relates to miRNAs as virulence markers or predictive markers, and more particularly, the invention relates to compositions for the determination or prediction of virulence, comprising an agent for measuring the level of expression of miRNA, a virgin identifying or predicting kit comprising said composition, and determining the level of expression of the miRNA.

Description

A marker for the determination of Mibyeong}

The present invention relates to miRNAs that are virulence markers or predictive markers, and more specifically, the present invention relates to compositions for identifying or predicting virulent diseases, comprising an agent for measuring the level of expression of miRNA, a virulence determining or predicting kit comprising said composition, And measuring the level of expression thereof. The present invention also relates to a method for providing information for a virion discrimination or prediction.

(JaeChul Lee et al., Eur . J. Intern. Med ., 2002). In this study, we have investigated the relationship between the severity of discomfort and body fatigue , 2013). Subtypes of infectious diseases include fatigue, pain, sleep disturbances, and indigestion (JaeChul Lee et al., Integr . Med . Res, 2014). In order to diagnose these infectious diseases, Korean medicine has developed devices and methods for measuring infectious disease index based on the responses to the questionnaire (Korean Patent No. 10-1578270).

Fatigue is classified into physical, mental, persistent fatigue according to the period that lasts indicated by the social factors (one month or more than 6 months old) and chronic fatigue (more than 6 months) (Epstein KR et al., Med. Clin. North Am. 1995). Chronic fatigue syndrome is classified as a disease, characterized by persistent fatigue symptoms lasting at least six months and is not ameliorated by rest, and the Centers for Disease Control and Prevention (CDC) It is diagnosed according to the definition of the case as amended in 1994.

MicroRNAs (miRNAs) are short single-stranded ribonucleic acids (RNAs) having 19 to 25 nucleotides in length and are known to regulate the expression of many genes in cells to be expressed (Bartel DP, Cell, 116 ): 281-297, 2004). Up to now, about 2,000 species have been found in humans, and it is predicted that about 30% of human genes will be regulated by miRNAs, as one miRNA is capable of modulating several types of mRNA (Bentwich, et al. Nat. Rev. Genet., 37 (7): 766-770, 2005).

miRNAs are produced naturally in cells and most of them are transcribed into primary miRNAs by RNA polymerase II and then transcribed in the nucleus using the RNase III enzymes drosha and pasha, DGCR8) or the like to a precursor miRNA (precursor miRNA). Subsequently, the precursor miRNAs are transferred from the nucleus to the cytoplasm by Exportin 5, and are produced in the cytoplasm by short and incomplete double stranded miRNAs by the RNase III enzyme dicer. The miRNA thus produced is separated into a single strand by binding to a risk (RISC, RNA-induced silencing complex) protein complex, and the isolated single strand miRNA is complementary to the base sequence of the 3'-UTR (untranslated region) of the target mRNA (Lee, et al., Nature, 425 (6956): 415 (1991)). In addition, it has been known that the expression of mRNA is inhibited by ribosome- -419, 2003).

It has been reported that miRNAs are very stable in serum and that the expression levels of cardiovascular disease and various types of cancer are significantly different from those of normal individuals. Therefore, it is suggested to be a useful biomarker for diagnosis of these diseases (Xi Chen et al., Cell Research , 2008). However, the use of miRNA as a virulence marking or prediction marker has not yet been disclosed.

Under these circumstances, the present inventors have made intensive efforts to develop a method for discriminating or predicting infectious diseases, and as a result, they have identified miRNAs whose expression levels are changed compared to normal individuals in adult male and female populations with persistent fatigue, Or predicted, and completed the present invention.

One object of the present invention is to provide a composition for in vitro diagnosis or prediction comprising an agent for measuring the expression level of a miRNA comprising SEQ ID NO: 1 or SEQ ID NO: 2.

It is another object of the present invention to provide a kit for determining or diagnosing microbial disease comprising said composition.

It is still another object of the present invention to provide a method for providing information for a microinvasive discrimination or prediction, which comprises the step of measuring the expression level of the miRNA.

In one aspect to achieve the above object, the present invention provides a composition for Infectious Disease Identification or Prediction, which comprises an agent for measuring the expression level of a miRNA comprising SEQ ID NO: 1 or SEQ ID NO: 2.

The term "miRNA" used in the present invention means that most introns on a chromosome are interrupted, and this is converted into a precursor-miRNA (precursor form) after being transcribed by Drosha . It is then excreted by exportin 5 and transformed into a mature form of miRNA of about 18 to 22 bp in length by Dicer. These mature miRNAs function by binding to RISC (RNA induced silencing complex) (Nat Rev Mol Cell Biol 6, 376-785; 2005). In the present invention, miRNAs can be used in combination with microRNAs or microRNAs.

For the purposes of the present invention, the miRNA is not limited as long as it can be used for the identification or prediction of an infectious disease, but may be a hsa-let-7f-5p or hsa-miR-3605-5p and exist in isoform form. Preferably a polynucleotide having the nucleotide sequence of SEQ ID NO: 1 or 2.

The sequence of the miRNA can be obtained from GenBank of NCBI, a well-known known database.

The term "miRNA isoform" refers to a sequence in which isomiR has a mutation to a reference miRNA sequence (Morin, R. D et al., Genome Research . (2008) 18 (4): 610-621.) Specifically, the core sequence of the miRNA is the same, but a portion of the nucleotide sequence is added or deleted before or after the core sequence. miRNA sequences can be obtained from miRBase, and isomiR expression profiles are known to be able to exhibit race, population, and gender dependence.

For the purposes of the present invention, miRNA isoforms are not limited as long as they can be used for the identification or prediction of infectious diseases as miRNAs. In the present invention, miRNA isoform may be used in combination with microRNA isoform or isomiR.

In addition, each of the miRNAs of the present invention has not only the miRNA sequence shown in each of the above-mentioned sequence numbers but also a sequence having 80% or more, specifically 90% or more, more specifically 95% or more, particularly, 97% Lt; RTI ID = 0.0 > miRNA < / RTI > As the sequence having such homology, any sequence that represents an miRNA that exhibits substantially the same or a corresponding effect as that of each miRNA is included without limitation. It is also apparent that miRNA sequences in which some sequences are deleted, modified, substituted, or added are also included within the scope of the present invention, if the miRNA sequences have such homology.

The term "infectious disease " as used in the present invention means a state in which there is no distinct disease condition but complaints are uncomfortable. Subtypes of infectious disease include fatigue, pain, sleep disturbance, and indigestion.

In the present invention, an individual who is not infected by a disease has an infectious disease state due to a cause such as a trauma or a metastatic disease of the cervical region, drug administration, pregnancy, lactation, The subjects were classified except for those who were suspected to be infected due to causes such as disability, depression, other medical history, medication, pregnancy, lactation. Integr med res 3, 60-66; According to 2014, the definition of infectious disease and infectious disease index are examined and classified as 'infectious' according to the score. For the purposes of the present invention, the infectious disease can be, but is not limited to, specifically exhibiting fatigue.

As used herein, the term "fatigue" means that the mind or body is exhausted or is in a state of difficulty. (Epstein KR et al. 1995), which is characterized by physical, mental, and social factors and is classified as persistent fatigue (less than 1 month to less than 6 months) and chronic fatigue (more than 6 months). Chronic fatigue syndrome is categorized as a disease, characterized by persistent fatigue symptoms lasting at least 6 months and not resolving when taken at rest. For the purpose of the present invention, the present inventors conducted studies on persons suffering from persistent fatigue symptoms other than chronic fatigue syndrome as described above.

Specifically, individuals having fatigue symptoms according to the present invention are those aged from 35 to 45 years old, having a constant occupation, complaining of continuous or repeated fatigue of 1 to 6 months, and Chalder Fatigue Scale (CFS) questionnaire ), And those individuals with a score of 14 or higher in the incontinence questionnaire (Fig. 2a and Fig. 2b) except for those suspected to exhibit fatigue symptoms due to sleep disturbances, depression, other medical history, drug abuse, pregnancy, Respectively.

The term " Chalder Fatigue Scale (CFS) questionnaire " is a self-administered questionnaire for measuring the degree and severity of fatigue in clinical and non-clinical and epidemiological studies. Originally developed to measure the severity of chronic fatigue symptoms in clinical populations, it is more commonly used to measure the severity of 'fatigue'. Fatigue is important in both academic and applied industrial medicine, and understanding of the link between employee health, performance, safety, and general workability continues to grow. A simple questionnaire that provides a simple tool to measure physical and mental fatigue using a simple response system. Korean Journal of Oriental Medicine, Vol. 28, No. 4, 791-796; According to 2007, the fatigue index is examined and classified according to the score as "fatigue".

As used herein, the term "discrimination or prediction" means identifying the presence or characteristic of a pathological condition. In the present invention, the discrimination or prediction can be interpreted as confirming whether the disease progresses or does not occur.

In the present invention, the miRNA may be a polynucleotide having a nucleotide sequence as set forth in SEQ ID NOS: 1 to 2, or a fragment thereof. The miRNA may be used as a discriminator or a predictive marker capable of discriminating or predicting the progression or incidence of an infectious disease, and the sequence may be modified to some extent in discriminating or predicting whether the infectious disease progresses or not. Those skilled in the art will appreciate that nucleotides that retain more than 80% homology, preferably more than 90% homology, more preferably more than 95% homology, and most preferably 98% homology, It will be easily understood that the nucleotide sequence of the present invention is equivalent to the nucleotide sequence of the present invention as long as the difference in the expression level between the normal individual and the onset subject is significantly compared as a virulence marking or prediction marker.

The term "agent for measuring the expression level of miRNA " used in the present invention means an agent used in a method for confirming expression of an miRNA, its isoform or a fragment thereof contained in a sample. For example, RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), northern blotting, , And a method for gene chip analysis. However, the present invention is not limited to these specific primers or probes.

The term "primer" as used herein refers to a nucleic acid sequence having a short free 3 'hydroxyl group and capable of forming a base pair with a complementary template, Refers to a short nucleic acid sequence that functions as a starting point. The primers can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures.

As used herein, the term "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to a few nucleotides or hundreds of nucleotides, which can specifically bind to a gene or mRNA. An oligonucleotide, A probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like, and can be labeled for easier detection.

The primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, capping, substitution of one or more natural nucleotides with one or more homologues, and modifications between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, (E.g., phosphoramidate, carbamate, etc.) or charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.).

Specifically, as a result of discriminating or predicting fatigue which is one of the diseases in the present invention, the expression level of the miRNA of SEQ ID NO: 1 is upregulated in men compared with that of a normal human, And the expression level was down-regulated (Table 3).

These results indicate that the expression levels of the miRNAs are significantly different from those of the normal group as the disease progresses, and that the use of the composition of the present invention effectively discriminates or predicts infectious diseases.

As another embodiment for achieving the above object, the present invention provides a kit for determining or diagnosing a disease-free disease comprising the composition for discriminating or preventing the disease-free disease.

The miRNA, its isoform, or its fragment expression level can be used to identify or predict a virulent disease by measuring the expression level of the miRNA from a sample of a suspected or suspected virulent patient of the present invention As well as one or more other component compositions, solutions or devices suitable for the assay method may be included.

As a specific example, a discrimination or prediction kit for measuring the level of expression of miRNA may be a kit containing the necessary elements necessary to perform RT-PCR. The RT-PCR kit can be used in combination with a test tube or other appropriate container, a reaction buffer (pH and magnesium concentration varies), deoxynucleotides (dNTPs), Taq polymerase and reverse transcriptase , DNase, RNAse inhibitor, DEPC-water, sterile water, and the like. In addition, it may contain a primer pair specific to a gene used as a quantitative control.

As another example, the kit of the present invention may contain necessary elements necessary for performing gene chip analysis. The kit for gene chip analysis may include a reagent, a preparation, an enzyme, and the like for producing a fluorescent-labeled probe in which a cDNA corresponding to a gene or a fragment thereof is attached as a probe.

In another aspect, the present invention provides a method for providing information for a microinfection or prediction using the composition or kit for discriminating or predicting or predicting or predicting, Measuring the expression level of the miRNA of 1 or 2; And (b) comparing the expression level of the measured miRNA with the expression level of the corresponding hsa-let-7f-5p and hsa-miR-3605-5p of the normal control sample.

In step (b), when the expression level of hsa-let-7f-5p of SEQ ID NO: 1 is elevated above the hsa-let-7f-5p expression level of male normal group, . ≪ / RTI >

The "upregulation" may refer to a case where the amount of miRNA expression is increased by 1 to 10 times, specifically 2 to 5 times as compared to a normal control.

In step (b), when the expression level of hsa-miR-3605-5p consisting of SEQ ID NO: 2 is regulated below the hsa-miR-3605-5p expression level in female normal group, . ≪ / RTI > For the purposes of the present invention, the infectious disease can be, but is not limited to, specifically fatigue.

The "down-regulation" may mean a decrease in the amount of miRNA expressed by 1 to 5 times, specifically 2 to 3 times, compared with a normal control.

The term "individual" of the present invention can refer to any animal including a group of persistent fatigue that continuously feels fatigued or a human being suspected to be infectious. The animal may be, but is not limited to, a mammal such as a cow, a horse, a sheep, a pig, a goat, a camel, a nutrient, a dog, a cat,

The term "control group " of the present invention refers to a normal individual who does not feel fatigued. For the purpose of the present invention, the subject may be a subject having no symptoms of fatigue and general health discomfort within 6 months based on the results of the questionnaire shown in Table 1.

The term "sample" of the present invention means blood isolated from the fatigue group in which persistent fatigue is felt. Specifically, it may be RNA isolated from serum and serum isolated from blood.

Since the miRNA and its isoform provided by the present invention can be used as a marker capable of discriminating or predicting infectious diseases, it can be widely used as a marker having an excellent effect in the development of an infectious disease determination or prediction kit and in the search for a disease inhibiting substance.

1 is a schematic diagram of an overall experiment.
Figures 2a and 2b are unattended questionnaires.
3 is a fatigue questionnaire showing fatigue index (CFS) for classifying fatigue groups.
4 is a box plot of the RT-qPCR verification box.
Figure 5 is a fatigue miRNA ROC analysis.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and the scope of the present invention is not construed as being limited by these embodiments.

Example  1. Selection of study subjects

Using the Chalder Fatigue Scale (CFS) questionnaire and the infirmity questionnaire score, 83 men and 114 women were selected as fatigue groups for those who visited the oriental medicine hospital due to fatigue symptoms. Five male and five female subjects were selected for the serum microRNA NGS (Next Generation Squencing), and five men and five women were selected for the control group (normal). Detailed selection criteria and exclusion criteria are as follows.

Example  1-1. Sustainability fatigue group selection criteria

- 35 years old and under 45 years old

- Persons who complain of persistent or recurrent fatigue of 1 to 6 months

- Chalder Fatigue Scale (CFS) Question 15 or above

- Those with a score of 14 or higher in the uninjured questionnaire (fatigue, pain, sleep, digestion, depression, anxiety, anger)

- Those with a certain occupation

The Chalder Fatigue Scale (CFS) questionnaire and the missing questionnaire were shown in FIGS. 2a, 2b and 2.

Example  1-2. Sustainability fatigue group exclusion criteria

- Those with a primary cause of sleep disturbance (sleep apnea, narcolepsy, etc.)

- Those who have been diagnosed with depression within the last 5 years

- A person with a physical cause that may cause fatigue on a history or current medical history

· Organ failure (heart failure, chronic kidney failure, emphysema, cirrhosis, etc.)

· Chronic infectious diseases (such as hepatitis B or C)

· Rheumatism / inflammatory diseases (SLE, rheumatoid arthritis, inflammatory bowel disease, chronic pancreatitis, etc.)

· Neurological diseases (multiple sclerosis, epilepsy, etc.)

· Endocrine / metabolic diseases (diabetes, thyroid disease, severe obesity with BMI> 45)

· Other diseases (anemia, tuberculosis, chronic lung disease, hypertension, etc.)

· Malignant tumor

- In the last 2 weeks, people taking medication such as antihypertensive, antidepressant, anti-anxiety, hypnotics, antihistamine

- Those who do not have symptoms of fatigue symptom

- Those who have received medical and oriental medicine for fatigue within the past month

- pregnant women, lactating women and those who have a pregnancy plan within 6 months

- Those who are working at night shift, shift work, and heavy redundancy

- Those who believe that they are unable to take medications that may affect their clinical studies or otherwise comply with the test requirements

Example  1-3. NGS  Analysis and RT- qPCR  Selection of verification target

- Five males and five females who are within the top 10% of the CFS questionnaire scores (Figure 3)

- The fatigue score in the incontinence questionnaire (Figures 2a and 2b) is ranked by summing the scores of the four fatigue related items (fatigue, discomfort, persistence, recovery)

- Twenty men and women were selected for the first RT-qPCR verification in the fatigue group excluding the NGS subjects, and 30 men and women were selected for the second RT-qPCR verification

In the fatigue group, the CFS questionnaire score (FIG. 3) is the top 10, and the fatigue score (sum of fatigue, discomfort, persistence, resilience 4 points) Five men and five women were selected.

Example  1-4. Criteria for Selection of Normal Control Group

Based on the results of the questionnaire shown in Table 1, healthy control subjects were selected from healthy subjects aged from 35 to 45 years who had no symptoms of fatigue and general health within 6 months.

① What is the degree of fatigue? None (none, little, medium, severe)
② Does physical pain interfere with normal life? There is no or little (none, little, some, many, very extreme)
③ What is the overall quality of sleep? Very good or generally good (Very good, Almost good, Almost poor, Very poor)
④ Is digestion well? Yes (yes or no)
⑤ What is your health condition? Best or Very Good (Best, Very Good, Good, Poor, Bad)
⑥ Was my heart so bad and depressed? Rarely or not at all (always, mostly, sometimes, rarely, not at all)
⑦ What if there were difficulties in social activities (eg visiting a friend or relative) due to physical health problems or emotional problems? Rarely or not at all (always, mostly, sometimes, rarely, not at all)

Specifically, the subjects were selected as healthy control subjects with no fatigue, pain, dyspepsia, and difficulty in social activities through the questionnaire, good sleep quality, little depression, and good health.

Example  2. Serum microRNA  analysis

Example  2-1: NGS  Analysis (excavation phase, candidate microRNA  Selection) and nucleotide sequence analysis

Five rabbits were selected from the fatigue group and normal control group selected in Example 1, and 1.5 ml of each of the 20 blood samples were separated from the blood. Then, total RNA was isolated from the serum using Mirvana Paris kit (Ambion). Separated RNAs were identified and isolated from microRNA-containing small RNAs using Bioanalyzer (Agilent Technologies). A cDNA library was prepared using 10 ng small RNA per sample using NEBNext® Multiplex Small RNA Library Prep Set. Sequence information of cDNA library constructed using Illumina Nextseq 500 (Illumina) was analyzed and sequence information showing unnecessary information (adapter dimmer generated when the library was produced) and low quality (less than Read length 15) was obtained by FASTQ Toolkit app (Illumina) Lt; / RTI > Qualitative analysis (mapping using human genome and microRNA database) and quantitative analysis (normalized read count analysis) of the analyzed base sequences were performed using BaseSpace® Small RNA v1.0.

Example  2-2. NGS  by microRNA  Quantitative difference analysis

The type, change and statistical significance (P-values) of microRNAs that were significantly changed compared to the normal control group were analyzed using DESeq2 (Love MI et al. (2014) Genome Biol ) program. Specifically, 8 microRNAs were selected for NGS analysis of 10 healthy control subjects and 10 fatigue control groups (5 males and 5 females), respectively, in which read count was significantly increased (P <0.05) or decreased in the fatigue group (Table 2) .

NGS analysis of fatigue-related microRNA
microRNA Women (fatigue = 5, health = 5) Male (fatigue = 5, health = 5) Overall (fatigue = 10, health = 10) Increase / decrease ratio P value Increase / decrease ratio P value Increase / decrease ratio P value One hsa-miR-122-5p decrease 3.0 times 8.8 x 10-3 increase 2.3 times 0.17 decrease 1.4 times 0.24 2 hsa-let-7f-5p increase 1.4 times 0.45 increase 4.4 times 1.0 x 10-3 increase 2.3 times 1.0 x 10-2 3 hsa-miR-24-3p increase 1.2 times 0.63 decrease 4.3 times 5.6 × 10 -5 decrease 1.5 times 0.17 4 hsa-miR-203a-3p increase 4.9 times 3.1 x 10-3 decrease 2.4 times 0.069 decrease 1.5 times 0.21 5 hsa-miR-409-3p increase 1.3 times 0.59 increase 3.0 times 1.7 x 10-2 increase 2.0 times 0.052 6 hsa-miR-3605-5p decrease 3.0 times 0.077 decrease 1.9 times 0.22 decrease 2.2 times 4.3 × 10-2 7 hsa-miR-4433b-5p increase 3.0 times 4.3 × 10-2 increase 1.1 times 0.81 increase 1.7 times 0.12 8 hsa-miR-30c-5p increase 3.5 times 3.9 × 10 -2 - - - increase 1.3 times 0.31

As shown in Table 2, the expression levels of two miRNAs (hsa-let-7f-5p and hsa-miR-3605-5p) in the fatigue group were significantly increased (P-value <0.05). The expression level of hsa-let-7f-5p registered in the miRBase database was found to be 4.4 times (P-value = 1.0 X 10-3) higher in the fatigue group (including the measles) than in the normal control group (control group).

On the other hand, the expression level of the other miRNA hsa-miR-3605-5p was 3.0 times lower (P-value = 0.077) in the fatigue group (including female) than the normal female group (control group).

These results indicate that the miRNAs of the present invention can be discriminated or predicted by detecting upregulation in males or downregulation in females compared with normal controls according to onset of disease.

Example  2- 3: Step 2  RT- qPCR  Analysis (sequential candidates microRNA  Verification)

We tried to perform two verification tests with RT-qPCR method in fatigue group and normal control group except NGS subjects. Specifically, in the first test, serum samples from 40 fatigue patients and 40 control samples were used. In the second test, serum samples from 60 fatigue patients and 40 control patients were used, and the male to female sex ratio was 1: 1 in each group.

Example  2-4: RT- qPCR  On by microRNA  Quantitative difference analysis

Real-time RT-qPCR experiments were carried out using primers capable of specifically binding to each of the candidate microRNAs, and the amplification of only the corresponding microRNAs was confirmed by melting curve analysis.

In addition, the Ct (threshold cycle) value of the candidate microRNAs was corrected to the control microRNA (Cel-miR-39) Ct value, and the significance of the quantitative difference between the fatigue group and the normal control group was analyzed by the Wilcoxon rank sum test. The results of the analysis are shown in Table 3.

Results of fatigue-related microRNA RT-qPCR assays microRNA female male Increase / decrease ratio P value Increase / decrease ratio P value NGS (10 fatigue vs. 10 normal) hsa-let-7f-5p increase 1.4 times 0.45 increase 4.4 times 1.0 x 10-3 hsa-miR-24-3p increase 1.2 times 0.63 decrease 4.3 times 5.6 × 10 -5 hsa-miR-3605-5p decrease 3.0 times 0.077 decrease 1.9 times 0.22 hsa-miR-30c-5p increase 1.8 times 3.9 × 10 -2 - - - RT-qPCR primary verification (40 fatigue patients vs. 40 normal patients) hsa-let-7f-5p increase 1.6 times 0.072 increase 5.1 times 6.2 × 10 -4 hsa-miR-24-3p decrease 1.4 times 0.14 decrease 1.7 times 4.7 x 10-3 hsa-miR-3605-5p decrease 3.2 times 6.0 × 10-7 decrease 1.5 times 2.0 x 10 -2 hsa-miR-30c-5p increase 1.2 times 0.17 increase 1.2 times 0.56 RT-qPCR secondary verification (fatigue 60 vs. normal 40) hsa-let-7f-5p increase 1.2 times 0.48 increase 2.0 times 2.4 x 10-3 hsa-miR-24-3p increase 1.2 times 0.40 increase 2.3 times 5.0 x 10-3 hsa-miR-3605-5p decrease 2.0 times 8.2 × 10 -4 increase 1.5 times 0.050 hsa-miR-30c-5p decrease 1.1 times 0.51 increase 5.3 times 1.7 × 10 -6

As shown in Table 3, 4 out of 8 microRNAs (hsa-let-7f-5p, hsa-miR-24-3p, hsa-miR-3605-5p, hsa-miR-30c-5p ) microRNAs in male and female fatigue groups were significantly increased or decreased in comparison with the control group, and 4 microRNAs with different tendency of NGS analysis were judged to be failed in verification.

However, at the same time comparing NGS with the first and second verification results, it was confirmed that two microRNAs were associated with fatigue. hsa-let-7f-5p was significantly increased in both fatigue males and NGS, and hsa-miR-3605-5p was significantly reduced in fatigue women in both NGS and first- (Table 3, Fig. 4)

Example  2-5. ROC analysis result ( microRNA  Persistent fatigue discrimination test)

The fatigue discrimination power of let-7f-5p was measured in 50 fatigue men and 40 normal control men. The fatigue discrimination power of miR-3605-5p was measured in 50 fatigue women and 40 normal control women.

The SPSS statistical program was used to calculate the fatigue prediction probability, sensitivity, and 1-specificity for each sample using a logistic regression model with age, BMI, and microRNA expression as variables . Based on the calculated result values, an ROC (Receiver Operating Curve) that sets the x axis to 1-specific sensitivity and the sensitivity to the y axis is determined using an SPSS program, and an Area Under the Curve (AUC) P value was calculated.

As a result, fatigue males were able to discriminate persistent fatigue with 76% accuracy using the amount of hsa-let-7f-5p serum expression. In the case of fatigue women, the expression level of hsa-miR-3605-5p was 82 % Fatigue (Fig. 5). This suggests that hsa-let-7f-5p and hsa-miR-3605-5p may be used as objective indicators to determine persistent fatigue.

In this study, serum microRNAs were tested for sex-specific changes in serum levels due to persistent fatigue. This finding is consistent with previous findings that the serum microRNA profile associated with metabolic syndrome varies according to sex (J Biomed Sci. 2013 Oct; 20 (1): 72).

Therefore, two microRNAs (hsa-let-7f-5p and hsa-miR-3605-5p), which were verified based on the above results, could be used as markers for fatigue state discrimination.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

<110> Korea Institute of Oriental Medicine <120> A marker for the determination of Mibyeong <130> KPA170830-KR <140> 10-2017-0098906 <141> 2017-08-04 <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 22 <212> RNA <213> Homo sapiens <400> 1 ugagguagua gauuguauag uu 22 <210> 2 <211> 23 <212> RNA <213> Homo sapiens <400> 2 ugaggaugga uagcaaggaa gcc 23

Claims (14)

1. A composition for the determination or prediction of fatigue comprising an agent for measuring the level of expression of an miRNA consisting of SEQ ID NO: 1.
delete 2. The composition of claim 1, wherein the agent further measures the level of expression of the miRNA isoform.
The method according to claim 1,
Wherein the agent comprises a primer or a probe that specifically binds to the miRNA.
The method according to claim 1,
Wherein the agent for measuring the expression level of the miRNA consisting of SEQ ID NO: 1 is for discriminating or predicting fatigue of a human male.
delete 6. A kit for fatigue discrimination or prediction comprising a composition according to any one of claims 1 to 5.
delete 8. The method of claim 7,
Wherein the kit is an RT-PCR kit or a gene chip kit.
(a) determining the level of expression of the miRNA of SEQ ID NO: 1 in a biological sample separated from the individual; And (b) comparing the expression level of the measured miRNA with the expression level of the corresponding miRNA of the normal control sample.
delete 11. The method of claim 10, wherein step (a) further measures the expression level of the miRNA isoform.
11. The method of claim 10,
The method further comprises (c) determining that fatigue has progressed if the expression level of the miRNA of SEQ ID NO: 1 in the step (b) is adjusted upward than the miRNA expression level of the male normal group. Delivery method.
delete

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