KR20220060615A - Method for diagnosing pre-eclampsia using ratio of expression levels of miR-155-5p and miR-1290-3p - Google Patents

Abstract

The present invention relates to a method for diagnosing preeclampsia using a ratio of expression levels of miR-155-5p and miR-1290-3p. More specifically, the present invention provides an information providing method for diagnosing preeclampsia, the method including a step of determining a ratio between the expression level of miR-155-5p and the expression level of miR-1290-3p in a sample isolated from a subject; and a composition and kit for diagnosing preeclampsia therefor. According to the present invention, preeclampsia can be accurately and easily diagnosed using an expression level ratio of miR-155-5p and miR-1290-3p. In particular, when the method of the present invention is used, reliability can be increased and clear diagnosis is possible compared to the case of using each miRNA alone. In addition, since the method of the present invention can obtain related information from blood, preeclampsia can be diagnosed very safely and easily compared to other methods that require tissues or cells such as the fetus and placenta.

Description

Method for diagnosing pre-eclampsia using ratio of expression levels of miR-155-5p and miR-1290-3p

The present invention relates to a method for diagnosing preeclampsia using the expression level ratio of miR-155-5p and miR-1290-3p. More specifically, the present invention provides an information providing method for diagnosing preeclampsia comprising determining the ratio between the expression level of miR-155-5p and the expression level of miR-1290-3p in a sample isolated from a subject, this It relates to a composition and kit for diagnosing preeclampsia.

Preeclampsia is a complex disease, the cause of which is not yet clear, but it is presumed that the cause is the inability to supply nutrients and oxygen from the mother to the fetus due to impaired blood flow due to abnormal blood vessel formation in the uterus and abnormal blood vessels in the uterus.

Preeclampsia is defined as a disease that develops after 20 weeks of pregnancy and is accompanied by high blood pressure (systolic 140mmHg, diastolic 90mmHg or more) and proteinuria (300mg or more/day urine). In addition, it shows the main symptoms of edema and weight gain, and is sometimes accompanied by pathological symptoms such as destruction of blood cells, decreased liver function, and decreased platelets. Preeclampsia occurs in 2 to 8% of all pregnant women worldwide, and is known to cause miscarriages and premature births, and is a major contributor to maternal and fetal mortality.

According to the analysis of the gestational maternal mortality rate in the United States, about 10 deaths per 100,000 live births in the early 1900s, 14.5 deaths between 1998 and 2005, 16.0 deaths between 2016 and 2010, and 17.0 deaths between 2011 and 2013. did According to these basic data, the pregnancy-related mortality rate is gradually increasing every year. Meanwhile, from 2011 to 2013, it is reported that 98 cases (4.9%) of fetal mortality among 2,009 pregnant women were reported. This mortality rate is highly related to the mother's age, and is rapidly increasing in the late 30s. The causes of death include bleeding, infection, pulmonary embolism due to thrombosis, cardiomyopathy, cardiovascular disease, non-cardiovascular disease, gestational hypertension, and the like, and each of these causes accounts for 10 to 13% of fetal deaths. Among these causes, gestational hypertension is known to be the leading cause of fetal death.

When preeclampsia develops, there is no clear treatment, and the only solution is to induce childbirth through vaginal delivery or cesarean section in an immature state. However, since the risk of pregnant women and the viability of the fetus are very vulnerable before the 34th week of pregnancy, it is necessary to delay childbirth to lower the risk to the pregnant woman and the fetus. Therefore, in the case of pregnant women who have risk factors for inducing preeclampsia, it is known that the best method is to prevent the onset of the disease through diet control and exercise. Therefore, early diagnosis of disease risk factors is necessary.

Meanwhile, recent research results have reported that specific miRNAs are involved in the onset and progression of various diseases, including preeclampsia, and since these miRNAs circulate through the blood, they can be detected relatively easily through blood tests. Therefore, many studies are being conducted to utilize them as biomarkers for diagnosing preeclampsia.

However, since there are cases where one miRNA is linked to multiple diseases, when such miRNA is used as a biomarker for preeclampsia, there is a problem that a situation related to another disease may be mistaken for a situation related to preeclampsia. There is also a problem that there are many cases where judgment is ambiguous because there is not much difference between the control group, that is, the normal person and the risk group for preeclampsia in the change of markers. Therefore, there is an urgent need to develop a technology that can further enhance reliability and ease of use of such miRNA as a biomarker for preeclampsia.

Phipps EA, Thadhani R, Benzing T, Karumanchi SA. Pre-eclampsia: pathogenesis, novel diagnostics and therapies. Nat Rev Nephrol. 2019, 15:275-289. English FA, Kenny LC, McCarthy FP. Risk factors and effective management of preeclampsia. Integr Blood Press Control. 2015, 8:7-12. Berg CJ, Callaghan WM, Syverson C, Henderson Z. Pregnancy-related mortality in the United States, 1998 to 2005. Obstet Gynecol. 2010, 116:1302-1209. Creanga AA, Syverson C, Seed K, Callaghan WM. Pregnancy-Related Mortality in the United States, 2011-2013. Obstet Gynecol. 2017, 130:366-373. Rana S, Lemoine E, Granger JP, Karumanchi SA. Preeclampsia: Pathophysiology, Challenges, and Perspectives. Circ Res. 2019. 124:1094-1112.

Therefore, the main object of the present invention is to provide a method that can further increase the reliability and ease of using miRNA as a biomarker of preeclampsia.

Another object of the present invention is to provide a means such as a composition and kit for diagnosing preeclampsia, which increases reliability and ease by applying such a method.

According to one aspect of the present invention, the present invention provides for the diagnosis of preeclampsia comprising determining the ratio between the expression level of miR-155-5p and the expression level of miR-1290-3p in a sample isolated from a subject. Information provision method is provided.

In the information providing method of the present invention, the ratio is the ratio of the expression level of miR-155-5p to the expression level of miR-1290-3p or the expression of miR-1290-3p to the expression level of miR-155-5p It is preferable that it is a ratio of the level.

In the information providing method of the present invention, the expression level is preferably a concentration.

In the information providing method of the present invention, the concentration is preferably a molar concentration.

In the information providing method of the present invention, if the ratio of the molar concentration of miR-155-5p to the molar concentration of miR-1290-3p is 2.11 or more, it is preferable to classify the group as a high-risk group for preeclampsia.

In the information providing method of the present invention, the sample is preferably blood or serum.

The information providing method of the present invention is preferably used for diagnosing preeclampsia in Korean women.

According to another aspect of the present invention, there is provided a composition for diagnosing preeclampsia, comprising a means for measuring the expression level of miR-155-5p and a means for measuring the expression level of miR-1290-3p.

In the composition of the present invention, the means for measuring the expression level of miR-155-5p is complementary to the nucleotide sequence of miR-155-5p, the nucleotide sequence of miR-155-5p cDNA, or the cDNA of miR-155-5p. It is a primer or probe that specifically binds to a specific nucleotide sequence, and the means for measuring the expression level of miR-1290-3p is the nucleotide sequence of miR-1290-3p, the nucleotide sequence of miR-1290-3p cDNA, or miR- Preferably, the primer or probe specifically binds to a nucleotide sequence complementary to the cDNA of 1290-3p.

The composition of the present invention is preferably used for diagnosis of preeclampsia in Korean women.

According to another aspect of the present invention, the present invention provides a kit for diagnosing preeclampsia comprising the composition.

In the kit of the present invention, when the ratio of the molar concentration of miR-155-5p to the molar concentration of miR-1290-3p is 2.11 or more, it is preferable to further include data suggesting criteria for classification as a high-risk group for preeclampsia.

The kit of the present invention is preferably used for diagnosis of preeclampsia in Korean women.

According to the present invention, it is possible to accurately and easily diagnose preeclampsia by utilizing the expression level ratio of miR-155-5p and miR-1290-3p. In particular, when the method of the present invention is used, reliability can be increased and a clear diagnosis is possible compared to the case where each miRNA is used alone. In addition, the method of the present invention has the advantage that it is very safe and easy to diagnose preeclampsia compared to other methods that require tissues or cells such as a fetus or placenta because related information can be obtained from blood.

1 is a result of analysis of the relative expression levels of miR-155-5p and miR-1290-3p in the serum of pregnant women who have paid off (HP) and patients with preeclampsia (PE).
2 is a result of analyzing the correlation between systolic blood pressure (SBP) and proteinuria and the expression levels of miR-155-5p and miR-1290-3p in patients with preeclampsia.
3 is a result of analyzing the sensitivity and specificity of miR-155-5p and miR-1290-3p as biomarkers for preeclampsia.
4 is a result of analyzing the sensitivity and specificity of miR-155-5p/miR-1290-3p ratio as a preeclampsia biomarker.
5 is a result of analyzing the correlation between systolic blood pressure (SBP) and proteinuria and the miR-155-5p/miR-1290-3p ratio of preeclampsia patients.
6 is a result of analyzing the miR-155-5p/miR-1290-3p ratio for each pregnancy period of preeclampsia patients.
7 is a molar concentration standard curve for quantification of miR-155-5p and miR-1290-3p and molar concentrations of miR-155-5p and miR-1290-3p in normal pregnant women and preeclampsia patients using the standard curve. is the result of deriving
8 is a result of analyzing the miR-155-5p/miR-1290-3p ratio of normal pregnant women and preeclampsia patients calculated based on the molar concentration derived through quantification, and the sensitivity and specificity of this ratio.

The present invention has been devised based on the following new research results.

1. The concentration of miR-155-5p in the blood of preeclampsia patients is significantly increased compared to that of normal pregnant women.

2. The concentration of miR-1290-3p in the blood of preeclampsia patients was significantly decreased compared to that of normal pregnant women.

3. There is a high correlation between the increase in the ratio of miR-155-5p/miR-1290-3p and hypertension and proteinuria, which are symptoms of preeclampsia, and the disease induction timing is very similar.

4. The ratio of miR-155-5p/miR-1290-3p has much higher sensitivity and specificity for the diagnosis of preeclampsia than each miRNA concentration.

Based on the research results as described above, the present invention provides for the diagnosis of preeclampsia comprising determining the ratio between the expression level of miR-155-5p and the expression level of miR-1290-3p in a sample isolated from a subject. Information provision method is provided.

In the present invention, the subject is a woman, preferably a Korean woman, preferably a pregnant woman, more preferably a Korean pregnant woman.

In the present invention, the sample may be, for example, a sample such as a tissue, cell, or body fluid isolated from a subject, preferably blood (whole blood) or serum. The present invention has the advantage that it can be safely and easily performed compared to the conventional method using a sample that is difficult to collect because blood or serum can be used as a sample, for example, a sample derived from a fetus or a sample such as placental tissue.

Information on miR-155-5p and miR-1290-3p is well known through the micro RNA database miRBase (http://www.mirbase.org). miR-155-5p is one of the mature miRNAs of miR-155, and the typical human ( Homo sapiens ) miR-155-5p sequence is shown in SEQ ID NO: 1. miR-1290-3p is a mature miRNA of miR-1290, and typically human ( Homo sapiens ) miR-1290-3p sequence is shown in SEQ ID NO: 2.

In the present invention, the expression level of miR-155-5p and the expression level of miR-1290-3p may be a relative expression level, for example, a relative expression level to a control (a sample isolated from a normal pregnant woman), and the absolute expression level; For example, it may be an absolute expression level such as a concentration in a sample. Preferably it is an absolute expression level, More preferably, it is a concentration, More preferably, it is a molar concentration.

Such expression level is, for example, a conventional method for determining the expression level of miRNA in a sample, for example, quantitative real-time PCR, microarray expression profiling (microarray expression profiling), Northern blot (northern blot), etc. methods can be determined through In the case of the relative expression level, the miRNA expression level of the sample isolated from the subject can be determined by using the above methods but determining the expression level relative to the expression level of the control group confirmed under equivalent conditions. For example, using quantitative real-time PCR to determine the expression level value (eg, the strength of the amplification signal) of the corresponding miRNA in the control group (eg, a sample isolated from normal pregnant women), and in the experimental group under equivalent conditions ( That is, the miRNA expression level of the sample isolated from the subject is determined by determining the expression level value of the corresponding miRNA in the sample isolated from the subject), and then determining the ratio of the expression level value of the experimental group to the expression level value of the control group. can be decided In the case of the absolute expression level, various methods for determining the concentration of miRNA in a sample are well known and can be determined based on this. For example, a standard curve is created by performing quantitative real-time PCR on a sample for each concentration of the corresponding miRNA, and the result derived by performing quantitative real-time PCR on a sample isolated from a subject under equivalent conditions (e.g., of the amplification signal intensity) can be substituted into the standard curve to determine the miRNA expression level of the sample isolated from the subject.

In the present invention, the ratio between the expression level of miR-155-5p and the expression level of miR-1290-3p is preferably the ratio of the expression level of miR-155-5p to the expression level of miR-1290-3p or miR- It is the ratio of the expression level of miR-1290-3p to the expression level of 155-5p. More preferably, the ratio of the concentration of miR-155-5p to the concentration of miR-1290-3p or the ratio of the concentration of miR-1290-3p to the concentration of miR-155-5p. More preferably, it is the ratio of the molar concentration of miR-155-5p to the molar concentration of miR-1290-3p or the ratio of the molar concentration of miR-1290-3p to the molar concentration of miR-155-5p. In this case, the expression level of miR-155-5p and the expression level of miR-1290-3p are preferably expression levels determined under equivalent conditions.

After the step of determining the ratio in the present invention, the method may further include classifying subjects based on the determined ratio. This step may include, for example, comparing the ratio with the corresponding ratio (ie, the ratio between the expression level of miR-155-5p and the expression level of miR-1290-3p) of a control (eg, a sample isolated from normal pregnant women) This can be achieved by way of comparison. For example, when the ratio is the ratio of the expression level of miR-155-5p to the expression level of miR-1290-3p, this ratio is the ratio of the control group (miR to the expression level of miR-1290-3p in the control group) -155-5p expression level ratio), the subject is classified as a risk group for preeclampsia. Conversely, when the ratio is the ratio of the expression level of miR-1290-3p to the expression level of miR-155-5p, this ratio is the ratio of the control group (miR-1290- If it is less than the ratio of the expression level of 3p), the subject can be classified as a risk group for preeclampsia.

More preferably, the step of classifying the subject is a step of classifying the subject into a high-risk group for preeclampsia when the ratio of the molar concentration of miR-155-5p to the molar concentration of miR-1290-3p is 2.11 or more. This is according to the experimental results of samples from normal pregnant women and patients with preeclampsia, and according to this, subjects can be classified with very high sensitivity and specificity without needing to compare the proportion of the control group.

According to the study results of the present invention, the difference in the ratio between the subject and the control group from the second trimester (2nd trimester, 14 to 26 weeks of pregnancy) appears more clearly. Therefore, in order to provide more reliable information, the subject's sample is preferably collected after 14 weeks of pregnancy, that is, after 14 weeks.

Based on the above study results, the present invention also provides a composition for diagnosing preeclampsia comprising a means for measuring the expression level of miR-155-5p and a means for measuring the expression level of miR-1290-3p.

The composition of the present invention is a composition for diagnosing preeclampsia of a woman, preferably a Korean woman, preferably a pregnant woman, more preferably a Korean pregnant woman.

The means for measuring the expression level of miR-155-5p in the composition of the present invention may be a miR-155-5p-specific substance required for the existing method for measuring the concentration of miR-155-5p, preferably miR A primer or probe that specifically binds to the nucleotide sequence of -155-5p, the nucleotide sequence of the cDNA of miR-155-5p, or the nucleotide sequence complementary to the cDNA of miR-155-5p. Similarly, the means for measuring the expression level of miR-1290-3p may be a miR-1290-3p-specific material required for the existing method for measuring the concentration of miR-1290-3p, preferably miR-1290-3p It is a primer or probe that specifically binds to the nucleotide sequence of , the nucleotide sequence of miR-1290-3p cDNA, or the nucleotide sequence complementary to the nucleotide sequence of miR-1290-3p cDNA. For example, it may be a commercial primer used to confirm the concentration of each miRNA through quantitative real-time PCR (qRT-PCR), and each miRNA such as a chip based on the sequence of each miRNA. Detection means may also be included here.

In the composition of the present invention, in addition to the means for measuring the expression level of miR-155-5p and the means for measuring the expression level of miR-1290-3p as described above, reagents necessary for measuring the expression level using these means are further included. can For example, reagents necessary for performing qRT-PCR, reagents required for performing microarray chip analysis, reagents required for performing Northern blot, and the like may be further included.

Based on the above research results, the present invention also provides a kit for diagnosing preeclampsia comprising the above composition.

The kit of the present invention preferably further includes data suggesting criteria for classification as a high-risk group for preeclampsia when the ratio of the molar concentration of miR-155-5p to the molar concentration of miR-1290-3p is 2.11 or more. This is according to the experimental results on samples of normal pregnant women and patients with preeclampsia, and according to this, subjects can be classified with very high sensitivity and specificity. There is no particular limitation on the form of the above data, and any form may be possible as long as information on the above standards can be presented. For example, it may be in the form of instructions or reference tables.

The kit of the present invention is a kit for diagnosing preeclampsia of a woman, preferably a Korean woman, preferably a pregnant woman, more preferably a Korean pregnant woman.

In addition to the composition of the present invention, the kit of the present invention may further include reagents, instruments or devices necessary for measuring the expression level as described above.

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

[Example]

1. Experimental method

1-1. Experimental Materials and Animal Ethics

Human blood and tissue samples used in the experiment were obtained according to the guidelines of the Biomedical Ethics Committee of Kangwon National University Hospital. Blood samples were obtained from normal pregnant women and from 92 patients with preeclampsia, respectively. Pregnant women's blood was collected in a vacuum tube containing an anticoagulant. The clinical criteria for preeclampsia patients are proteinuria ≥300mg/day and blood pressure ≥140/90mmHg.

1-2. Measurement of blood miRNA

Serum from blood obtained from normal pregnant women and patients with preeclampsia is centrifuged at 3,000rpm for 15 minutes in a refrigerated microcentrifuge to remove impurities, and the entire miRNA is purified using miRNeasy Serum/Plasma kit (Qiagen, Cat.#: 217184). separated. After adding 5 times the volume of Qiazol to serum (200 μl) and mixing it well, the same amount of chloroform was added, mixed vigorously for 1 minute, stored at room temperature for 3 minutes, and centrifuged for 15 minutes at 13,000 rpm in a refrigerated microcentrifuge. An aqueous solution layer in which RNA is present was taken, 1.5 times the amount of 100% EtOH was added and mixed slowly, 700 μl was taken, loaded on an RNeasy MinElute spin column, and centrifuged at 13,000 rpm for 1 minute. 700 μl of RWT buffer, 700 μl of RPE buffer, and 500 μl of 80% EtOH were sequentially added to the column and centrifuged to wash the membrane. After centrifugation once more to dry the membrane, 14 μl of RNase free water was added to the column and centrifuged to dissolve miRNA. After polyadenylation of the 3'-end of the miRNA isolated using HiSpec buffer, cDNA was synthesized using oligo-dT and reverse transcriptase (Qiagen, Cat.#: 218161), followed by quantitative real-time PCR (quantitative). real-time PCR: qRT-PCR). Using the synthesized cDNA as a template, miScript SYBR Green PCR kit (Qiagen, Cat.#: 218073), primers specific for miR-155-5p or miR-1290-3p (Qiagen, Cat.#: MS00031486, MS00014518) were used. The concentration of each miRNA was measured through qRT-PCR.

1-3. statistical analysis

Statistical analysis of all results obtained from blood pressure, proteinuria, and blood of normal pregnant women and preeclampsia patients was performed using Prism 6 (Graphpad Software, Inc.). Statistical significance test was analyzed by two-tailed Student's t test, and P <0.05 was considered as having statistical significance. On the other hand, the accuracy (sensitivity and specificity) and characteristic evaluation of the diagnostic test using the concentration of miRNA in the blood was performed using the receiver operating characteristic (ROC) curve analysis method.

2. Experimental results

2-1. Confirmation of relative levels of miR-155-5p and miR-1290-3p in serum of normal pregnant women and patients with preeclampsia

The relative levels of miR-155-5p and miR-1290-3p in the serum of normal pregnant women and preeclampsia patients were measured by qRT-PCR and statistically compared and analyzed. The level of miR-155-5p in the serum of preeclampsia patients increased by 3.1 fold (2.69±0.02 fold vs. 0.86±0.06 fold, P <0.0001) compared to that of normal pregnant women ( FIG. 1A ). In the case of miR-1290-3p, it was reduced by 3.9-fold (0.36±0.02 fold vs. 1.01±0.05 fold, P <0.0001) in patient serum ( FIG. 1B ). These results suggest that miR-155-5p is significantly increased and miR-1290-3p is significantly decreased in the serum of patients with preeclampsia.

2-2. The correlation between the levels of miR-155-5p and miR-1290-3p in the serum of preeclampsia patients and the symptoms of preeclampsia

Preeclampsia is known as a disease that accompanies hypertension and proteinuria. Therefore, we analyzed the correlation between the blood levels of miR-155-5p and miR-1290-3p in the patient's blood, blood pressure and proteinuria. As a result, there was a positive correlation between miR-155-5p ( r = 0.478, P <0.0001) levels and systolic blood pressure, and a negative correlation between miR-1290-3p ( r = -0.397, P <0.0001) and systolic blood pressure. It was confirmed that this exists (FIGS. 2A and 2B). In addition, there was a positive correlation between the level of miR-155-5p ( r = 0.618, P < 0.0001) and proteinuria, and there was a negative correlation between miR-1290-3p ( r = -0.376, P <0.0001) and proteinuria. It was confirmed that there is (FIGS. 2C to 2D). These results suggest that tracking changes in the level of these mRNAs in the blood of pregnant women can diagnose preeclampsia and can be used as positive and negative diagnostic biomarkers.

2-3. Validation of the utility of miR-155-5p and miR-1290-3p as biomarkers for the diagnosis of preeclampsia

In order to verify the possibility of use as a preeclampsia biomarker by utilizing these miRNAs showing changes in levels in the blood of preeclampsia patients, a ROC (receiver operating characteristic) curve and statistical analysis using the same were performed. The closer the AUC (area under the ROC curve) value is to 1.0, the higher the potential for use as a diagnostic biomarker. The AUC values of miR-155-5p and miR-1290-3p were found to be 0.931 (95% confidence interval: 0.895-0.967, P <0.0001) and 0.957 (95% confidence interval: 0.931-0.984, P <0.0001), respectively. (FIGS. 3A-3C). Therefore, it is judged that these miRNAs have very high utility value as biomarkers for diagnosing preeclampsia. The sensitivity of miR-155-5p, which is highly likely to be used as a positive diagnostic biomarker, was 89.13%, and the specificity was 88.04%, and the sensitivity of miR-1290-3p, which can be used as a negative diagnostic biomarker, was 94.57%, specific The degree was 84.78% (Figure 3C). These results suggest that each of these miRNAs has a high potential for use as a biomarker for the diagnosis of preeclampsia.

On the other hand, as a result of checking the cut-off (cut-off value or threshold value) values for individual biomarkers, miR-155-5p and miR-1290-3p were 1.365 and 0.595, respectively. Therefore, the level of miR-155-5p in the blood of pregnant women is 1.365 times higher than that of the control group (the level of miR-155-5p in the blood of normal pregnant women), or the level of miR-1290-3p is higher than that of the control group (miR in the blood of normal pregnant women). If it is 0.595 times or less of the level of -1290-3p), it can be determined that it corresponds to the reliability section of the onset of preeclampsia. Conversely, if the positive diagnostic miR-155-5p level is less than 1.365 times that of the control group or the negative diagnostic miR-1290-3p level exceeds 0.595 times that of the control group, the reliability of the risk of developing preeclampsia is a rule-out. can be judged as

2-4. Validation of the possibility of diagnosing preeclampsia using the miR-155-5p/miR-1290-3p ratio

It has been reported that miR-155-5 is involved in the onset and progression of diseases other than preeclampsia. In order to improve the specificity of the preeclampsia diagnosis, it is necessary to utilize multiple biomarkers, particularly the ratio values of the biomarker levels with positive and negative specificity. Therefore, the possibility of diagnosing preeclampsia using the miR-155-5p/miR-1290-3p ratio value was verified.

The ratio values of miR-155-5p/miR-1290-3p were statistically compared and analyzed in the serum of normal pregnant women and patients with preeclampsia. The ratio of miR-155-5p/miR-1290-3p in the serum of preeclampsia patients was increased by 10.9-fold (10.53±1.02 vs. 0.97±0.08, P <0.0001) compared to that of normal pregnant women ( FIG. 4A ). These results suggest that the ratio value of miR-155-5p/miR-1290-3p in the serum of patients with preeclampsia significantly increased than the value of miRNA alone (10.53±1.02 vs. 2.69±0.02). As a result of performing the ROC curve and statistical analysis using it, the AUC value of the miR-155-5p/miR-1290-3p ratio value was confirmed to be 0.990 (95% confidence interval: 0.981-1.000, P <0.0001) (Fig. 4B). and 4C). The sensitivity of this ratio value was 93.48%, and the specificity was 97.83% (Fig. 4C). These results mean that when the miR-155-5p/miR-1290-3p ratio is used, the sensitivity and specificity for diagnosing preeclampsia can be improved than when using a single miRNA level.

On the other hand, as a result of confirming the cut-off value for the miR-155-5p/miR-1290-3p ratio, it was 2.767 (FIG. 4C). Therefore, if the value of these ratios in the blood of pregnant women is 2.767 or more, it can be judged that it corresponds to the reliability section of the onset of preeclampsia, and, conversely, if it is less than the cut-off value, it is judged that the reliability of the risk of preeclampsia is rule-out. can do.

2-5. Verification of correlation between miR-155-5p/miR-1290-3p ratio and symptoms of preeclampsia

To confirm the potential of this miRNA ratio as a marker for diagnosing preeclampsia, the correlation with clinical symptoms was analyzed. As a result, the patient's blood miR-155-5p/miR-1290-3p ratio had a high positive correlation with systolic blood pressure ( r = 0.547, P <0.0001) and proteinuria ( r = 0.688, P <0.0001) ( 5A and 5B). These results suggest that the miR-155-5p/miR-1290-3p ratio is closely related to preeclampsia.

2-6. Validation of correlation between miR-155-5p/miR-1290-3p ratio and gestational organs

Preeclampsia develops after 20 weeks of pregnancy (2nd trimester). Therefore, the level of miRNA in the blood of the patient was measured for each pregnancy period, and based on this, the change in the miR-155-5p/miR-1290-3p ratio was analyzed. As a result, the miR-155-5p/miR-1290-3p ratio was significantly increased in the second trimester of pregnancy, and further increased in the third trimester ( FIG. 6 ). That is, the ratio of miR-155-5p/miR-1290-3p was found to increase in a very similar manner to the period when the symptoms of preeclampsia were induced, and a clear change was also observed at the beginning of the onset of preeclampsia. These results suggest that this ratio and preeclampsia are closely related, and this ratio can be used to effectively diagnose preeclampsia at an early stage.

2-7. Quantification of the concentrations of miR-155-5p and miR-1290-3p in the blood of normal pregnant women and patients with preeclampsia

In order to quantify the concentrations of miR-155-5p and miR-1290-3p in the blood, each commercially available miRNA was first purchased and a standard curve was created ( FIGS. 7A and 7B ). As a result of quantifying the concentration of miRNA in the blood using these curves, the molar concentration of miR-155-5p in the blood of normal pregnant women was 0.587±0.044 pM, and the concentration in the blood of the patient was 1.832±0.092 pM ( p <0.0001). (Fig. 7C). On the other hand, in the case of miR-1290-3p, the molar concentration in the blood of normal pregnant women was 0.784±0.042 pM and the molar concentration in the blood of the patient was 0.277±0.014 pM ( p <0.0001) ( FIG. 7C ).

2-8. Determination of preeclampsia diagnostic value using miR-155-5p/miR-1290-3p molar concentration ratio

As a result of calculating the miR-155-5p/miR-1290-3p ratio using the blood miRNA molar concentrations of normal pregnant women and patients with preeclampsia, it was 0.852±0.069 for normal pregnant women and 9.275±0.894 for patients with preeclampsia ( p <0.0001). ) was (Fig. 8A). Meanwhile, in order to effectively diagnose preeclampsia, the cut-off calculated from the ROC curve was 2.11 ( FIGS. 8B and 8C ). This means that when the molar concentration ratio of miR-155-5p to miR-1290-3p in the blood of pregnant women is 2.11 or higher, the probability of developing preeclampsia is more than 95%.

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Claims (13)

A method for diagnosing preeclampsia, comprising determining a ratio between the expression level of miR-155-5p and the expression level of miR-1290-3p in a sample isolated from a subject. According to claim 1,
The method, wherein the ratio is the ratio of the expression level of miR-155-5p to the expression level of miR-1290-3p or the ratio of the expression level of miR-1290-3p to the expression level of miR-155-5p. According to claim 1,
The expression level is a concentration, the method. 4. The method of claim 3,
wherein the concentration is molarity. 5. The method of claim 4,
A method of classifying as a high-risk group for preeclampsia if the ratio of the molar concentration of miR-155-5p to the molar concentration of miR-1290-3p is 2.11 or more. According to claim 1,
The method of claim 1, wherein the sample is blood or serum. According to claim 1,
The method is for the diagnosis of preeclampsia in Korean women, the method. A composition for diagnosing preeclampsia, comprising a means for measuring the expression level of miR-155-5p and a means for measuring the expression level of miR-1290-3p. 9. The method of claim 8,
The means for measuring the expression level of miR-155-5p is specifically for the nucleotide sequence of miR-155-5p, the nucleotide sequence of miR-155-5p cDNA, or the nucleotide sequence complementary to the cDNA of miR-155-5p. It is a primer or probe that binds,
The means for measuring the expression level of miR-1290-3p is specifically to the nucleotide sequence of miR-1290-3p, the nucleotide sequence of miR-1290-3p cDNA, or the nucleotide sequence complementary to the cDNA of miR-1290-3p. A composition that is a primer or probe that binds. 9. The method of claim 8,
The composition is for use in the diagnosis of preeclampsia in Korean women, the composition. A kit for diagnosing preeclampsia comprising the composition of any one of claims 8 to 10. 12. The method of claim 11,
When the ratio of the molar concentration of miR-155-5p to the molar concentration of miR-1290-3p is 2.11 or more, the kit further includes data suggesting criteria for classification as a high-risk group for preeclampsia. 12. The method of claim 11,
The kit is for the purpose of diagnosing preeclampsia in Korean women, the kit.

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