CN113736877A - Pre-eclampsia diagnostic use and composition based on micro RNA27a - Google Patents
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Abstract
The invention provides a method for diagnosing whether a subject has preeclampsia or is at risk of preeclampsia, and also provides application of microRNA and/or a specific probe/detection primer of the microRNA in preparing a diagnostic composition and the diagnostic composition for detecting the preeclampsia, wherein the expression of the microRNA 27a in blood plasma is obviously reduced in a preeclampsia patient, and the method can be used as a valuable, simple and convenient diagnostic index for dynamically monitoring the preeclampsia; meanwhile, the reduction of the microRNA 27a in the placenta tissue is also a potential diagnostic index for predicting preeclampsia.
Description
Technical Field
The invention belongs to the technical field of biological medicine and molecular biology, and particularly relates to application of peripheral blood micro RNA27a in diagnosis and treatment of preeclampsia.
Background
Preeclampsia (Pre-eclampsia, PE) is a hypertensive disorder that occurs during pregnancy affecting about 5-8% of pregnant women, with PE/eclampsia associated with direct death of 10-15% of pregnant women. PE is associated with eclampsia and HELLP syndrome in mothers, as well as preterm delivery, intrauterine growth restriction and perinatal death of fetuses. PE has adverse effects on the health and quality of life of the parturient and the offspring after delivery. Women with a history of PE have a 2-fold increased risk of cardiovascular complications later in life and a 5-12-fold increased risk of end-stage renal disease. Preeclampsia can be subdivided into two phenotypes, Early Onset (EOPE) and Late Onset (LOPE), before and after 34 weeks of diagnosis. However, these two phenotypic studies have found different etiologies and pathogenesis, with impaired placental development and impaired helical arterial remodeling in the early stages of pregnancy often associated with EOPE, and LOPE possibly associated with maternal endothelial dysfunction. The disease can be classified into patients with mild preeclampsia and patients with severe preeclampsia according to the severity of the disease. In recent years, non-invasive biomarkers have been sought to diagnose and predict preeclampsia. Of these, the most important are one of the angiogenesis-related markers, members of the VEGF family, which are expected to be evaluated as effective biomarkers for the prediction and diagnosis of PE. Although the sFlt-1/placental growth factor (PIGF) ratio has been considered a new marker in the last few years, patients who normally show a change in this ratio in the late part of pregnancy before eclampsia and cannot predict the risk of preeclampsia early, we still need to find new non-invasive markers.
Micrornas (miRNAs/miRs) are endogenous small non-coding RNA molecules, typically 22-24 nucleotides in length, that regulate expression of target genes by pairing with a 3' -untranslated region (UTR), thereby regulating translation of downstream mrnas. miRNAs play important roles in a variety of cellular processes, such as differentiation, proliferation, apoptosis, and homeostatic regulation, while miRNAs are highly stable in the circulation, allowing them to exist as biomarkers in a variety of diseases, such as cancer, cardiovascular diseases, and the like. Previous researches report that miR-210 inhibits trophoblast cell proliferation, migration and promotes trophoblast apoptosis through KCMF1, Notch1 and MAPK signal paths so as to induce preeclampsia. At present, few reports are reported on micro RNA in peripheral blood for predicting preeclampsia.
Disclosure of Invention
Therefore, the present invention aims to overcome the defects in the prior art and provide a micro-RNA 27 a-based preeclampsia diagnosis application and a composition.
Before setting forth the context of the present invention, the terms used herein are defined as follows:
the term "PE" refers to: preeclampsia.
The term "EOPE" refers to: early onset epilepsy.
The term "LOPE" refers to: late onset preeclampsia.
The term "EDTA" means: ethylene diamine tetraacetic acid.
The term "PCR" refers to: polymerase chain reaction.
The terms "Real-time PCR", "qPCR" refer to: real-time fluorescent quantitative PCR.
The term "Trizol reagent" refers to: a novel total RNA extraction reagent can directly extract total RNA from cells or tissues. It contains phenol, guanidinium isothiocyanate, etc., and can rapidly break cells and inhibit nuclease released from cells.
The term "LSD" refers to: least significant difference method.
The term "ROC curve" means: subject work characteristic curve.
To achieve the above objects, a first aspect of the present invention provides a method of diagnosing whether a subject has or is at risk of pre-eclampsia, comprising determining the level of microrna 27a in a test sample from said subject, wherein an alteration in the level of microrna in the test sample, relative to the level of corresponding microrna 27a in a control sample, is indicative of the subject having or being at risk of pre-eclampsia.
In a second aspect, the invention provides the use of a microrna and/or a probe/detection primer specific for said microrna in the preparation of a diagnostic composition for diagnosing whether a subject has or is at risk of pre-eclampsia by determining the level of the aforementioned microrna 27a in a test sample from said subject, wherein a change in the level of said microrna 27a in the test sample relative to the level of the corresponding microrna 27a in a control sample indicates that the subject has or is at risk of pre-eclampsia.
The method/use according to the first or second aspect of the invention wherein the level of microrna 27a in the test sample is lower than the level of the corresponding microrna 27a in the control sample.
The method/use according to the first or second aspect of the invention, wherein the subject is a pre-eclamptic patient.
The method/use according to the first or second aspect of the invention, wherein the subject is a pregnant or parturient subject during pregnancy, preferably the subject is a pregnant woman presenting with a systolic pressure ≥ 140mmHg and/or a diastolic pressure ≥ 90mmHg after 20 weeks of gestation.
The method/use according to the first or second aspect of the invention wherein the microRNA 27a is selected from one or more of miRNA-27a-1, miRNA-27a-2, miRNA-27a-5p and miRNA-27a-3p, more preferably the microRNA is miRNA-27a-5 p.
The method/use according to the first or second aspect of the invention, wherein the test sample is from a tissue and/or body fluid, wherein: the tissue is preferably placental tissue and the body fluid is preferably plasma.
In a third aspect, the invention provides a diagnostic composition for the detection of preeclampsia, wherein the diagnostic composition comprises a probe/detection primer specific for microrna 27 a; wherein the microRNA 27a is selected from one or more of miRNA-27a-1, miRNA-27a-2, miRNA-27a-5p and miRNA-27a-3p, more preferably the microRNA is miRNA-27a-5 p.
The use according to the second aspect of the invention or the diagnostic composition according to the third aspect of the invention, wherein the nucleic acid sequence of the specific probe/detection primer comprises SEQ ID No. 1.
In a fourth aspect, the present invention provides the use of a microrna and/or a probe/detection primer specific for said microrna in the preparation of a diagnostic composition for diagnosing whether a subject has or is at risk of pre-eclampsia by determining the level of such a microrna in a test sample from a pregnant woman, wherein a change in the level of said microrna in the test sample, relative to the level of a corresponding microrna in a control sample, is indicative of the subject having or being at risk of pre-eclampsia, said microrna being miRNA-27a-5 p; wherein the level of miRNA-27a-5p in the test sample is lower than the level of the corresponding microRNA in the control sample.
The pre-eclampsia diagnostic use and composition based on microRNA 27a of the present invention may have the following beneficial effects, but is not limited to:
1. microrna 27a was reduced in expression in placental tissue of PE and plasma of PE.
2. Micro RNA27a showed a clear negative correlation with systolic, diastolic, and mean arterial pressures.
3. The results of ROC curve analysis and calculation of the area under the ROC curve (AUC) show that the micro RNA27a has a certain diagnostic value for preeclampsia.
4. The expression of the microRNA 27a in the plasma of the preeclampsia patient is obviously reduced, and the method is suggested to be a valuable, simple and convenient diagnostic index for dynamically monitoring preeclampsia. Meanwhile, the results of the invention also indicate that the reduction of the microRNA 27a in the placenta tissue is also a potential diagnostic index for predicting preeclampsia.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 shows amplification curves for miR-27a-5p in placenta samples of example 7.
FIG. 2 shows amplification curves for miR-27a-5p in plasma samples of example 7.
FIG. 3 shows the expression levels of miR-27a-5p in a pre-eclamptic patient in a placental sample from example 9.
FIG. 4 shows the expression levels of miR-27a-5p in a pre-eclamptic patient in a plasma sample of example 9.
FIG. 5 shows the statistical differences of miR-27a-5p of example 9 between a group of patients with severe preeclampsia and a control group and between a group of patients with mild preeclampsia and a control group.
FIG. 6 shows the statistical differences that exist for example 9miR-27a-5p between early-onset preeclampsia, late-onset preeclampsia and the control group, between early-onset preeclampsia and the control group, and between late-onset preeclampsia and the control group.
FIG. 7 shows AUC values for miR-27a-5p in plasma for diagnosing preeclampsia in example 9.
Detailed Description
The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
This section generally describes the materials used in the testing of the present invention, as well as the testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible. It will be apparent to those skilled in the art that the materials and methods of operation used in the present invention are well within the skill of the art, provided that they are not specifically illustrated.
Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations.
The reagents and instrumentation used in the following examples are as follows:
reagent:
trizol reagent, TaKaRa Mir-X miRNA First-Strand Synthesis kit, TBGreenTM premix ExTaqTM kit, purchased from TaKaRa, Dalian and China.
The instrument comprises the following steps:
high speed refrigerated centrifuge, Microfuge 21R, available from semer fly, shanghai, china.
A full-automatic medical PCR analysis system, SLAN-96S, was purchased from Hongshi medical science and technology Co., Ltd, Shanghai, China.
EXAMPLE 1 study subjects
In this study, a total of 200 cases of medical record data collected in the prenatal diagnosis laboratory were divided into a Preeclampsia (PE) group and a normal control group from 2019 to 2021, month 06, and the case data, placental tissue samples, and venous blood were collected.
Example 2 collection of placental tissue specimens
After the placenta is delivered by caesarean section, two pieces of placenta with the size of about 1cm multiplied by 1cm and a small amount of decidua tissues are taken from the center of the maternal surface of the placenta in a sterile state, a sample is placed into a sterile Eppendorf tube and is placed in liquid nitrogen for storage within 15min until RNA is extracted. The miR-27a-5p level in each group of placenta tissues is detected by Real-time PCR.
EXAMPLE 3 venous blood Collection
The pregnant woman draws 5ml of venous blood before giving birth and collects it in an EDTA vacuum tube. The extracted venous blood was centrifuged for 15 minutes in a centrifuge set at 4 ℃ and 1500g for 30 minutes, and then plasma was separated for RNA extraction.
EXAMPLE 4 extraction of RNA
According to the instructions of the reagent, Trizol reagent (TaKaRa) is used for separating total RNA, and the specific steps are as follows: adding a proper amount of Trizol reagent into placenta tissues, homogenizing, adding a proper amount of Trizol reagent into the leucocyte layer and the blood plasma respectively, fully mixing, standing for 5 minutes, adding 1/5 initial amount of chloroform, standing for 5 minutes at room temperature after violent shaking, transferring supernate into a new centrifuge tube after centrifugation of 12000 Xg for 15 minutes, adding isopropanol with the same volume as that of the supernate, fully mixing, standing for 10 minutes, centrifuging for 10 minutes at 12,000 Xg, washing precipitates with 75% ethanol with the same volume, centrifuging for 5 minutes at 7,500 Xg, discarding supernate, retaining the precipitates, drying, and dissolving in a proper amount of RNase-free water.
Example 5 reverse transcription to obtain RNA
The reverse transcription reaction uses TaKaRa Mir-X miRNA First-Strand Synthesis kit (TaKaRa, Dalian, China). 5 microliters of mRQ buffer, 3.75 microliters of total RNA, and 1.25 microliters of mRQ enzyme were mixed, and the mixture in a total volume of 10 microliters was incubated at 37 degrees Celsius for 1 hour, and then stopped at 85 degrees Celsius for 5 minutes to inactivate the enzyme.
Example 6 real-time quantitative polymerase chain reaction
Real-time quantitative polymerase chain reaction (qRT-PCR) was performed using a TBGreenTM premix ExTaqTM kit (TaKaRa, Dalian, China). Adding 10 microliters of TB Green Premix Ex TaqII, 0.8 microliters of front primer and rear primer, 2 microliters of DNA template and 6.4 microliters of water into each well, uniformly mixing, configuring U6 internal reference mixed liquor with the same volume, pre-denaturing the mixed liquor with the total volume of 20 microliters at 95 ℃ for 30 seconds, then reacting at 95 ℃ for 5 seconds and 60 ℃ for 20 seconds, simultaneously carrying out 40 cycles on the two PCR reactions, and finally reacting at 95 ℃ for 5 seconds and 60 ℃ for 30 seconds.
Example 7 analysis of primer specificity and amplification efficiency
And judging the reaction specificity of the primer according to the dissolution curve. Obtaining Ct values from amplification curves, FIG. 1 shows the amplification curves for miR-27a-5p in the placenta sample of example 7; FIG. 2 shows amplification curves for miR-27a-5p in plasma samples of example 7. The relative expression level of the target gene was analyzed by the relative amount method and the reference U6.
The calculation formula is as follows: 2^ (-. DELTA.Ct), and [ Delta ] Ct is Ct gene-Ct control.
Primer information, details are shown in table 1:
NCBI Reference Sequence:NR_029501.1
TABLE 1 primer information
| Primer name | Primer sequence |
Tm value |
| hsa-miR-27a-5p-F | GGCUUAGCUGCUUGUGAGCA | 58 |
Example 8 data processing
Statistical analysis was performed using SPSS 20.0 and GraphPad Prism 9.0 software. Normally distributed measurement data is expressed by mean + -standard deviation (x + -s), single-factor variance analysis is adopted for comparison among multiple groups, LSD-t test is adopted for pairwise comparison, and count data is expressed in frequency or rate (%) form and chi2And (6) checking. And analyzing the correlation between the relative expression quantity of the miRNA and the related indexes by using a Pearson analysis method, and analyzing the diagnostic value of the miRNA on the PE by using ROC. P < 0.05 is statistically significant.
Example 9 results of the study:
MiR-27a-5p expression reduction in placental tissue of PE
As shown in FIG. 1, qPCR in placental samples from 30 control groups and 30 preeclamptic patients confirmed that the expression level of miR-27a-5p was significantly reduced in preeclamptic patients.
Decreased expression of miR-27a-5p in plasma of PE
As shown in FIG. 2, qPCR in plasma samples of 100 healthy pregnant women and 100 preeclamptic patients confirmed that the expression level of miR-27a-5p was significantly reduced in preeclamptic patients.
Correlation between miR-27a-5p and blood pressure
As shown in Table 2, miR-27a-5p and systolic, diastolic, and mean arterial pressures exhibited significant negative correlations in all samples by Pearson correlation analysis.
TABLE 2 correlation of miR-27a-5p and blood pressure values in all samples
| —— | Systolic pressure | Diastolic blood pressure | Mean arterial pressure |
| MiR-27a-5p Pearson correlation | -0.211 | -0.255 | -0.195 |
| P value | 0.005** | 0.001** | 0.015* |
4. Differential expression of miR-27a-5p among various groups
As shown in FIG. 3, miR-27a-5p has a statistical difference between not only the patients with severe preeclampsia and the control group, but also the patients with mild preeclampsia and the control group have a significant difference (p < 0.01).
As shown in FIG. 4, there were statistical differences (p < 0.01) for miR-27a-5p between the early onset preeclampsia, the late onset preeclampsia and the control group, between the early onset preeclampsia and the control group, and between the late onset preeclampsia and the control group.
5. Diagnostic value of miR-27a-5p in plasma on preeclampsia
As shown in FIG. 5, the result of ROC curve analysis and calculation of the area under the ROC curve (AUC) showed that the AUC value for diagnosing preeclampsia by plasma miR-27a-5p was 0.6140 (95% CI: 0.4673-0.6364, p: 0.0158). The result shows that the miR-27a-5p has a certain diagnostic value for preeclampsia.
In conclusion, the research of the invention shows that the expression of miR-27a-5p in plasma is obviously reduced in preeclampsia patients, and the suggestion that the miR-27a-5p can be used as a valuable, simple and convenient diagnostic index for dynamically monitoring preeclampsia easily. Meanwhile, the result of the invention also indicates that the reduction of miR-27a-5p in placenta tissue is a potential diagnostic index for predicting preeclampsia.
Although the present invention has been described to a certain extent, it is apparent that appropriate changes in the respective conditions may be made without departing from the spirit and scope of the present invention. It is to be understood that the invention is not limited to the described embodiments, but is to be accorded the scope consistent with the claims, including equivalents of each element described.
Sequence listing
<110> affiliated Hospital of Qingdao university
<120> use and composition for the diagnosis of preeclampsia based on microRNA 27a
<130> YZDI-210065
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA/RNA
<213> Intelligent (Homo sapiens)
<400> 1
Claims (10)
1. A method of diagnosing whether a subject has or is at risk of pre-eclampsia, comprising determining the level of microrna 27a in a test sample from the subject, wherein an alteration in the level of microrna in the test sample, relative to the level of corresponding microrna 27a in a control sample, indicates that the subject has or is at risk of pre-eclampsia.
2. Use of a microrna and/or a probe/detection primer specific for said microrna in the preparation of a diagnostic composition for diagnosing whether a subject has or is at risk of pre-eclampsia by determining the level of the aforementioned microrna 27a in a test sample from said subject, wherein an alteration in the level of said microrna 27a in the test sample relative to the level of the corresponding microrna 27a in a control sample indicates that the subject has or is at risk of pre-eclampsia.
3. The method/use according to claim 1 or 2 wherein the level of microrna 27a in the test sample is lower than the level of the corresponding microrna 27a in the control sample.
4. A method/use according to any one of claims 1 to 3 wherein the subject is a pre-eclamptic patient.
5. The method/use according to any of claims 1-4, wherein the subject is a pregnant maternal subject, preferably the subject is a pregnant woman presenting with a systolic pressure ≥ 140mmHg and/or a diastolic pressure ≥ 90mmHg after 20 weeks of gestation.
6. The method/use according to any of claims 1-5, wherein said microRNA 27a is selected from one or more of miRNA-27a-1, miRNA-27a-2, miRNA-27a-5p and miRNA-27a-3p, more preferably said microRNA is miRNA-27a-5 p.
7. The method/use of any of claims 1-6, wherein said test sample is from a tissue and/or body fluid, wherein: the tissue is preferably placental tissue and the body fluid is preferably plasma.
8. A diagnostic composition for detecting preeclampsia, wherein said diagnostic composition comprises a probe/detection primer specific for microrna 27 a; wherein the microRNA 27a is selected from one or more of miRNA-27a-1, miRNA-27a-2, miRNA-27a-5p and miRNA-27a-3p, more preferably the microRNA is miRNA-27a-5 p.
9. The use according to any one of claims 2 to 7, or the diagnostic composition according to claim 8, wherein the nucleic acid sequence of the specific probe/detection primer comprises SEQ ID No. 1.
10. Use of a microrna and/or a probe/detection primer specific for said microrna in the preparation of a diagnostic composition for diagnosing whether a subject has or is at risk of pre-eclampsia by determining the level of such a microrna in a test sample from a subject pregnant woman, wherein a change in the level of said microrna in the test sample, relative to the level of a corresponding microrna in a control sample, is indicative of the subject having or being at risk of pre-eclampsia, said microrna being miRNA-27a-5 p; wherein the level of miRNA-27a-5p in the test sample is lower than the level of the corresponding microRNA in the control sample.
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