Application of reagent for detecting molecular marker in preparation of product for predicting preeclampsia

Abstract

The invention discloses an application of a detection reagent for detecting a molecular marker related to preeclampsia in preparation of a product for predicting the preeclampsia disease risk. The molecular marker comprises at least one of tRF-19-BZOS4YE2 and tRF-29-86J8WPMN1EJ3. The invention further discloses an application of the detection reagent for detecting the molecular marker related to preeclampsia in preparation of a product for predicting the preeclampsia disease risk. Whether a patient is in preeclampsia or not is judged by detecting the relative expression level of the molecular marker in a plasma sample of a subject. Through subject working curve analysis, the area AUC under a curve of combined diagnosis of the two is equal to 0.95, the molecular marker has extremely high diagnosis sensitivity, specificity and diagnosis efficacy, the expression stability of the molecular marker is good, the problems that a traditional marker is poor in detection sensitivity and specificity and unstable in expression are solved, and the molecular marker has good application prospects. And whether the pregnant woman is in the preeclampsia state can be effectively predicted.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, and in particular to the application of a reagent for detecting molecular markers in the preparation of a product for predicting preeclampsia. Background Art

[0002] Preeclampsia (PE) is a serious complication of gestational hypertension that is unique to pregnancy. It is characterized by hypertension and proteinuria after 20 weeks of pregnancy. It can lead to maternal and neonatal death, seriously endangering the health of mothers and infants. If preeclampsia is not managed carefully in clinical practice, it can easily induce eclampsia, accompanied by aspiration pneumonia, cerebral hemorrhage, renal failure or cardiac arrest, with a high mortality rate, seriously threatening the safety of mothers and infants. Early identification and management of preeclampsia are crucial to reducing the health risks of pregnant women and perinatal infants. However, because the etiology and specific pathogenesis of preeclampsia are very complex and have not yet been fully elucidated, there is a lack of effective clinical treatment methods. Early termination of pregnancy is still regarded as the most direct treatment measure for patients with severe preeclampsia whose disease continues to progress.

[0003] One of the difficulties in early diagnosis of preeclampsia lies in the heterogeneity of its course and the diversity of clinical manifestations. The pathological mechanism of preeclampsia involves multiple factors, such as placental dysfunction, immune system abnormalities, and vascular endothelial damage. Preeclampsia may begin to develop weeks before symptoms appear, but biomarkers in the early stages are not obvious, and traditional clinical indicators such as hypertension and proteinuria may not appear in the early stages of the disease, which makes early identification difficult. In addition, the course of preeclampsia varies from person to person. Some patients develop rapidly, while others progress slowly and are difficult to predict.

[0004] Early diagnosis and risk screening remain the key to reducing adverse outcomes of preeclampsia. In recent years, researchers are actively exploring more accurate methods for early diagnosis of preeclampsia. Currently, the main methods for early diagnosis of preeclampsia include assessments based on serum markers and placental function.

[0005] The etiology of preeclampsia is not fully understood, but the disease is thought to result from abnormal remodeling of the maternal spiral arteries leading to poor placental perfusion. As an antagonist of placental growth factor (PlGF) and vascular endothelial growth factor, soluble fms-like tyrosine kinase-1 (sFlt-1) causes vasoconstriction and endothelial damage, which may lead to fetal growth restriction and the development of preeclampsia. Reference 1 (Harald Zeisler, Stefan Verlohren, Predictive Value of the sFlt-1:PlGF Ratio in Women with Suspected Preeclampsia. N Engl J Med., 374, 13-22, 2016, doi: 10.1056 / NEJMoa1414838) discloses that in the plasma of pregnant women with preeclampsia, an abnormal increase in the sFlt-1 / PlGF concentration ratio determined by magnetoluminescence can indicate the occurrence of preeclampsia four weeks in advance, with a sensitivity of 66.2%, a specificity of 83.1%, and a positive predictive value of 36.7%. However, the detection sensitivity and specificity of serum markers are poor, which is insufficient to meet clinical needs, and the marker levels of different races and individuals vary greatly, which limits their wide application.

[0006] Publication No. CN110785499A discloses a method for detecting preeclampsia and / or determining an increased risk of preeclampsia in a pregnant woman, the method comprising identifying a plurality of circulating RNA (C-RNA) molecules in a biological sample obtained from the pregnant woman. However, the method is based on analysis of a confirmed population, and a model is constructed using these C-RNAs for classification, rather than prediction.

[0007] In addition, some emerging imaging technologies (such as placental blood flow Doppler ultrasound) also show potential in the early diagnosis of preeclampsia. However, imaging technology requires high operating skills and equipment support, which is difficult to promote in areas with limited resources. Therefore, it is urgent to develop more sensitive and specific biomarkers and early diagnostic methods to provide pregnant women with a better early intervention time window.

[0008] tRNA-derived fragments (tRFs) are a new class of regulatory noncoding RNAs with unique biological functions in tumors and stress diseases. In recent years, they have been considered to have potential for application in a variety of diseases, including cancer and cardiovascular disease. tRFs are relatively stable in plasma because they are often bound to protective proteins or extracellular vesicles (such as exosomes) to avoid degradation by ribonucleases. This stability is very important for reliable detection in clinical settings. For preeclampsia, tRFs can participate in the regulation of cell proliferation, apoptosis, and immune response, and can produce changes in expression in the early stages of the disease, reflecting pathological changes associated with inflammation, oxidative stress, and abnormal placental development. Given these advantages, tRFs in plasma can be used as potential biomarkers for the diagnosis of preeclampsia. Summary of the invention

[0009] In order to solve the above technical problems, the present invention provides a detection reagent for detecting molecular markers related to preeclampsia for use in the preparation of a product for predicting the risk of preeclampsia. By detecting the relative expression levels of molecular markers in the plasma samples of the subjects, it is determined whether the patient is in preeclampsia. The reagent has high sensitivity and specificity and good expression stability, and can effectively predict whether a pregnant woman is in a preeclampsia state.

[0010] The use of a detection reagent for detecting molecular markers associated with preeclampsia in the preparation of a product for predicting the risk of preeclampsia, wherein the molecular markers include at least one of tRF-19-BZOS4YE2 and tRF-29-86J8WPMN1EJ3, the nucleotide sequence of tRF-19-BZOS4YE2 is shown in SEQ ID NO.1, and the nucleotide sequence of tRF-29-86J8WPMN1EJ3 is shown in SEQ ID NO.2.

[0011] The present invention detected that when the early stage patients had no clinical symptoms, the expression level of the above molecular markers was significantly higher than that of normal pregnant women. Through the receiver operating curve analysis (ROC), the area under the curve of the combined diagnosis of the two was AUC = 0.95, which has extremely high diagnostic sensitivity, specificity and diagnostic efficacy, and the expression stability of this group of molecular markers is good, which solves the problem of poor sensitivity and specificity of traditional marker detection and unstable expression, and can effectively predict whether a pregnant woman is in a state of preeclampsia.

[0012] Preferably, the product for predicting the risk of preeclampsia is a kit.

[0013] Preferably, the detection sample of the detection reagent is a human plasma sample.

[0014] In the present invention, the plasma sample to be tested is a plasma sample to which EDTA anticoagulation is added.

[0015] Preferably, the molecular markers associated with preeclampsia are upregulated in plasma samples of preeclampsia patients.

[0016] Preferably, the detection reagent for the molecular marker associated with preeclampsia comprises specific primers for amplifying tRF-19-BZOS4YE2 and / or tRF-29-86J8WPMN1EJ3.

[0017] Further preferably, the forward primer sequence of tRF-19-BZOS4YE2 is shown as SEQ ID NO.3, and the reverse primer sequence of tRF-19-BZOS4YE2 is shown as SEQ ID NO.4.

[0018] Further preferably, the forward primer sequence of tRF-29-86J8WPMN1EJ3 is shown as SEQ ID NO.5, and the reverse primer sequence of tRF-29-86J8WPMN1EJ3 is shown as SEQ ID NO.6.

[0019] The forward primer sequence and the reverse primer sequence selected in the present invention are both subjected to primer specificity comparison analysis and optimization using the "Primer-Blast" tool, and the primer sequences are obtained by comprehensively considering the GC content and the Tm value.

[0020] The present invention also provides a method for using the above detection reagent, by comparing the ΔCt values ​​of tRF-19-BZOS4YE2 and tRF-29-86J8WPMN1EJ3 in plasma samples of the subject and normal pregnant women, thereby predicting the occurrence of preeclampsia.

[0021] The method for using a detection reagent for detecting a molecular marker associated with preeclampsia comprises the following steps:

[0022] (1) Collect plasma samples from subjects and extract total RNA from the plasma samples;

[0023] (2) The extracted total RNA is pre-treated and then reverse transcribed into cDNA;

[0024] (3) The reverse transcribed cDNA was subjected to qRT-PCR, and the Ct value of tRF-19-BZOS4YE2 and / or tRF-29-86J8WPMN1EJ3 was detected after the reaction;

[0025] (4) Based on the Ct value, the difference between the Ct value of tRF-19-BZOS4YE2 and / or tRF-29-86J8WPMN1EJ3 in the subject's plasma sample and the internal reference gene, i.e., the ΔCt value, was calculated;

[0026] If the ΔCt value of tRF-19-BZOS4YE2 and / or tRF-29-86J8WPMN1EJ3 in the subject's plasma sample is less than the cut-off value of the ROC curve, it indicates that the subject is in the preeclampsia stage.

[0027] In the present invention, by detecting the expression level of tRF-19-BZOS4YE2 and / or tRF-29-86J8WPMN1EJ3 in the plasma sample of the subject, if the expression level of the molecular marker in the plasma sample of the subject is higher than that of normal pregnancy, it means that the subject is likely to be in preeclampsia.

[0028] Preferably, in step (2), the pretreatment is to demethylate the extracted total RNA, and add a 3' linker and a 5' linker to both ends of the demethylated RNA, respectively, wherein the 3' linker is 5'-AAAAAAAAAA-3' (whose nucleotide sequence is SEQ ID NO.7), and the 5' linker is 5'-AGAUCGGAAGAGC-3' (whose nucleotide sequence is SEQ ID NO.8).

[0029] In the present invention, a 3' linker (ie, a poly(A) tail) and a 5' linker are added to both ends of the demethylated RNA, respectively, to obtain an artificially extended RNA, which is convenient for subsequent reverse transcription reactions.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention provides the use of a detection reagent for detecting molecular markers tRF-19-BZOS4YE2 and / or tRF-29-86J8WPMN1EJ3 associated with preeclampsia in the preparation of a product for predicting the risk of preeclampsia. Both molecular markers have high sensitivity and specificity and good expression stability. When used in combination, they have extremely high diagnostic sensitivity, specificity and diagnostic efficacy, and can effectively predict whether a pregnant woman is in a state of preeclampsia. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the ROC curve of tRF-19-BZOS4YE2 for the diagnosis of preeclampsia.

[0033] Figure 2 This is the ROC curve of tRF-29-86J8WPMN1EJ3 for the diagnosis of preeclampsia.

[0034] Figure 3 This is the ROC curve of the combined diagnosis of preeclampsia by tRF-19-BZOS4YE2 and tRF-29-86J8WPMN1EJ3.

[0035] Figure 4 This is a stability test chart of tRF-19-BZOS4YE2 and tRF-29-86J8WPMN1EJ3 in a 4°C environment. DETAILED DESCRIPTION

[0036] The present invention will be further described in detail below in conjunction with the examples, but the embodiments of the present invention are not limited to the following examples.

[0037] Unless otherwise specified, the experimental materials used in the following examples can be purchased from conventional biochemical reagent companies.

[0038] Example 1

[0039] 1. Sample Collection

[0040] (1) Collect 2 ml of EDTA anticoagulated blood from 25 normal pregnant women and 2 ml of EDTA anticoagulated blood from 25 patients with preeclampsia;

[0041] (2) The EDTA-anticoagulated sample was centrifuged at 600-1000 g for 20 min at 4°C to remove impurities such as cell debris and separate the plasma.

[0042] 2. Extraction of Total RNA

[0043] Carefully take 200 μL of plasma supernatant with a pipette, add TRIzol LS reagent (purchased from INVITROGEN, product number 10296010CN) and mix by pipetting, then add 100 fmol of exogenous microRNA (cel-miR-39) (purchased from NORGEN, Product#59000), pipet and mix, ensure that the plasma and TRIzol LS reagent are fully in contact, and fully lyse the cells and proteins. Let the mixture stand at room temperature for 5 minutes to ensure that the cells are completely lysed and RNA is released. Add 200 μL of chloroform to each 1 mL of TRIzol LS-plasma mixture, close the tube cap tightly, shake vigorously for 15 seconds, ensure sufficient mixing, and let it stand at room temperature for 3 minutes to separate RNA and protein phases. Place the mixture in a centrifuge and centrifuge at 4°C and 12000xg for 15 minutes. After centrifugation, the sample should be divided into three layers: the upper aqueous phase (containing RNA), the middle white protein layer, and the lower phenol phase (containing DNA). Carefully transfer the upper aqueous phase to a new 1.5 mL RNase-free centrifuge tube, avoiding aspirating the protein layer. Add 100% ethanol to the aqueous phase in a 1:1 volume ratio (e.g., 500 μL of 100% ethanol to 500 μL of aqueous phase) and mix well. Let stand on ice for 10 minutes to promote RNA precipitation. Centrifuge at 4°C, 12,000 x g for 10 minutes to precipitate RNA. Gently wash the RNA pellet with 750 μL of pre-cooled 75% ethanol. Gently mix and centrifuge at 44°C, 7,500 x g for 5 minutes, carefully pouring off the ethanol and removing as much residual ethanol as possible. Open the tube cap and let the RNA pellet air dry at room temperature or cold for 2-3 minutes. Dissolve the RNA pellet by adding 20 μL of RNase-free water and gently mix the RNA on ice to ensure complete dissolution.

[0044] 3. Pre-treatment

[0045] (1) Demethylation

[0046] Prepare the following reaction system in an RNase-free centrifuge tube.

[0047]

[0048] (2) Gently mix the reaction solution to avoid violent shaking that may cause RNA degradation. Incubate the sample in a 37°C water bath or metal bath for 1 hour. This can be extended to 2 hours to improve demethylation efficiency depending on experimental requirements.

[0049] (3) Stop the reaction. Add EDTA to a final concentration of 2 mM to chelate Fe2+ and stop the reaction. Alternatively, heat the sample to 65°C and incubate for 5 minutes to inactivate the enzyme activity. Use an RNA cleaning column to remove enzyme and reagent residues.

[0050] (2) Adding a 3' linker

[0051] After the methylation is removed, a 3' linker is added. First, a 3' linker ligation reaction system (as shown in Table 1) is prepared, incubated at 25°C for 1 to 2 hours, and the reaction is stopped by adding proteinase K.

[0052] Table 1: 3' linker ligation reaction system

[0053] 3' linker ligation reaction system Addition amount (μL) 10X T4 RNA Ligase Reaction Buffer 2 50% PEG8000 solution 4 Demethylated RNA (10 μM) 2 5' pre-adenylated DNA adapter (20 μM) 2 T4 RNA Ligase 2 (NEB#M0242) 1 Nuclease-free water Top up to 20

[0054] (3) Adding a 5' linker

[0055] Take the 3' adapter-ligated RNA and add the 5' adapter (5'-AGAUCGGAAGAGC-3') to the reaction system shown in Table 2 and incubate at 16°C for 1 hour. Clean up the reaction using an RNA purification kit or ethanol precipitation to remove excess adapters, enzymes, and ATP.

[0056] Table 2: 5' linker ligation reaction system

[0057] 3' linker ligation reaction system Addition amount RNA sample after 3' adapter ligation 500ng 5' adapter 10pmol T4 RNA Ligase 1 (NEB#M0204) 1μL ATP(10mM) 1μL RNase inhibitors 0.5μL RNA Ligase Buffer Make up to 20 μL

[0058] (4) Digest gDNA and prepare for reverse transcription.

[0059] 4. Reverse Transcription

[0060] Reverse transcription was performed using the miRCURY LNA RT Kit (purchased from QIAGEN, catalog number 339340). The reaction system was as shown in Table 3. The amplification program was 42°C for 60 minutes and 95°C for 5 minutes. After completion, 60 μL of RNase-free water was added for dilution and stored at -20°C for later use.

[0061] Table 3: Reverse transcription reaction system

[0062] Reverse transcription reaction system Addition amount (μL) miRCURY LNA RT Reaction Buffer 2 miRCURY LNA RT Enzyme Mix 1 RNA sample obtained in step 3 4 RNase-free water Make up to 10

[0063] 5. qRT-PCR

[0064] use miRNA PCR Assays (purchased from QIAGEN, catalog number 339306) were used for amplification. 5 μL of miRCURY LNA SYBR Green Master Mix, 4 μL of cDNA template, and 1 μL of primers (tRF-19-BZOS4YE2, tRF-29-86J8WPMN1EJ3, and internal reference cel-miR-39) were mixed. The primer sequences are shown in Table 5 and were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0065] The PCR reaction conditions are: initial 95°C, 2 minutes; PCR cycle (40 cycles): denaturation: 95°C, 10 seconds; annealing and extension: 56°C, 60 seconds; after completing qPCR, a melting curve analysis can be performed to ensure amplification specificity and check for nonspecific amplification or primer dimers. The 480qPCR instrument software analyzed the results and obtained the CT value for relative quantitative analysis of tRF.

[0066] Table 5: Primers and their corresponding sequences

[0067]

[0068]

[0069] 6. Data Analysis

[0070] After qRT-PCR, the ΔCt method was used to calculate the relative expression levels of the targets tRF-19-BZOS4YE2 and tRF-29-86J8WPMN1EJ3 in plasma.

[0071] Calculate the ΔCt value: subtract the Ct value of the reference gene from the Ct value of the target tRF to obtain the ΔCt value. Analyze expression differences: The lower the ΔCt value, the higher the expression level of the target tRF, and vice versa. Generally, the ΔCt values ​​of different samples can be compared to evaluate the relative expression differences of tRFs.

[0072] Sample analysis

[0073] 1. Relative expression levels of tRF-19-BZOS4YE2 and tRF-29-86J8WPMN1EJ3 in plasma

[0074] The ΔCt value was calculated according to the above method to obtain the relative expression level of tRF. The results are shown in Tables 6 and 7.

[0075] Table 6: Relative expression levels of tRF-19-BZOS4YE2 in patients in different groups

[0076]

[0077] Table 7: Relative expression levels of tRF-29-86J8WPMN1EJ3 in patients in different groups

[0078]

[0079] As shown in Tables 1 and 2, there were significant differences in the expression of tRF-19-BZOS4YE2 and tRF-29-86J8WPMN1EJ3 in the preeclampsia group and the control group. The expression levels of the two in the preeclampsia group were significantly higher than those in the control group, and the P values ​​were all <0.001.

[0080] 2. Diagnostic efficacy of tRF-19-BZOS4YE2 and tRF-29-86J8WPMN1EJ3 in preeclampsia

[0081] Determination of cut-off value: In this example, the cut-off value of tRF-19-BZOS4YE2 is 4.39; the cut-off value of tRF-29-86J8WPMN1EJ3 is 5.07.

[0082] The receiver operating characteristic (ROC) curve was drawn and the area under the curve (AUC) was used to evaluate the diagnostic performance. Figure 1 to Figure 3 shown.

[0083] The area under the curve of tRF-19-BZOS4YE2 was AUC = 93.88%, P < 0.001 ( Figure 1 ), with reliable diagnostic efficacy. The area under the curve of tRF-29-86J8WPMN1EJ3 was AUC = 90.00%, P < 0.001 ( Figure 2 ), and also has reliable diagnostic efficacy. If the two molecules are used for combined diagnosis, the area under the curve is AUC = 95.83%, P < 0.001 ( Figure 3 ) suggests that combined diagnosis has better diagnostic efficacy.

[0084] III. Stability of tRF-19-BZOS4YE2 and tRF-29-86J8WPMN1EJ3

[0085] The EDTA anticoagulated plasma of the same sample was stored in a 4°C refrigerator for 7 days and tested for tRF-19-BZOS4YE2 and tRF-29-86J8WPMN1EJ3. The results were as follows Figure 4 shown.

[0086] Figure 4 This is a stability test chart of tRF-19-BZOS4YE2 and tRF-29-86J8WPMN1EJ3 in a 4°C environment. Both tRF-19-BZOS4YE2 and tRF-29-86J8WPMN1EJ3 can be stably stored in a 4°C environment for 7 days, and there is no significant difference in the expression levels of the same tRF (P=0.456 and P=0.665, respectively), indicating that this is conducive to the arrangement of the time window for routine clinical testing and shows the potential for clinical application.

[0087] In summary, tRF-19-BZOS4YE2 and tRF-29-86J8WPMN1EJ3 in plasma can exist stably at 4°C for 7 days. Detecting the expression levels of tRF-19-BZOS4YE2 and tRF-29-86J8WPMN1EJ3 in plasma can be used for screening of patients with preeclampsia. The area under the curve of the combined diagnosis of the two molecular markers is AUC = 95.83%, indicating that the combined diagnosis has better diagnostic efficacy and is a better set of clinical markers for early diagnosis of preeclampsia.

[0088] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a detection reagent for detecting molecular markers associated with preeclampsia in the preparation of a product for predicting the risk of preeclampsia, characterized in that: The molecular marker includes at least one of tRF-19-BZOS4YE2 and tRF-29-86J8WPMN1EJ3, the nucleotide sequence of the tRF-19-BZOS4YE2 is shown in SEQ ID NO.1, and the nucleotide sequence of the tRF-29-86J8WPMN1EJ3 is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: The product for predicting the risk of preeclampsia is a kit.

3. The use according to claim 1, characterized in that: The detection sample of the detection reagent is a human plasma sample.

4. The use according to claim 1, characterized in that: The molecular markers associated with preeclampsia are upregulated in plasma samples of preeclampsia patients.

5. The use according to claim 1, characterized in that: The detection reagent for the molecular markers associated with preeclampsia comprises specific primers for amplifying tRF-19-BZOS4YE2 and / or tRF-29-86J8WPMN1EJ3.

6. The use according to claim 5, characterized in that: The forward primer sequence of tRF-19-BZOS4YE2 is shown in SEQ ID NO.3, and the reverse primer sequence of tRF-19-BZOS4YE2 is shown in SEQ ID NO.

4.

7. The use according to claim 5, characterized in that: The forward primer sequence of tRF-29-86J8WPMN1EJ3 is shown in SEQ ID NO.5, and the reverse primer sequence of tRF-29-86J8WPMN1EJ3 is shown in SEQ ID NO.

6.

8. A method for using a detection reagent for detecting molecular markers associated with preeclampsia, characterized in that: The following steps are involved: (1) Collect plasma samples from subjects and extract total RNA from the plasma samples; (2) The extracted total RNA is pre-treated and then reverse transcribed into cDNA; (3) The reverse transcribed cDNA was subjected to qRT-PCR, and the Ct value of tRF-19-BZOS4YE2 and / or tRF-29-86J8WPMN1EJ3 was detected after the reaction; (4) Based on the Ct value, the difference between the Ct value of tRF-19-BZOS4YE2 and / or tRF-29-86J8WPMN1EJ3 in the subject's plasma sample and the internal reference gene, i.e., the ΔCt value, was calculated; If the ΔCt value of tRF-19-BZOS4YE2 and / or tRF-29-86J8WPMN1EJ3 in the subject's plasma sample is less than the cut-off value defined by the ROC curve, it indicates that the patient is in the preeclampsia stage.

9. The method for using the detection reagent according to claim 8, characterized in that: In step (2), the pretreatment is to demethylate the extracted total RNA, and add a 3' linker and a 5' linker to both ends of the demethylated RNA, respectively. The 3' linker is 5'-AAAAAAAAAA-3', and the 5' linker is 5'-AGAUCGGAAGAGC-3'.

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