Molecular marker for preeclampsia diagnosis and product and application thereof

Abstract

The invention relates to a molecular marker for preeclampsia diagnosis as well as a product and application thereof, and belongs to the field of genetic engineering, MAPK8, CSNK1E and NOTUM genes or encoding proteins thereof are taken as the molecular marker for preeclampsia diagnosis, and clinical experiment data shows that when the MAPK8 gene shows significant low expression in a PE sample and the CSNK1E and NOTUM genes show significant high expression, the MAPK8 gene is not expressed in the PE sample, and the CSNK1E and NOTUM genes show significant high expression in the PE sample. The diagnosed AUC value exceeds 0.7, which proves that these markers have high sensitivity and specificity, and can effectively distinguish normal pregnant women from PE patients.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and in particular, relates to a molecular marker for diagnosing preeclampsia and a product and application thereof. Background Art

[0002] Preeclampsia (PE) is a common and serious pregnancy complication that is known to pose a major threat to the health of mothers and infants. The pathogenesis of PE has not yet been fully elucidated, and its current management is mostly focused on symptom relief and supportive treatment, lacking effective early diagnosis methods and treatments. Existing diagnostic methods mainly rely on clinical symptoms, such as hypertension and proteinuria, but these symptoms only appear when the disease develops more seriously and cannot provide early warning. Therefore, there is an urgent need to develop new early diagnostic markers.

[0003] At present, many studies have found that certain biomarkers are associated with the occurrence and development of PE, including some serum markers and gene expression patterns. Existing studies, such as those based on proteomics and genomics, mainly focus on some specific biomolecules, such as placental proteins, cytokines, miRNA, etc. However, the application of these markers in clinical practice is not yet mature. The specificity and sensitivity of many markers are low, and a set of early diagnostic criteria that can be widely used has not yet been formed. In addition, the expression of some markers varies greatly in different populations, which leads to unstable predictive effects in different patient groups.

[0004] In the existing technology, biomarker detection based on blood and urine has become a more common detection method. However, most of the current detection methods rely on a single marker or a limited combination of markers, which usually cannot fully reflect the complexity and diversity of PE. For example, some protein markers may be interfered with by other factors, resulting in large fluctuations in their expression in PE patients. In addition, the existing detection methods also have problems such as high cost, complex operation and unstable results. Therefore, the development of biomarkers that can accurately and sensitively diagnose PE, that is, to identify and diagnose PE in the early stages, can effectively reduce the threat of PE to the health of mothers and infants. Summary of the invention

[0005] In order to overcome the problems existing in the background technology, the present invention provides a molecular marker for the diagnosis of preeclampsia and its product and application, using MAPK8, CSNK1E and NOTUM genes as molecular markers for the diagnosis of preeclampsia. These markers have high sensitivity and specificity and can effectively distinguish normal pregnant women from PE patients.

[0006] To achieve the above object, the present invention is implemented through the following technical solutions: The molecular marker for diagnosing preeclampsia is at least one of MAPK8, CSNK1E and NOTUM genes or their encoded proteins.

[0007] The present invention also protects a drug for treating preeclampsia, wherein the drug treats preeclampsia by up-regulating MAPK8, or down-regulating the expression levels of CSNK1E and NOTUM genes or their encoded proteins.

[0008] The present invention also provides a reagent comprising the above-mentioned marker, wherein the reagent comprises a primer pair for specifically amplifying MAPK8, CSNK1E and NOTUM genes, and the nucleotide sequence of the primer pair is shown as SEQ ID NO. 1-6.

[0009] Furthermore, the reagent also includes an internal reference primer having a nucleotide sequence as shown in SEQ ID NO. 7-8.

[0010] The present invention also provides a product comprising the above reagent, wherein the product is a kit, a preparation, a chip or a nucleic acid membrane strip.

[0011] The invention provides an application of the above product in the diagnosis of preeclampsia.

[0012] Beneficial Effects of the Invention The present invention discovers for the first time that the gene expression levels of MAPK8, CSNK1E and NOTUM are abnormal in patients with preeclampsia, and then uses these three genes or their encoded proteins as molecular markers for the diagnosis of preeclampsia.

[0013] Through experimental verification, the MAPK8, CSNK1E and NOTUM genes or their encoded proteins were used as molecular markers for the diagnosis of preeclampsia. The diagnostic results were accurate, and the AUC value of the diagnosis was 0.920, which proved that these markers have high sensitivity and specificity and can effectively distinguish normal pregnant women from PE patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is the comparison of MAPK8 expression levels between healthy people and PE patients; Figure 2 is the comparison of CSNK1E expression levels between healthy people and PE patients; Figure 3 is the comparison of NOTUM expression levels between healthy people and PE patients; Figure 4 are the amplification curve and melting curve of MAPK8; Figure 5 are the amplification and melting curves of CSNK1E; Figure 6 It is the amplification curve and melting curve of NOTUM; Figure 7 It is the amplification curve and melting curve of the internal reference control group GAPDH on the machine; Figure 8 is the ROC curve of biomarkers in the PE blood training set; Fig. 9 is the ROC curve of biomarkers in the PE GSE75010 validation set; Fig.10 is the total expression score of the three biomarkers in the blood training set; Fig.11 It is the calibration curve of the nomogram prediction model based on the three biomarkers in the blood training set; Fig.12 is the decision curve of the three biomarkers in the blood training set; Fig.13 This is the ROC curve of the three biomarkers for the joint diagnosis of PE. DETAILED DESCRIPTION

[0015] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention. Example 1

[0016] This embodiment provides biomarkers for the diagnosis of preeclampsia, wherein the biomarkers are MAPK8, CSNK1E and NOTUM genes or their encoded proteins. The expression levels of MAPK8, CSNK1E and NOTUM genes or their encoded proteins are detected for the diagnosis of preeclampsia. Specifically, the in vitro blood of the subject is used as the detection object. If the expression level of MAPK8 is downregulated and the expression levels of CSNK1E and NOTUM are upregulated, preeclampsia can be diagnosed. Example 2

[0017] This example provides a primer pair for specifically amplifying MAPK8, CSNK1E and NOTUM genes, and the nucleotide sequences of the primer pair are shown in SEQ ID NOs. 1-6.

[0018]

[0019] The primers provided in this example can be used to prepare a reagent for diagnosing preeclampsia, and the reagent also includes an internal reference primer whose nucleotide sequence is shown as SEQ ID NO. 7-8.

[0020] This embodiment also provides a product for diagnosing preeclampsia, which includes the above reagents and is a preparation, a kit chip or a nucleic acid membrane strip. Example 3

[0021] This embodiment provides a drug for treating eclampsia, which has a therapeutic effect on eclampsia by upregulating the expression level of MAPK8 protein and inhibiting the expression levels of CSNK1E and NOTUM proteins. Example 4

[0022] The present invention protects the molecular markers MAPK8, CSNK1E and NOTUM genes used for the diagnosis of preeclampsia, and diagnoses preeclampsia by the expression levels of these three genes. This is the first application in the diagnosis of preeclampsia.

[0023] In order to demonstrate the accuracy of the method of the present invention, healthy people and patients with preeclampsia were used as demonstration objects to verify the method of the present invention.

[0024] Ten volunteers were randomly selected, including five healthy subjects (as a blank control group) and five patients clinically diagnosed with PE. The expression levels of biomarkers MAPK8, CSNK1E and NOTUM in the blood of healthy volunteers and PE patients were compared to verify the accuracy of using MAPK8, CSNK1E and NOTUM as biomarkers for early diagnosis of PE. The specific experimental steps are as follows: (1) Extraction of total RNA 10 freshly collected blood (human) samples were added to 15ml centrifuge tubes with an equal volume of PBMC separation solution (blood < 3ml, add 3ml), and then slowly added to the mixed whole blood. Centrifuge at 2000g for 20min. The liquid can be seen to be divided into four layers, and PBMC is present in the second ring-shaped milky white layer. Carefully aspirate the PBMC layer into a new centrifuge tube, add PBS to 15ml, and resuspend the cells. Centrifuge at 1000g for 10min, carefully discard the supernatant, add 1ml TRIZol and let stand at room temperature for 10min to resuspend the lysed cells (or freeze in a -80° refrigerator). Add 200ul chloroform, shake vigorously for 30 seconds, let stand at room temperature for 10 minutes, let the liquid separate, and centrifuge at 12000g and 4℃ for 15 minutes. The liquid can be seen to be divided into three layers, and RNA is retained in the colorless upper aqueous phase. Carefully draw the upper aqueous phase into another EP tube, being careful not to draw the middle and lower layers (if you do draw them accidentally, you must squeeze them out gently), then add an equal volume of ice isopropanol, invert and mix, let stand for 10 minutes (when the sample amount is small, you can put it in a -20 degree refrigerator overnight to improve the RNA extraction rate), then centrifuge at 12000g and 4℃ for 10 minutes, and you can see that there is a white RNA precipitate at the bottom of the tube (when the sample amount is small, the precipitate is invisible to the naked eye and does not affect normal operation). Gently tilt the tube to discard the supernatant, be careful not to pour out the precipitate, dry the tube with absorbent paper, add 1ml of 75% ethanol to the precipitate, invert a few times to make the precipitate float, let stand for 2 minutes, centrifuge at 7500g and 4℃ for 5 minutes, and let the precipitate adhere to the bottom of the tube again, and repeat this step twice. Discard the supernatant, turn the centrifuge tube upside down on absorbent paper, carefully absorb the remaining liquid with a 10ul pipette tip, be careful not to absorb the precipitate, dry it naturally for 20 minutes or put it in a clean bench to blow dry, so that the ethanol and water can evaporate as much as possible, and the RNA precipitate becomes transparent (note that it should not be too dry, otherwise it will affect the subsequent dissolution of RNA). Add 20-50ul of RNase-free water to the dried RNA precipitate, let it stand for 15 minutes to completely dissolve the RNA, and take 1ul for concentration detection with Nano drop, record the RNA purity / concentration to calculate the amount of sample for the subsequent reverse transcription step, and immediately reverse transcribe the remaining RNA or freeze it in a -80℃ refrigerator.

[0025] (2) RNA concentration detection Take 1ul RNA and detect it using NanoPhotometer N50.

[0026] (3) Reverse transcription For reverse transcription of mRNA, use the Hifair® Ⅲ 1st Strand cDNA SynthesisSuperMix for qPCR kit from Yisheng Company. Specifically, take out the components of the reverse transcription kit, melt them at room temperature, centrifuge briefly, place on ice, and add the following reagents and solutions in the following order on ice:

[0027] After a brief centrifugation, reverse transcription was performed on a common PCR instrument according to the following conditions:

[0028] (4) On-machine testing First, dilute the reverse transcription product cDNA by 5-20 times with ddH2O (RNase / DNase free). Then perform qPCR reaction according to the following reaction system:

[0029] During the sample loading operation, you should check whether there are bubbles in the gun tip each time you draw up liquid. After blowing the liquid into the hole, you should also check whether there is any liquid residue in the gun tip. If there is any residue, the residual liquid should be blown into the corresponding hole by the instrument. The sample loading operation must be completed in one go. You cannot perform other experimental operations or do irrelevant things at the same time to ensure the consistency of repeated holes.

[0030] After a brief centrifugation, 40 cycles of reaction were performed in a CFX96 real-time quantitative fluorescence PCR instrument under the following conditions. The introduced sequences are shown in the following table.

[0031]

[0032] The amplification conditions are as follows:

[0033] Make amplification curve and melting curve, first look at the melting curve, if it is a smooth single peak, it can be judged that the primer specificity is good, if the curve is not smooth but has only one large single peak, it means that the primer specificity is average and the data is still usable, if the curve is double or multi-peaked, the primer specificity is too poor, and the primers need to be redesigned and the experiment repeated.

[0034] The amplification curve and melting curve obtained in this example are shown in the attached Figures 4 to 7 , it can be seen that the biomarkers MAPK8, CSNK1E and NOTUM all have good specificity.

[0035] Read the Ct value and use the 2–△△Ct method to calculate the relative expression of the gene. Specifically, the first step is to calculate △Ct=Ct (target gene)-Ct (reference gene); △△CT=△Ct (experimental group)-△Ct (control group); finally calculate the 2-△△CT value, and use Graphpad Prism 10 to draw a graph and calculate the P value. The results are as follows: Figures 1 to 3As shown, the results showed that: compared with the Control group (healthy group), CSNK1E and NOTUM were highly expressed, and MAPK8 was lowly expressed in the PE group, and the difference was statistically significant (P<0.05), which indicates that the present invention is accurate in diagnosing PE through the expression levels of CSNK1E, NOTUM and MAPK8.

[0036] Explanation on the use of MAPK8, CSNK1E and NOTUM genes as molecular markers for the diagnosis of preeclampsia: In order to screen out markers that are stably expressed in PE patients, two different data sets (both data sets are from the GEO public database) were used for intersection verification, such as Figure 8 As shown in the figure, except for LEF1, the AUC values ​​of the intersection genes are all greater than 0.7, indicating that these 7 intersection genes have a good diagnostic effect on PE. Next, in order to verify the universality of this intersection gene, we plotted the ROC curve in the external validation set GSE75010 dataset, as shown in the figure below. Fig. 9 As shown in the figure, the AUC values ​​of three genes are greater than 0.7, indicating that these three genes have good diagnostic value for PE and good universality. Therefore, we selected genes with AUC values ​​greater than 0.7 in both the blood training set and the validation set as biomarkers, namely: MAPK8, CSNK1E, and NOTUM.

[0037] Through experimental verification, MAPK8, CSNK1E and NOTUM genes or their encoded proteins were used as molecular markers for the diagnosis of preeclampsia. This study used the R language rms package to score the three biomarkers according to their expression in the blood training set. Each factor corresponds to a score, and the sum of the scores of each factor corresponds to the total score (TotalPoint). Then, the probability of disease diagnosis is predicted based on the total score. The higher the score, the greater the probability of diagnosis of PE. Finally, we plotted the prediction results into a nomogram, such as Fig.10 As shown, the nomogram regression equation is: y=-70.6421+(-2.0131)*MAPK8+(5.4648)*CSNK1E+(3.9017)*NOTUM.

[0038] Based on the nomogram prediction model, we draw the calibration curve corresponding to the nomogram ( Fig.11 ), the horizontal axis of the calibration curve is the predicted probability, and the vertical axis is the actual probability. The closer the slope of the calibration curve is to 1, the more accurate the model prediction is. Fig.11 As can be seen in the figure, the slope of the curve is close to 1 in the diagnosis of PE, indicating that these three biomarkers have good diagnostic ability for PE. Then the R package "ggDCA" is used to draw the decision curve, as shown in Fig.12As shown in the figure, the net benefit value is greater than 0, indicating that the model has a good prediction effect. Then the ROC curve is drawn using the R package "pROC", as shown in Fig.13 As shown in the figure, the combined diagnosis result of the three biomarkers showed AUC = 0.920, and the model was considered accurate. These markers have high sensitivity and specificity and can effectively distinguish normal pregnant women from PE patients.

[0039] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A molecular marker for the diagnosis of preeclampsia, characterized in that: The molecular marker is at least one of MAPK8, CSNK1E and NOTUM genes or their encoded proteins.

2. A drug for treating preeclampsia, characterized in that: The drug treats eclampsia by up-regulating MAPK8, or down-regulating the expression levels of CSNK1E and NOTUM genes or their encoded proteins.

3. A reagent for detecting the expression level of the molecular marker according to claim 1, characterized in that: The reagents include primer pairs for specifically amplifying MAPK8, CSNK1E and NOTUM genes, and the nucleotide sequences of the primer pairs are shown as SEQ ID NO.1-6.

4. The reagent according to claim 3, characterized in that The reagents also include internal reference primers whose nucleotide sequences are shown as SEQ ID NO.7-8.

5. A product for diagnosing preeclampsia, characterized in that: The product comprises the reagent as claimed in claim 3 or 4.

6. The product according to claim 5, characterized in that The products include kits, preparations, chips or nucleic acid membrane strips.

7. Use of the product as claimed in claim 6 in the diagnosis of preeclampsia.

Images