Pre-eclampsia piRNA non-invasive diagnostic marker, diagnostic kit and application

Abstract

The invention discloses a preeclampsia piRNA noninvasive diagnostic marker, a diagnostic kit and application, and belongs to the technical field of molecular biology, the marker is piR-002094, and the nucleotide sequence of the marker is shown as SEQ ID NO: 1. The marker disclosed by the invention can be applied to preparation of kits and preparation of therapeutic drugs of the kits. The kit comprises a specific primer and a standardized reagent, and can be used for sensitively detecting the expression (AUC = 0.779) of piR-002094 in plasma. The piR-002094 inhibitor (SEQ ID NO: 7) provides a new strategy for preeclampsia treatment by inhibiting the piR-002094 to reduce the secretion of IL-6 (Interleukin-6).

Description

Technical Field

[0001] This invention relates to the field of molecular biology technology, specifically to a non-invasive diagnostic biomarker for preeclampsia using piRNA, a diagnostic kit, and its applications. Background Technology

[0002] Preeclampsia (PE) is a multisystem progressive disease specific to pregnancy, characterized by new-onset hypertension and proteinuria after 20 weeks of gestation. As one of the most serious pregnancy complications, it currently ranks second globally as the leading cause of maternal mortality. In my country, the prevalence of PE is 2.3%, with severe cases accounting for 68.1%. Despite continuous improvements in perinatal care, the incidence of PE and perinatal mortality continue to rise, making prevention and control a serious challenge. PE can lead to eclampsia and HELLP syndrome in pregnant women, as well as serious complications such as premature birth, intrauterine growth restriction, and perinatal death, significantly threatening maternal and infant health and safety, and is a major contributing factor to maternal and perinatal mortality. Because the pathogenesis of PE is not yet fully understood, new risk factor assessment criteria are constantly being updated, and its initial symptoms are complex and clinically heterogeneous, current early prediction and intervention methods are very limited, consuming significant medical resources and imposing a heavy burden on patients' families and society.

[0003] Real-time monitoring of PE progression faces significant challenges due to the difficulty in obtaining molecular characteristics of the primary affected organ (placenta). Against this backdrop, peripheral blood biomarkers have demonstrated significant potential for non-invasive monitoring. Among non-invasive biomarkers, angiogenesis-related biomarkers are particularly noteworthy, with members of the VEGF family considered promising biomarkers for predicting and diagnosing PE. Although the sFlt-1 / PIGF ratio has been considered an effective indicator in recent years, it suffers from the following drawbacks: 1. Insufficient sensitivity and specificity: ① Risk of false positives and false negatives: Some pregnant women may have an elevated ratio but not develop preeclampsia (false positive), or a normal ratio but develop the disease later (false negative). For example, patients with chronic hypertension, fetal growth restriction, or kidney disease may exhibit similar ratio changes. Furthermore, biochemical levels are easily affected by various factors such as diet and stress. ② Cross-reactivity with other pregnancy complications: HELLP syndrome, placental abruption, etc., may also be accompanied by elevated sFlt-1, leading to reduced specificity. 2. Strong gestational age dependence: The ratio threshold for early-onset (<34 weeks) preeclampsia is higher (e.g., >85), while the threshold is lower for late-onset (≥34 weeks) preeclampsia (e.g., >38-40). Failure to adjust the threshold according to gestational age may lead to misjudgment of risk. 3. High biological variability: Factors such as maternal weight, race, and multiple pregnancies may affect the baseline levels of sFlt-1 and PlGF. For example, multiple pregnancies themselves have higher sFlt-1 levels, which may lead to a falsely elevated ratio. 4. Unclear guidance for treatment and prognosis: It cannot directly guide treatment; even with an elevated ratio, clinical management still mainly relies on traditional methods such as controlling blood pressure and terminating pregnancy, lacking targeted therapy. 5. Strict technical requirements: It requires standardized laboratory procedures, which may not be feasible in primary care hospitals. Therefore, there is an urgent need to explore novel non-invasive biomarkers.

[0004] Piwi-interacting RNAs (piRNAs) are a newly discovered class of small non-coding RNAs (23-32 nt) found in germ cells and somatic cells, playing important roles in biological processes such as embryonic development regulation, maintenance of germline genome stability, translation regulation, and mRNA stability regulation. Recent studies have shown that a large number of piRNAs exhibit organ-specific expression patterns in the human placenta, and these placenta-specific piRNAs participate in maintaining normal placental physiological function through epigenetic regulation. Research has found that abnormally expressed piRNAs in the PE placenta may impair the proliferation and invasion capacity of trophoblast cells through mechanisms associated with PE placental dysfunction, such as regulating PI3K-Akt, mTOR, Apelin, Wnt signaling pathways, and autophagy, thereby leading to abnormal spiral artery remodeling. This suggests that piRNA expression levels may reflect the degree of PE placental damage and have dual value as a non-invasive diagnostic biomarker and therapeutic target for PE.

[0005] However, there is currently a lack of diagnostic studies for preeclampsia based on plasma piRNA expression levels, and a clinically validated standardized testing system has not been established. Therefore, developing novel non-invasive biomarkers for preeclampsia has become a key scientific issue that urgently needs to be addressed in this field. Summary of the Invention

[0006] This invention provides a non-invasive diagnostic biomarker for preeclampsia using piRNA, a diagnostic kit, and its applications. The biomarker can be used in the preparation of the kit and therapeutic drugs. The kit contains specific primers and standardized reagents, and can sensitively detect the expression of piR-002094 in plasma (AUC=0.779). Targeting the piR-002094 inhibitor (SEQ ID NO:7) reduces IL-6 secretion by inhibiting piR-002094, providing a new strategy for the treatment of preeclampsia.

[0007] To achieve the above objectives, a first aspect of this application provides a preeclampsia biomarker, namely piR-002094, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0008] A second aspect of this application provides a specific reverse transcription primer for reverse transcription of the aforementioned piR-002094, the sequence of which is shown in SEQ ID NO:2.

[0009] A third aspect of this application provides a primer pair for amplifying the piR-002094 described above, comprising: an upstream primer, the sequence of which is shown in SEQ ID NO:3; and a downstream primer, the sequence of which is shown in SEQ ID NO:4.

[0010] The fourth aspect of this application provides the use of a reagent for detecting piR-002094 levels in the preparation of products for diagnosing preeclampsia.

[0011] The fifth aspect of this application provides a product for detecting preeclampsia, including a reagent for detecting the expression level of piR-002094.

[0012] The sixth aspect of this application provides a preeclampsia detection kit comprising the following components: 2-20 μM / μL of the reverse transcription primers described above, 2-20 μM / μL of the amplification primer pairs described above, 4×gDNA wiper Mix, 10×RTMix, HiScript III Enzyme Mix, and 2×ChamQ Universal SYBR qPCR Master Mix.

[0013] As a preferred embodiment of the present invention, the preeclampsia detection kit comprises the following components: 10 μM / μL of the above-described reverse transcription primers, 10 μM / μL of the above-described amplification primer pairs, 4×gDNA wiper Mix, 10×RTMix, HiScript III Enzyme Mix, and 2×ChamQ Universal SYBR qPCR Master Mix.

[0014] The seventh aspect of this application provides a piR-002094 inhibitor molecule for inhibiting preeclampsia-related vascular endothelial dysfunction, characterized in that it targets the aforementioned piR-002094, the sequence of which is shown in SEQ ID NO:7.

[0015] The eighth aspect of this application provides the use of the piR-002094 inhibitor molecule in the preparation of a drug for treating preeclampsia, by inhibiting piR-002094 expression to reduce IL-6 secretion.

[0016] In a preferred embodiment of the present invention, the reagent is used to detect plasma samples by real-time quantitative PCR.

[0017] The beneficial effects of the present invention are as follows: (1) The kit of the present invention contains specific primers (SEQ ID NO: 2-4) and standardized reagents (Novizan product number R312-01, Q311-02 / 03), which can accurately detect the expression level of piR-002094 in plasma. Through clinical verification, the area under the ROC curve (AUC) is 0.779, which is significantly better than traditional protein markers (such as sFlt-1 / PlGFAUC≈0.7), and can realize early non-invasive screening for preeclampsia.

[0018] (2) The piR-002094 inhibitor (SEQ ID NO:7) designed in this invention for piR-002094 can significantly reduce its expression level (inhibition rate 70%). P =0.0035), and reduced the secretion of the inflammatory factor IL-6 (inhibition rate 20%). P =0.0170), providing a safe and effective new strategy for the treatment of preeclampsia, with highly effective inhibitory effects of targeted therapy drugs.

[0019] (3) The kit of the present invention is suitable for plasma sample detection, is easy to operate (the whole process takes less than 4 hours), and is compatible with the hospital's conventional qPCR platform.

[0020] (4) The core components of the kit of the present invention are domestically produced reagents (such as Novizan enzyme preparations), which reduce the cost by 50% compared with imported products; the synthesis process of piR-002094 inhibitor is mature (Shanghai Jima Pharmaceutical), with high batch-to-batch stability (CV<5%), and it has the conditions for large-scale production. Attached Figure Description

[0021] Figure 1 This is a graph showing the expression of the preeclampsia piRNA diagnostic marker piR-002094 in the plasma of the discovery cohort (71 patients with preeclampsia and 53 healthy pregnant women). Example 1.

[0022] Figure 2 This is a graph showing the expression of the preeclampsia piRNA diagnostic marker piR-002094 in the plasma of the validation cohort (129 patients with preeclampsia and 107 healthy pregnant women). Example 1.

[0023] Figure 3 This is the ROC curve of piR-002094, a piRNA diagnostic marker for preeclampsia, in Example 1.

[0024] Figure 4 This is a graph evaluating the transfection effect of piR-002094 mimics, a mimic of the preeclampsia piRNA diagnostic marker piR-002094, in HUVEC cells, as shown in Example 2.

[0025] Figure 5 This is an evaluation diagram of the transfection effect of piR-002094 inhibitor, the inhibitor of the preeclampsia piRNA diagnostic marker piR-002094, in HUVEC cells, as shown in Example 2.

[0026] Figure 6 This is a graph showing the effect of overexpression of the preeclampsia piRNA diagnostic marker piR-002094 on the proliferation capacity of HUVEC cells in Example 2.

[0027] Figure 7 This is a graph showing the effect of inhibiting the preeclampsia piRNA diagnostic marker piR-002094 on the proliferation capacity of HUVEC cells in Example 2.

[0028] Figure 8 This is a graph from Example 3 showing the effect of the preeclampsia piRNA diagnostic marker piR-002094 on the migration ability of HUVEC cells (cell scratch assay).

[0029] Figure 9 This is a bar chart (cell scratch assay) showing the effect of the preeclampsia piRNA diagnostic marker piR-002094 on the migration ability of HUVEC cells in Example 3.

[0030] Figure 10 Example 3 describes the effect of the preeclampsia piRNA diagnostic marker piR-002094 on the migration ability of HUVEC cells (Transwell cell migration assay).

[0031] Figure 11 This is a bar chart (Transwell cell migration assay) showing the effect of the preeclampsia piRNA diagnostic marker piR-002094 on the migration ability of HUVEC cells in Example 3.

[0032] Figure 12 This is a graph from Example 4 showing the effect of the preeclampsia piRNA diagnostic marker piR-002094 on IL-6 mRNA expression in HUVEC cells.

[0033] Figure 13 This is a graph from Example 4 showing the effect of the preeclampsia piRNA diagnostic marker piR-002094 on IL-6 secretion in HUVEC cells. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0036] In this invention, numerical ranges are involved. Unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe features or characteristics, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0037] In this invention, there are no particular limitations on the specific dispersion and stirring methods.

[0038] Unless otherwise specified, all reagents or instruments used in this invention are commercially available conventional products. Unless otherwise specified, the raw materials used in each comparative example and the parallel experiments of each embodiment are the same commercially available products.

[0039] Example 1 This invention extracts plasma RNA from patients with preeclampsia, then reverse transcribes it using primers for reverse transcription of the preeclampsia diagnostic marker piR-002094. Subsequently, it uses primers to amplify piR-002094 for real-time quantitative PCR amplification to detect the expression level of piR-002094 in the plasma of patients with preeclampsia, thus achieving a non-invasive diagnosis of preeclampsia.

[0040] The sequence of piR-002094, a piRNA diagnostic biomarker for preeclampsia, is SEQ ID NO:1.

[0041] The expression level of piR-002094, a diagnostic marker of preeclampsia, in the plasma of patients with preeclampsia and healthy pregnant women was detected using the following method: (1) Blood sample pretreatment: Before delivery, collect at least 3 ml of venous blood using sterile blood collection tubes anticoagulated with EDTA-K2. Centrifuge rapidly at 3000 rpm for 15 min at room temperature. Aspirate and aliquot the plasma into EP tubes, each containing approximately 500 μL of plasma. Add RNAiso Blood lysis buffer at a 1:1 ratio, mix thoroughly by pipetting, and proceed with subsequent experiments immediately or store at -80°C. For venous blood samples collected from other locations, secure with ice packs and ship on the same day, maintaining a temperature below 4°C throughout transport. Upon receipt, process the samples immediately according to the above procedure and store at -80°C.

[0042] (2) Plasma RNA extraction: S1. Remove the RNAiso Blood lysis buffer sample from the -80℃ freezer, thaw at 4℃, quickly add 200 μL of chloroform, mix by inverting, and incubate at 4℃ for 10 min. Centrifuge at 11200 rpm at room temperature for 15 min, remove the EP tube, and transfer the entire upper aqueous phase to a new EP tube.

[0043] S2. Add an equal volume of isopropanol pre-cooled to -20℃, mix thoroughly by inverting the container, and let stand at -20℃ for 20 min. Centrifuge at 11200 rpm at room temperature for 10 min; a white precipitate will be visible. Discard the supernatant.

[0044] S3. Add 1 mL of 75% ethanol, gently tap the bottom of the EP tube to suspend the precipitate, invert the tube several times, and let it stand at -20℃ for 5 min. Centrifuge at 12000 rpm at room temperature for 5 min, discard the supernatant, and let the precipitate dry at room temperature for 5 min.

[0045] S4. Add 20 μL of DEPC water and repeatedly pipette to dissolve the precipitate. Use a nucleic acid and protein analyzer to detect RNA concentration and purity. RNA samples with an OD260 / OD280 ratio between 1.8 and 2.2 are considered to have acceptable purity. To prevent RNA degradation, use the samples immediately for subsequent reverse transcription experiments.

[0046] (3) RNA is reverse transcribed into cDNA: RNA reverse transcription was performed using the HiScript III RT SuperMix for qPCR kit (Nanjing Novizan Co., Ltd.). The specific steps are as follows: Prepare the reaction mixture in an RNase-free centrifuge tube according to the following ratio: 1 μg total RNA; 2 μL 5×g DNA Wiper Mix. Add RNase-free ddH2O to a final volume of 10 μL. Gently mix the mixture in the centrifuge tube using a pipette. Transfer the mixture to a PCR amplification instrument and set the program to 42℃, 2 min → 4℃. Add 2 μL 10×RTMix; 2 μL HiScript II Enzyme Mix; 1 μL reverse transcription primer (SEQ ID NO. 2, synthesized by Qingdao Deloitte Biotechnology Co., Ltd.); and add RNase-free ddH2O to a final volume of 20 μL. Mix well and place in the PCR amplification instrument. Set the reaction program to 37℃ for 15 min, then 85℃ for 5 s. The cDNA product obtained from reverse transcription can be used immediately for RT-qPCR or stored at -20℃, avoiding repeated freeze-thaw cycles.

[0047] (4) RT-qPCR: The reverse transcribed cDNA product obtained in step (3) was used for amplification and detection. The following reaction system was prepared in an octet: 0.4 μL of upstream primer (SEQ ID NO:3, synthesized by Qingdao Deloitte Biotechnology Co., Ltd.); 0.4 μL of downstream primer (SEQ ID NO:4, synthesized by Qingdao Deloitte Biotechnology Co., Ltd.); 2 μL of cDNA; 7.2 μL of RNase-free ddH2O; and 10 μL of 2×ChamQ Universal SYBR qPCR Master Mix (Nanjing Novizan Co., Ltd.).

[0048] The amplification reaction was carried out under the following conditions: denaturation: 95℃ 30 s → cyclic reaction (40 cycles): 95℃ 10 s, 60℃ 30 s → melting curve analysis: 95℃ 15 s, 60℃ 60 s, 95℃ 15 s. (4) Result analysis: Samples with a single peak in the melting curve are specific. While amplifying piR-002094, U6 internal control was amplified as a control and 2 -ΔΔCtThe relative expression level of piR-002094 was evaluated using the method.

[0049] Plasma samples from 71 patients with preeclampsia and 53 healthy pregnant women were collected as the discovery cohort, with each sample collected three times. Results are as follows: Figure 1 As shown.

[0050] Figure 1 The results showed that, in the discovery cohort, the preeclampsia piRNA diagnostic marker piR-002094 was significantly upregulated in the peripheral blood of preeclampsia patients compared with healthy pregnant women. P <0.0001).

[0051] Plasma samples from 129 patients with preeclampsia and 107 healthy pregnant women were collected as the discovery cohort, with each sample collected three times. Results are as follows: Figure 2 As shown.

[0052] Figure 2 The results showed that, in the validation cohort, the preeclampsia piRNA diagnostic marker piR-002094 was significantly upregulated in the peripheral blood of preeclampsia patients compared with healthy pregnant women. P <0.0001).

[0053] (5) ROC curve analysis: ROC curves were plotted based on the relative expression levels of piR-002094 in the plasma of preeclampsia patients and healthy pregnant women, such as... Figure 3 As shown, the area under the curve (AUC) is 0.779.

[0054] As is known to those skilled in the art, the AUC value ranges from 0.5 to 1.0, with AUC > 0.5 and an AUC value approaching 1.0 indicating superior diagnostic efficacy. Specifically, an AUC between 0.5 and 0.7 indicates low diagnostic discrimination for the target; an AUC between 0.7 and 0.9 indicates clinically acceptable discriminative ability; and an AUC value above 0.9 confirms excellent diagnostic efficacy. A value greater than 0.7 indicates that the target has clinically acceptable discriminative ability as a diagnostic marker for preeclampsia.

[0055] Example 2 Example 2 of this invention examines the effect of overexpression and knockdown of the preeclampsia piRNA diagnostic marker piR-002094 on the proliferation of human umbilical vein endothelial cells (HUVECs). The specific method is as follows: (1) Based on the sequence information of piR-002094 (SEQ ID NO:1), mimics piR-002094 mimics (SEQ ID NO:5), mimic control mimics NC (SEQ ID NO:6), inhibitor piR-002094 inhibitor (SEQ ID NO:7) and inhibitor control inhibitor NC (SEQ ID NO:8) were designed and synthesized. mimics NC, piR-002094 mimics, inhibitor NC and piR-002094 inhibitor were all synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd.

[0056] (2) Cell culture: HUVEC cells in normal growth state were seeded into 6-well plates one day in advance so that the cell density reached 80% at the time of transfection the next day.

[0057] (3) Transfection: Prepare two RNase-free EP tubes (tube A and tube B). Add 3 μL of the RNA to be transfected and 50 μL of serum-free DMEM high-glucose medium to tube A. Add 3 μL of Lipofectamine 3000 and 50 μL of serum-free DMEM high-glucose medium to tube B. Incubate at room temperature for 5 min. Mix the two tubes together gently by pipetting. Incubate at room temperature for 20 min, then add the RNA to the transfection wells. Transfection efficiency was assessed by RT-qPCR. The experiment was repeated three times, and the results are shown below. Figure 4-5 As shown.

[0058] Figure 4 The results showed that, compared with the mimics NC group, the expression level of piR-002094 in the mimics group was significantly increased ( P =0.0304).

[0059] Figure 5 The results showed that, compared with the inhibitor NC group, the expression level of piR-002094 in the piR-002094 inhibitor group was significantly reduced. P =0.0035).

[0060] (4) CCK-8 cell proliferation experiment: The OD values ​​of cells in each group were measured at 450 nm at 24 h, 48 h, and 72 h after transfection, and proliferation curves were plotted. The experiment was repeated three times, and the results are as follows: Figure 6-7 As shown.

[0061] Figure 6The results showed that overexpression of the preeclampsia piRNA diagnostic marker piR-002094 significantly inhibited HUVEC cell proliferation (48h: P=0.0104, 72h: P=0.0104). P =0.0033).

[0062] Figure 7 The study showed that inhibition of the preeclampsia piRNA diagnostic marker piR-002094 significantly promoted HUVEC cell proliferation. P =0.0143).

[0063] Example 3 Example 3 of this invention examines the effect of the preeclampsia piRNA diagnostic marker piR-002094 on the migration ability of HUVEC cells by overexpressing and knocking down the marker. The specific method is as follows: (1) Cell scratch test: Cells were cultured in 6-well plates 24 h after transfection by cross-scraping. Images were taken under an inverted microscope at 0 h and 24 h post-culture. The scratch area at 0 h and 24 h was calculated using ImageJ software, and the cell migration rate for each group was calculated. Migration rate (%) = (0 h scratch area - 24 h scratch area) / 0 h scratch area × 100%. The experiment was repeated three times, and the results are as follows: Figure 8 and Figure 9 As shown.

[0064] Figure 8 and Figure 9 The results showed that overexpression of the preeclampsia piRNA diagnostic marker piR-002094 significantly inhibited HUVEC cell migration. P =0.0335), knockdown limited the promotion of HUVEC cell migration ( P =0.0239).

[0065] (2) Transwell cell migration assay: S1. Place the chambers in 24-well plates containing DMEM medium with 20% fetal bovine serum. Cells transfected for 24 h are then cultured at 6 × 10⁻⁶ cells / well. 4 Inject each cell into the chamber, and then fill the chamber with DMEM medium containing 2% fetal bovine serum to a total volume of 300 μL before culturing for 24 h.

[0066] S2. Aspirate the culture medium from the chamber, fix with 4% paraformaldehyde for 30 min, and then stain with 1% crystal violet for 30 min.

[0067] S3. After cleaning the chamber until there is no obvious purple color, gently wipe away the unpenetrated cells in the upper chamber with a moistened cotton swab.

[0068] S4. Observe and photograph under a microscope, and use ImageJ software to calculate the number of cells in each group. The experiment was repeated three times, and the results are as follows: Figure 10 and Figure 11 As shown.

[0069] Figure 10 and Figure 11 The results showed that overexpression of the preeclampsia piRNA diagnostic marker piR-002094 significantly inhibited HUVEC cell migration. P =0.0309), knockdown significantly promoted HUVEC cell migration ( P =0.0340).

[0070] Example 4 Example 4 of this invention investigated the effect of the preeclampsia piRNA diagnostic marker piR-002094 on IL-6 expression and secretion in HUVEC cells by overexpressing and knocking down the marker. The specific method is as follows: (1) RT-qPCR detection of IL-6 mRNA expression: piR-002094 was overexpressed and knocked down in HUVECs, and the expression level of IL-6 mRNA in each group was detected by RT-qPCR. Results are as follows: Figure 12 As shown.

[0071] Figure 12 The results showed that overexpression of the preeclampsia piRNA diagnostic marker piR-002094 significantly promoted the expression of IL-6 mRNA in HUVEC cells. P =0.0315), knockdown significantly inhibited IL-6 mRNA expression ( P =0.0055).

[0072] (2) ELISA detection of IL-6 secretion: S1. Take the cell culture base of each group 48h after transfection into 15 mL centrifuge tubes, centrifuge at 3000 rpm at room temperature for 20 min to remove cell debris and impurities, and aliquot the supernatant into EP tubes for experiments.

[0073] S2. Follow the instructions to perform IL-6 detection using the IL-6 ELISA kit (Wuhan Beinlai Biotechnology Co., Ltd.).

[0074] S3. Zero the instrument using a blank well and measure the OD value of each well at a wavelength of 450 nm. Plot a standard curve with the standard concentration on the x-axis and the OD value on the y-axis using four-parameter fitting. Substitute the sample OD value into the curve equation to calculate the sample concentration. Repeat the experiment three times. The results are as follows: Figure 13 As shown.

[0075] Figure 13 The results showed that overexpression of the preeclampsia piRNA diagnostic marker piR-002094 significantly promoted IL-6 secretion in HUVEC cells. P =0.0010), knockdown significantly inhibited IL-6 secretion ( P =0.0170).

[0076] Example 5: Composition and Performance Verification of the piR-002094 Detection Kit 1. The reagent kit components are shown in Table 1. Table 1

[0077] 2. Testing Process (1) Collect 3 mL of EDTA-anticoagulated venous blood and centrifuge at 3000 rpm for 15 min to separate the plasma; (2) Extract total RNA according to the RNAiso Blood instructions; (3) Reverse transcription to cDNA was performed using the HiScript III kit (catalog number R312-01); (4) Prepare the qPCR reaction system (including SEQ ID NO:3-4 primer pair and ChamQ SYBR Mix); (5) Amplification program: 95℃ 30 s → 40 cycles (95℃ 10 s → 60℃ 30 s) → melting curve analysis.

[0078] Example 6: Verification of the effect of piR-002094 inhibitor on inhibiting piR-002094 1. Design of piR-002094 inhibitor The piR-002094 inhibitor sequence targeting piR-002094 (SEQ ID NO:7).

[0079] 2. Cell transfection (1) HUVEC cells were seeded in 6-well plates and transfected with piR-002094 inhibitor (Lipofectamine 3000) when the density reached 80%. (2) Cells were collected 24 h after transfection and the expression of piR-002094 was detected.

[0080] 3. Results RT-qPCR: piR-002094 expression was reduced by 70% in the piR-002094 inhibitor group. P=0.0035).

[0081] Finally, it should be noted that the above embodiments are used to illustrate the technical solutions of the present invention and not to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A preeclampsia marker, characterized by, The marker is piR-002094, the nucleotide sequence of which is shown as SEQ ID NO:

1.

2. A specific reverse transcription primer for reverse transcription of the piR-002094 of claim 1, characterized by, The sequence of which is shown as SEQ ID NO:

2.

3. A primer pair for amplifying the piR-002094 amplification according to claim 1, characterized in that, Comprising: an upstream primer, the sequence of which is shown as SEQ ID NO: 3; a downstream primer, the sequence of which is shown as SEQ ID NO:

4.

4. Use of a reagent for detecting the level of piR-002094 in the preparation of a product for diagnosing preeclampsia.

5. A product for detecting preeclampsia, characterized by, Comprising a reagent for detecting the expression level of piR-002094.

6. A pre-eclampsia test kit characterized by, Comprising the following components: 2~20 μM / μL reverse transcription primer according to claim 2, 2~20 μM / μL amplification primer pair according to claim 3, 4×gDNA wiper Mix, 10×RT Mix, HiScript III Enzyme Mix and 2×ChamQ Universal SYBR qPCR Master Mix.

7. A piR-002094 inhibitor molecule for use in inhibiting preeclampsia-associated vascular endothelial dysfunction, characterized in that, Targeting piR-002094 according to claim 1, the sequence of which is shown as SEQ ID NO:

7.

8. Use of the piR-002094 inhibitor molecule of claim 7 for the manufacture of a medicament for the treatment of preeclampsia, characterized in that, Decreasing IL-6 secretion by inhibiting the expression of piR-002094.

9. Use of the piR-002094 according to claim 1 for the preparation of a test for preeclampsia, characterized in that, The reagent is used for detecting plasma samples by real-time fluorescent quantitative PCR.

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