Use of PIK3C3 as target in monitoring fetal intrauterine development

Abstract

The invention relates to the technical field of biology, in particular to application of PIK3C3 serving as a target in monitoring fetal intrauterine development, and particularly relates to application of PIK3C3 in monitoring fetal intrauterine dysplasia. According to the invention, the sequencing data of the maternal blood sample with abnormal intrauterine development is mined, integrated and analyzed, and the high expression of the PIK3C3 in the fetal intrauterine development abnormal sample is verified by designing the specific primer to carry out qPCR experiment, so that the PIK3C3 is suitable for rapid, economical and effective fetal intrauterine development monitoring.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the use of lipid kinase PIK3C3 as a target in monitoring fetal intrauterine development. Background Art

[0002] The earliest stage of human development is the stage of intrauterine development of the baby before birth. During this period, the fertilized egg develops into an embryo and then continues to develop into a mature fetus. It takes about 10 months from conception to delivery, about 280 days. Intrauterine fetal growth abnormalities refer to various abnormalities that occur during the development of the fetus in the mother's uterus, including growth retardation, structural malformations, and chromosomal abnormalities. The "developmental origins of health and disease" theory suggests that the growth of the fetus during intrauterine development is highly correlated with perinatal outcomes and even adult health. However, the mechanism of intrauterine fetal growth abnormalities is complex and may be the combined influence of maternal nutrition, placenta, demographic characteristics, and social environmental factors. This has also led to the fact that research on intrauterine fetal growth abnormalities has not made significant progress. Given the importance and urgency of the problem of intrauterine fetal growth abnormalities, its etiology and pathogenesis have always been a hot topic in the perinatal field and a problem that needs to be solved urgently. It is urgent to find a highly sensitive and rapid target for intrauterine development monitoring.

[0003] Autophagy is a highly conserved lysosomal degradation pathway in eukaryotes to maintain the body's metabolic adaptation and energy cycle. Therefore, the process of embryonic development and fetal growth must be regulated by autophagy. In recent years, it has been found that autophagy plays an important role in the formation and development of the human placenta. Current studies have confirmed that autophagy is related to pathological pregnancy. Abnormal autophagy can lead to abnormal biological behaviors such as trophoblast proliferation, differentiation, apoptosis and autophagy, destroy the balance of the maternal-fetal interface, and thus lead to a series of fetal intrauterine developmental abnormalities such as fetal growth restriction. The PIK3C3 gene (phosphatidylinositol 3-kinase catalytic subunittype 3), also known as Vps34 or hVps34, is located in band 3, region 12 of the long arm of chromosome 18. The protein encoded by this gene is part of the phosphatidylinositol 3-kinase (PI3K) family, specifically the catalytic subunit type 3. The PI3K family plays an important role in cell signaling, especially in regulating cell growth, survival and metabolism. PIK3C3 is an important protein kinase in the process of autophagy initiation and formation. Currently, there are no reports on the relationship between PIK3C3 and fetal intrauterine development. Summary of the invention

[0004] The purpose of the present invention is to provide the use of PIK3C3 as a target in the preparation of a product for monitoring fetal intrauterine development. The present invention found that PIK3C3 was significantly differentially expressed in the blood of mothers with abnormal fetal intrauterine development.

[0005] The technical solution of the present invention is as follows:

[0006] The present invention provides the use of PIK3C3 as a target in the preparation of a product for monitoring fetal intrauterine development. The specific sequence of PIK3C3 (Gene ID: 5289) of the present invention can be found in the international public nucleic acid sequence database GeneBank. The present invention finds that PIK3C3 is significantly differentially expressed in mothers with fetal intrauterine developmental abnormalities, and can be used as a molecular marker for fetal intrauterine developmental abnormalities.

[0007] The product for monitoring fetal intrauterine development of the present invention monitors the intrauterine development of the fetus by detecting the expression level of PIK3C3 in the body of the tested person and comparing it with the normal level.

[0008] As an embodiment, the present invention detects the expression level of PIK3C3 in the tissue or body fluid of the test subject. If the expression of PIK3C3 is higher than the normal level, the risk of abnormal intrauterine fetal development is high. The test subject of the present invention refers to the mother pregnant with a fetus. The PIK3C3 source for monitoring intrauterine fetal development of the present invention includes but is not limited to the tissue or body fluid of the test subject, and the body fluid includes blood, tissue fluid, and other in vivo liquid components in which DNA exists. In a specific embodiment of the present invention, the source of PIK3C3 for monitoring intrauterine fetal development is the blood of the test subject. The normal level of the present invention refers to the expression level of PIK3C3 in the body of the test subject pregnant with a normally developed fetus.

[0009] In the present invention, the product includes detecting the expression level of PIK3C3 of the test subject by reverse transcription PCR, real-time fluorescence quantitative PCR, in situ hybridization, chip or high-throughput sequencing platform to monitor the intrauterine development of the fetus. As an embodiment, the product includes a chip, a reagent, a kit or a test paper, and the kit includes a PCR kit or an in situ hybridization kit.

[0010] The present invention also provides the use of a substance for detecting the expression level of PIK3C3 in the preparation of a product for monitoring abnormal intrauterine fetal development.

[0011] In the present invention, the expression level of PIK3C3 in the test subject with abnormal intrauterine fetal development is increased compared with that in the test subject with normal intrauterine fetal development.

[0012] The present invention also provides a reagent or kit for monitoring fetal intrauterine development, wherein the reagent or kit comprises at least one pair of primers for specifically amplifying PIK3C3, and the expression level of PIK3C3 is detected by reverse transcription PCR or real-time quantitative PCR to monitor fetal intrauterine development.

[0013] As an embodiment, the sequences of the primers are shown as SEQ ID NO.1 and SEQ ID NO.2.

[0014] Preferably, the reagent or kit further comprises RT-PCR reagent or real-time quantitative PCR reagent.

[0015] As one embodiment, the kit is an in situ hybridization kit, comprising a probe that hybridizes with a PIK3C3 nucleic acid sequence, wherein the probe detects the expression level of PIK3C3 by in situ hybridization to monitor fetal intrauterine development.

[0016] The present invention also provides a chip for monitoring fetal intrauterine development, the chip comprising a solid phase carrier and an oligonucleotide probe fixed on the solid phase carrier, the oligonucleotide probe comprising a probe hybridizing with the nucleic acid sequence of PIK3C3 for detecting the transcription level of PIK3C3, and the expression level of PIK3C3 is detected by the chip to monitor fetal intrauterine development.

[0017] In the present invention, the probe hybridized with the PIK3C3 nucleic acid sequence can be DNA, RNA, DNA-RNA chimera, PNA or other derivatives. The length of the probe is not limited, as long as specific hybridization is completed and specifically combined with the target nucleotide sequence, any length is OK. The length of the probe can be as short as 25, 20, 15, 13 or 10 bases. Similarly, the length of the probe can be as long as 60, 80, 100, 150, 300 base pairs or longer, or even the entire gene. Due to different probe lengths, the hybridization efficiency and signal specificity have different effects, the length of the probe is usually at least 14 base pairs, the longest is generally no more than 30 base pairs, and the length complementary to the target nucleotide sequence is preferably 15-25 base pairs. The probe self-complementary sequence is preferably less than 4 base pairs, so as not to affect the hybridization efficiency.

[0018] In the context of the present invention, "monitoring intrauterine fetal development" refers to determining whether the intrauterine development of a subject's fetus is normal.

[0019] Compared with the prior art, the present invention has the following technical effects:

[0020] The present invention found that PIK3C3 was significantly differentially expressed in the blood of mothers with intrauterine developmental abnormalities through RNA sequencing, and further verified the high expression of PIK3C3 in patients with fetal developmental abnormalities by designing specific primers for qPCR experiments. This makes PIK3C3 suitable for rapid and cost-effective early monitoring of intrauterine developmental abnormalities.

[0021] The discovery of PIK3C3 helps us understand the mechanism of action of mRNA in intrauterine fetal developmental abnormalities. It also provides a research direction for screening new biomarkers related to intrauterine fetal developmental abnormalities and provides new targets for monitoring and intervention treatment of intrauterine fetal developmental abnormalities. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Volcano plot of gene expression differences in maternal blood samples for monitoring intrauterine growth restriction and normal intrauterine development of fetuses using RNA sequencing;

[0023] Figure 2 GeneOntology enrichment analysis bar graph of differentially expressed genes in maternal blood of fetuses with intrauterine growth restriction and normal intrauterine development;

[0024] Figure 3 KEGG Pathway enrichment analysis of differentially expressed genes in maternal blood of fetuses with intrauterine growth restriction and normal intrauterine development;

[0025] Figure 4 This is a statistical diagram of the difference in expression of PIK3C3 in maternal blood between fetuses with intrauterine growth restriction and normal intrauterine development using qPCR;

[0026] Figure 5 This is the ROC curve diagram for evaluating intrauterine growth restriction of fetus using PIK3C3 expression level. DETAILED DESCRIPTION

[0027] The present invention performs RNA sequencing analysis on maternal blood samples of 4 fetuses with intrauterine developmental abnormalities and 4 fetuses with normal intrauterine development, and finds differentially expressed mRNAs. Through functional enrichment analysis, an mRNA expressed by a gene related to cell autophagy, PIK3C3, is found. PIK3C3 is significantly differentially expressed in maternal blood samples of fetal intrauterine developmental abnormalities and normal intrauterine development. The present invention further verifies by designing specific primers for qPCR experiments in another 52 maternal blood samples of fetal intrauterine developmental abnormalities and 52 maternal blood samples of normal intrauterine development. The expression level of PIK3C3 in maternal blood samples of fetal intrauterine developmental abnormalities is significantly higher than that in maternal blood samples of normal intrauterine development (P<0.05). PIK3C3 can be used as a target predictive marker for monitoring fetal intrauterine development for rapid and cost-effective fetal intrauterine development monitoring.

[0028] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. The technical solution of the present invention includes but is not limited to the following embodiments.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0030] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0031] The reagents and equipment used in the following examples are as follows:

[0032] Table 1 Reagents used

[0033] Reagents company Universal RNA extraction kit ONREW 1-Bromo-3-chloropropane Sigma-Aldrich Reverse transcription kit abmgood PCR kits KAPA DEPC water Shanghai Bio-Tech Biotechnology Co., Ltd. Anhydrous ethanol Shanghai Lingfeng Chemical Reagent Co., Ltd.

[0034] Table 2 Equipment used

[0035]

[0036]

[0037] Example 1

[0038] Screening for differentially expressed genes in fetal intrauterine developmental abnormalities

[0039] 1. Specimen source

[0040] Blood sequencing samples were selected from 4 women with fetal growth restriction and 4 women with normal fetal development who delivered naturally in the hospital. All patients with fetal growth restriction were first-time mothers and had no genetic diseases or pregnancy complications before pregnancy.

[0041] 2. RNA Extraction

[0042] 1) The instruments used in the extraction process are placed in 84 disinfectant for one day for disinfection, then washed with detergent, dried, and soaked in acid overnight. After washing and drying, they are wrapped in aluminum foil and sterilized under high pressure, and then baked at 180℃ for 4 hours before RNA extraction.

[0043] 2) Before RNA extraction, the magnetic beads should be soaked in ONRol Reagent overnight, and the EP tubes used in the experiment should be pre-cooled on ice. Take out the samples that have been prepared and frozen in advance from the -80℃ refrigerator, and put two pre-cooled magnetic beads in a 2ml enzyme-free centrifuge tube.

[0044] 3) Add 0.8 ml of whole blood and 1 mL of ONRol Reagent to a 2 ml enzyme-free centrifuge tube and incubate at room temperature for 5 min to fully lyse the cells.

[0045] 4) RNA isolation: Add 200 μL 1-bromo-3-chloropropane (200 μL / 1 ml ONRol Reagent), shake vigorously by hand for 15 seconds, let stand at room temperature for 5 minutes, centrifuge at 13,000g, 4°C for 5 minutes, and pipette the upper aqueous phase into another clean enzyme-free centrifuge tube.

[0046] 5) Add 750 μL of anhydrous ethanol to the supernatant and vortex for 15 seconds.

[0047] 6) Place the NAExtraction Mini Columns in a 2 mL collection tube, transfer 750 μL of the mixture from step 5) to the filter column, and centrifuge at 13,000 g for 1 min.

[0048] 7) Discard the filtrate and transfer the remaining mixed solution to the filter column and centrifuge at 13,000 g for 1 min.

[0049] 8) Discard the filtrate, add 500 μL Buffer W1R to the filter column, and centrifuge at 13,000 g for 10 seconds.

[0050] 9) Discard the filtrate, add 600 μL Buffer W2R to the filter column, and centrifuge at 13,000 g for 10 s.

[0051] 10) Repeat step 9.

[0052] 11) Discard the filtrate and centrifuge again at 13,000 g for 2 min.

[0053] 12) Transfer the filter column to a 1.5 mL enzyme-free EP tube, add 30-100 μL enzyme-free water in the middle of the column, let stand at room temperature for 2 min, and centrifuge at 13,000 g for 1 min.

[0054] 13) Detection of extracted RNA quality: Take 2 μL of dissolved RNA and use a micro-nucleic acid quantitative analyzer to detect the concentration and purity of RNA. The OD value of RNA with good quality is 260 With OD 280 The ratio should be between 1.8 and 2.0.

[0055] 3. Transcriptome sequencing: The extracted RNA is randomly interrupted into short fragments, and the first cDNA chain is synthesized from the RNA fragments using reverse transcriptase and random hexamer primers. The second cDNA chain is then synthesized using DNA polymerase, RNase H and dNTPs. Finally, the ends of the cDNA are repaired using an end repair enzyme to give it a complete phosphorylated 3' end and 5' end. The adapter containing the sequence recognition site is connected to both ends of the cDNA, amplified by PCR, and the cDNA fragments of a specific size range are purified using gel electrophoresis or magnetic beads, and sequenced using the Illumina platform.

[0056] 4. Screening of differentially expressed genes: Quality control of sequencing results was performed, and data were normalized using the TMM algorithm in edgeR software. The p-value was calculated based on the hypothesis test model, and finally the p-value was corrected for multiple hypothesis tests. Differentially expressed genes were screened under the conditions of p-value < 0.05 and |log2(Fold-change)|>=1.

[0057] Figure 1 This is a volcano plot of gene expression differences in maternal blood samples of intrauterine growth restriction and normal intrauterine development. The horizontal axis represents the expression fold change of genes in different samples, and the vertical axis represents the significance level of expression differences. Up-regulated genes are indicated by red dots, and down-regulated genes are indicated by green dots. A total of 2292 differential mRNA genes were screened, of which 657 were up-regulated genes and 1635 were down-regulated genes.

[0058] The differential gene data corresponding to the three levels of cell components, molecular functions, and biological processes were annotated and functionally enriched in the GeneOntology database. GO entries with a p-value less than 0.05 were screened as significantly enriched GO entries, and the top 10 GO entries with the highest enrichment were displayed in a bar chart. Figure 2 This is a Gene Ontology enrichment analysis bar chart of differentially expressed genes in maternal blood of intrauterine growth restriction and normal intrauterine development. The horizontal axis is the specific GO entry name. The GO entries corresponding to the colors of the columns in the figure belong to the three major categories of the GO database, and the height of the column indicates the number of genes mapped to a certain GO entry. The top three GO entry genes are all related to PIK3C3.

[0059] The number of differentially expressed genes in each KEGG pathway category was counted. Pathways with a p-value less than 0.05 were screened as significantly enriched pathways, and the top 10 pathways with enrichment were displayed in a bubble chart. Figure 3 This is a KEGG Pathway enrichment analysis of differentially expressed genes in maternal blood of fetuses with intrauterine growth restriction and normal intrauterine development. The vertical axis is the specific KEGG Pathway name, and the horizontal axis is the enrichment score. The size of the point indicates the number of genes, and the color of the point corresponds to the size of the -log10 (P-value). Figure 3 The results showed that the genes with the largest enrichment and the most significant difference were those related to PIK3C3.

[0060] Example 2

[0061] Validation of differential expression of PIK3C3 in maternal blood samples with fetal growth restriction (FGR)

[0062] The specimens in this example were selected from 52 women with fetal growth restriction and 52 women with normal fetal development who delivered naturally in the hospital, and the pregnant women were aged 28-33 years old. The case screening criteria were the same as in Example 1.

[0063] The RNA in the maternal blood sample was extracted by the same extraction method as in Example 1. The extracted RNA of good quality was subjected to reverse transcription and qPCR detection as follows:

[0064] The first strand of cDNA was synthesized by reverse transcription, and the reverse transcription conditions were set as follows: 25°C for 10 min, 42°C for 15 min, 85°C for 5 min, and stored at 4°C. The reverse transcription system was: 2 μL of 5×All-In-One RT MasterMix, 500 ng of RNA solution, and RNaseFree H2O to make up to 10 μL.

[0065] The qRT-PCR (QPCR) program was: 95°C for 3 min; 95°C for 3 s, 60°C for 30 s, for a total of 40 cycles. The reaction system was: SYBR Primix Ex TaqⅡ 10 μL, 0.4 μL upstream primer, 0.4 μL downstream primer, 7.2 μL dH2O, 2 μL cDNA. According to the Ct value of each reaction, 2 -△△Ct Methods The expression level of the target gene was calculated, and the internal reference was ACTIN. The primer sequences are shown in Table 3.

[0066] Table 3 Primer sequences

[0067]

[0068] The experimental data were analyzed using GraphPad Prism 10.2.3 software. The quantitative data were expressed as mean ± standard deviation. One-way analysis of variance was used to compare differences among multiple groups, and paired t-test was used to compare between groups. P < 0.05 was considered statistically significant.

[0069] The expression difference of PIK3C3 in maternal blood was detected by qPCR. Figure 4 Compared with maternal blood samples with normal fetal development, the average expression level of PIK3C3 in maternal blood samples of FGR was significantly upregulated, and the difference was statistically significant (***: P < 0.001).

[0070] The ROC curve for evaluating intrauterine growth restriction was drawn based on the relative expression of PIK3C3 in the blood of 52 clinically normal mothers and 52 mothers with intrauterine growth restriction. Figure 5 The area under the curve (AUC) = 0.7544, indicating that the single PIK3C3 index has good diagnostic efficacy for intrauterine growth restriction of the fetus. The sensitivity and specificity of the test were analyzed by ROC curve analysis. The analysis showed that the relative expression of PIK3C3 was 1.800 as the cut-off value, which can take into account both sensitivity and specificity. In the process of fetal development, when there is inevitable interference from the maternal environment, the PIK3C3 index has a high diagnostic efficacy.

[0071] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of PIK3C3 as a target in the preparation of products for monitoring fetal intrauterine development.

2. The use according to claim 1, characterized in that: The product for monitoring fetal intrauterine development monitors fetal intrauterine development by detecting the expression level of PIK3C3 in the body of the tested person and comparing it with the normal level.

3. The use according to claim 2, characterized in that: The expression level of PIK3C3 in the tissues or body fluids of the tested person is detected. If the expression level of PIK3C3 is higher than the normal level, the risk of intrauterine developmental abnormalities of the fetus is high.

4. The use according to claim 2, characterized in that: The product includes detecting PIK3C3 expression levels through reverse transcription PCR, real-time quantitative PCR, in situ hybridization, chip or high-throughput sequencing platform.

5. Application of substances for detecting PIK3C3 expression levels in the preparation of products for monitoring fetal intrauterine developmental abnormalities.

6. The use according to claim 5, characterized in that: Compared with normal fetal intrauterine development, the expression level of PIK3C3 in the test subjects with abnormal intrauterine fetal development was increased.

7. A reagent or kit for monitoring fetal intrauterine development, characterized in that: It includes at least one pair of primers that specifically amplify PIK3C3, or includes a probe that hybridizes with a PIK3C3 nucleic acid sequence.

8. The reagent or kit according to claim 7, characterized in that The sequences of the primers are shown in SEQ ID NO.1 and SEQ ID NO.

2.

9. The reagent or kit according to claim 7 or 8, characterized in that Also included are RT-PCR reagents or real-time quantitative PCR reagents.

10. A chip for monitoring fetal intrauterine development, characterized in that: The chip comprises a solid phase carrier and an oligonucleotide probe fixed on the solid phase carrier, wherein the oligonucleotide probe comprises a probe hybridizing with a nucleic acid sequence of PIK3C3 for detecting the transcription level of PIK3C3.

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