Use of PIK3C3 as a target for monitoring fetal intrauterine development

Abstract

The present invention relates to the field of biotechnology, specifically to the use of PIK3C3 as a target in monitoring fetal intrauterine development, and specifically to the application of PIK3C3 in monitoring fetal intrauterine developmental abnormalities. The present invention mines and integratively analyzes the sequencing data of maternal blood samples with intrauterine developmental abnormalities, and verifies the high expression of PIK3C3 in samples of fetal intrauterine developmental abnormalities through qPCR experiments designed with specific primers, which makes PIK3C3 applicable to rapid and cost-effective monitoring of fetal intrauterine development.

Description

Technical Field

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

[0002] The earliest stage of human development is the stage of intrauterine development in the mother's body before the baby is born. During this period, the fertilized egg develops into an embryo and then continues to develop into a mature fetus. It takes about 10 months, approximately 280 days, from conception to delivery. Abnormal fetal intrauterine development refers to various abnormal situations that occur during the development of the fetus in the mother's uterus, including growth retardation, structural malformations, and chromosomal abnormalities, etc. The theory of "developmental origins of health and disease" suggests that the growth situation during fetal intrauterine development is highly correlated with perinatal outcomes and even adult health. However, the mechanism of abnormal fetal intrauterine development is intricate and complex, and may be the combined effect of maternal nutrition, placenta, demographic characteristics, and social environmental factors, etc. This has also led to no obvious progress in the research work on abnormal fetal intrauterine development. Given the importance and urgency of the problem of abnormal fetal intrauterine development, its etiology and pathogenesis have always been the research hotspots in the perinatal field and are also difficult problems that need to be solved urgently. Finding a highly sensitive and rapid target for monitoring intrauterine development is extremely urgent.

[0003] Cell autophagy is a highly conserved lysosomal degradation pathway in eukaryotes to maintain the body's metabolic adaptation and energy cycle. Therefore, there must be regulation of cell autophagy during embryonic development and fetal growth. In recent years, it has been found that autophagy plays an important role in the generation and development of the human placenta. Current research has confirmed that cell autophagy is related to pathological pregnancy, and abnormal autophagy can lead to abnormal biological behaviors such as trophoblast proliferation, differentiation, apoptosis, and autophagy, disrupting the balance of the maternal-fetal interface and thus resulting in a series of abnormal fetal intrauterine developments such as fetal growth restriction. The PIK3C3 gene (phosphatidylinositol 3-kinase catalytic subunit type 3), also known as Vps34 or hVps34, is located at region 3, band 12, 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 signal transduction, especially in regulating cell growth, survival, and metabolism. PIK3C3 is an important protein kinase in the process of autophagy initiation formation, and there is currently no relevant report on the relationship between PIK3C3 and fetal intrauterine development. Summary of the Invention

[0004] The object of the present invention is to provide the application of PIK3C3 as a target in the preparation of products for monitoring fetal intrauterine development. The present invention discovers that PIK3C3 is 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 application of PIK3C3 as a target in the preparation of products for monitoring fetal intrauterine development. The specific sequence of PIK3C3 (Gene ID: 5289) of the present invention can be queried in the international public nucleic acid sequence database GeneBank. The present invention discovers that PIK3C3 is significantly differentially expressed in mothers with abnormal fetal intrauterine development and can be used as a molecular marker for abnormal fetal intrauterine development.

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

[0008] As an implementation manner, the present invention detects the expression level of PIK3C3 in the tissue or body fluid of the tested person. If the expression level of PIK3C3 is higher than the normal level, the risk of abnormal fetal intrauterine development is high. The tested person referred to in the present invention means a mother pregnant with a fetus. The sources of PIK3C3 for monitoring fetal intrauterine development in the present invention include but are not limited to the tissue or body fluid of the tested person. The body fluid includes blood, tissue fluid and other in-vivo liquid components containing DNA. In a specific implementation manner of the present invention, the source of PIK3C3 for monitoring fetal intrauterine development is the blood of the tested person. The normal level in the present invention refers to the expression level of PIK3C3 in the tested person pregnant with a normally developing fetus.

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

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

[0011] In the present invention, compared with those with normal fetal intrauterine development, the expression level of PIK3C3 in the tested persons with abnormal fetal intrauterine development increases.

[0012] The present invention also provides a reagent or kit for monitoring fetal intrauterine development, and the reagent or kit includes at least a 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 as shown in SEQ ID NO.1 and SEQ ID NO.2.

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

[0015] As an embodiment, the kit is an in-situ hybridization kit, including a probe hybridizing with the PIK3C3 nucleic acid sequence, and the expression level of PIK3C3 is detected by in-situ hybridization to monitor fetal intrauterine development.

[0016] The present invention also provides a chip for monitoring fetal intrauterine development, and the chip includes a solid-phase carrier and oligonucleotide probes fixed on the solid-phase carrier. The oligonucleotide probes include probes 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 hybridizing with the PIK3C3 nucleic acid sequence can be DNA, RNA, DNA-RNA chimera, PNA or other derivatives. There is no limitation on the length of the probe, and any length can be used as long as specific hybridization is completed and specific binding to the target nucleotide sequence is achieved. The length of the probe can be as short as 25, 20, 15, 13 or 10 base lengths. 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. Since different probe lengths have different effects on hybridization efficiency and signal specificity, the length of the probe is usually at least 14 base pairs, and generally does not exceed 30 base pairs at most, and the length complementary to the target nucleotide sequence is optimally 15-25 base pairs. The self-complementary sequence of the probe is preferably less than 4 base pairs to avoid affecting hybridization efficiency.

[0018] In the context of the present invention, "monitoring fetal intrauterine development" means judging whether the intrauterine development of the fetus of the subject is normal.

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

[0020] Through RNA sequencing, the present invention found that PIK3C3 was significantly differentially expressed in the maternal blood with abnormal intrauterine fetal development. The qPCR experiment was further carried out by designing specific primers to verify the high expression of PIK3C3 in patients with abnormal fetal development. This makes PIK3C3 applicable to the rapid and cost-effective monitoring of early intrauterine fetal development abnormalities.

[0021] The discovery of PIK3C3 helps to understand the mechanism of mRNA in abnormal intrauterine fetal development. At the same time, it also provides a research direction for screening new biomarkers related to abnormal intrauterine fetal development, and provides new targets for the monitoring and intervention treatment of abnormal intrauterine fetal development. Brief Description of the Drawings

[0022] Figure 1 It is a volcano plot of gene expression differences in maternal blood samples with intrauterine growth restriction and normal intrauterine fetal development monitored by RNA sequencing;

[0023] Figure 2 It is a bar chart of Gene Ontology enrichment analysis of differentially expressed genes in maternal blood with intrauterine growth restriction and normal intrauterine fetal development;

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

[0025] Figure 4 It is a statistical chart of the expression difference of PIK3C3 detected by qPCR in maternal blood with intrauterine growth restriction and normal intrauterine fetal development;

[0026] Figure 5 It is an ROC curve graph using the PIK3C3 expression level to evaluate intrauterine growth restriction. Detailed Embodiments

[0027] The present invention performed RNA sequencing analysis on the maternal blood samples of 4 cases with abnormal intrauterine fetal development and 4 cases with normal intrauterine fetal development, and found differentially expressed mRNAs. Through functional enrichment analysis, an mRNA—PIK3C3 related to autophagy gene expression was found, and PIK3C3 was significantly differentially expressed in the maternal blood samples of abnormal intrauterine fetal development and normal intrauterine fetal development. In another 52 cases of maternal blood samples with abnormal intrauterine fetal development and 52 cases of maternal blood samples with normal intrauterine fetal development, the present invention further verified by designing specific primers for qPCR experiments that the expression level of PIK3C3 in the maternal blood of abnormal intrauterine fetal development was significantly higher than that in the maternal blood samples of normal intrauterine fetal development (P<0.05). This enables PIK3C3 to be used as a target prediction marker for monitoring intrauterine fetal development for rapid and cost-effective monitoring of intrauterine fetal development.

[0028] The following further details the technical solutions of the present invention with specific embodiments. The technical solutions of the present invention include but are not limited to the following embodiments.

[0029] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified.

[0030] The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

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

[0032] Table 1 Reagents Used

[0033] reagent company Universal RNA Mini Kit ONREW 1-Bromo-3-chloropropane Sigma-Aldrich Reverse Transcription Kit abmgood PCR Kit KAPA DEPC water Shanghai Beyotime Biotechnology Co., Ltd. absolute ethanol Shanghai Lingfeng Chemical Reagent Co., Ltd.

[0034] Table 2 Equipment Used

[0035]

[0036]

[0037] Example 1

[0038] Screening of Differentially Expressed Genes in Abnormal Intrauterine Fetal Development

[0039] 1. Specimen Source

[0040] The blood sequencing specimens were selected from 4 women with fetal growth restriction and 4 women with normal fetal development who gave natural birth in the hospital. All patients with fetal growth restriction were primiparas and had no hereditary diseases or pregnancy complications before pregnancy.

[0041] 2. RNA Extraction

[0042] 1) The instruments used in the extraction process are soaked in 84 disinfectant solution for one day, then washed with dishwashing detergent, dried, soaked in acid overnight, washed, dried, wrapped with aluminum foil, autoclaved, and baked at 180 °C for 4 hours before RNA extraction can be carried out.

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

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

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

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

[0047] 6) Install 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, transfer all the remaining mixture to the filter column, and centrifuge at 13,000 g for 1 min.

[0049] 8) Discard the filtrate, add 500 μL of BufferW1R to the filter column, and centrifuge at 13,000 g for 10 s.

[0050] 9) Discard the filtrate, add 600 μL of BufferW2R 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 de-enzymed EP tube, add 30 - 100 μL of de-enzymed 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 the quality of extracted RNA: Take 2 μL of the dissolved RNA and use a micro nucleic acid quantifier to detect the concentration and purity of the RNA. For RNA with better quality, the ratio of OD 260 to OD 280 should be between 1.8 and 2.0.

[0055] 3. Transcriptome sequencing: Randomly fragment the extracted RNA into short fragments. Use reverse transcriptase and random hexamer primers to synthesize the first strand of cDNA from the RNA fragments, then use DNA polymerase, RNase H, and dNTPs to synthesize the second strand of cDNA. Finally, use end repair enzymes to repair both ends of the cDNA to make it have complete phosphorylated 3' and 5' ends. Connect adapters containing sequence recognition sites to both ends of the cDNA, amplify by PCR method, use gel electrophoresis or magnetic beads to purify cDNA fragments within a specific size range, and perform sequencing using the Illumina platform.

[0056] 4. Screening of differentially expressed genes: Perform quality control on the sequencing results, use the TMM algorithm in the edgeR software for data normalization, then calculate the p-value according to the hypothesis testing model, and finally perform multiple hypothesis testing correction on the p-value. Screen differentially expressed genes with 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 with intrauterine growth restriction and normal intrauterine development of the fetus. The abscissa represents the fold change in gene expression in different samples, and the ordinate represents the significance level of expression differences. Upregulated genes are shown as red dots, and downregulated genes are shown in green. A total of 2292 differentially expressed mRNA genes were screened, including 657 upregulated genes and 1635 downregulated genes.

[0058] Perform annotation and functional enrichment of the GeneOntology database on the differentially expressed gene data corresponding to the three levels of cellular components, molecular functions, and biological processes. Screen GO terms with p-value less than 0.05 as significantly enriched GO terms, and display the top 10 GO terms with the highest enrichment degree in a bar chart. Figure 2 This is a bar chart of Gene Ontology enrichment analysis of differentially expressed genes in maternal blood with intrauterine growth restriction and normal intrauterine development of the fetus. The abscissa is the specific name of the GO term. The color of the column on the graph corresponds to the three major classifications of the GO database for the GO terms, and the height of the column represents the number of genes mapped to a certain GO term. The genes of the top three GO terms are all related to PIK3C3.

[0059] Statistically analyze the number of differentially expressed genes in each major category of KEGG pathways. Select pathways with a p-value less than 0.05 as significantly enriched pathways, and display the top 10 enriched pathways in a bubble chart. Figure 3 This is an enrichment analysis chart of KEGG Pathways for differentially expressed genes in the maternal blood of fetuses with intrauterine growth restriction and normal intrauterine development. The vertical axis represents the specific KEGG Pathway names, and the horizontal axis represents the enrichment score. The size of the dots represents the number of genes, and the color of the dots corresponds to the magnitude of the -log10(P-value). Figure 3 It shows that the genes related to PIK3C3 have the largest enrichment amount and the most significant difference.

[0060] Example 2

[0061] Verify the differential expression of PIK3C3 in the maternal blood samples of fetuses with growth restriction (FGR)

[0062] In this example, the specimens were selected from 52 women with fetal growth restriction and 52 women with normal fetal development who gave natural birth in the hospital. The pregnant women's ages were 28 - 33 years old. The case screening criteria were the same as in Example 1.

[0063] Extract the RNA from the above-mentioned maternal blood samples using the same method as in Example 1. Perform reverse transcription and qPCR detection on the RNA with good quality obtained, and the methods are as follows:

[0064] Reverse transcribe to synthesize the first strand of cDNA. The reverse transcription conditions are set as: 25°C for 10 min, 42°C for 15 min, 85°C for 5 min, and store at 4°C. The reverse transcription system is: 2 μL of 5×All-In-One RT MasterMix, 500 ng of RNA solution, and make up to 10 μL with RNaseFree H2O.

[0065] The qRT-PCR (QPCR) program is: 95°C for 3 min; 95°C for 3 s, 60°C for 30 s, for a total of 40 cycles. The reaction system is: 10 μL of SYBRPrimix Ex TaqⅡ, 0.4 μL of upstream primer, 0.4 μL of downstream primer, 7.2 μL of dH2O, and 2 μL of cDNA. According to the Ct value of each reaction, use the 2 -△△Ct method to calculate the expression level of the target gene, with ACTIN as the internal reference. 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. Use of a substance for detecting the expression level of PIK3C3 in the preparation of a product for monitoring fetal intrauterine developmental abnormalities, characterized in that, Compared with those with normal intrauterine fetal development, the expression level of PIK3C3 in the blood of the tested subjects with abnormal intrauterine fetal development is increased.

2. The application according to claim 1, characterized in that, The product includes detecting the expression level of PIK3C3 through reverse transcription PCR, real-time quantitative PCR, in situ hybridization, microarray or high-throughput sequencing platform.

3. The application according to claim 1, characterized in that The product is a reagent or kit for monitoring intrauterine fetal development, including at least a pair of primers specifically amplifying PIK3C3, or including a probe hybridizing with the nucleic acid sequence of PIK3C3.

4. The application according to claim 3, wherein The sequences of the primers are as shown in SEQ ID NO.1 and SEQ ID NO.

2.

5. The application according to claim 3 or 4, characterized in that, It also includes RT-PCR reagent or real-time quantitative PCR reagent.

6. The application according to claim 1, wherein The product is a microarray for monitoring intrauterine fetal development. The microarray includes a solid-phase carrier and oligonucleotide probes fixed on the solid-phase carrier. The oligonucleotide probes include probes hybridizing with the nucleic acid sequence of PIK3C3 for detecting the transcription level of PIK3C3.

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