Application of HMGB3 gene for regulating and controlling HLA-G expression in maternal and fetal immune tolerance

Abstract

The invention relates to the technical field of gene engineering, and particularly discloses application of an HMGB3 gene to regulation of HLA-G expression in maternal and fetal immune tolerance. The maternal and fetal immune tolerance level is evaluated by detecting the expression quantity of the HMGB3 gene, the operation is simple and rapid, the required detection samples are few and are easy to obtain, and the method has certain theoretical significance in research on the maternal and fetal immune tolerance mechanism.

Description

Technical Field

[0001] The present application relates to the field of genetic engineering technology, and in particular to a use of HMGB3 gene to regulate HLA-G expression in maternal-fetal immune tolerance. Background Art

[0002] During early human pregnancy, the endometrium undergoes decidual changes and the fetal placenta implants in the decidua. During this period, the trophectoderm of the blastocyst rapidly proliferates and produces trophoblasts (cytotrophoblasts, CTB). Trophoblasts differentiate into two major subpopulations: syncytiotrophoblasts (SCT) and extravillous trophoblasts (EVT). SCT is a multinucleated epithelial cell in the villi responsible for the exchange and production of hormones, and EVT fixes the placenta to the maternal decidua and transforms into maternal spiral arteries. Abnormal differentiation of CTB into EVT can lead to insufficient vascular remodeling and insufficient placental blood supply, leading to the occurrence of some pregnancy complications, such as preeclampsia, maternal hypertension, fetal growth restriction, premature birth or late miscarriage.

[0003] Human leucocyte antigen-G (HLA-G) is a non-classical major histocompatibility complex class I molecule that is only expressed on EVT. HLA-G plays a core role in the interaction between EVT and iris natural killer cells (dNK) and is considered to be one of the key maternal-fetal immune tolerance molecules. During the implantation process, EVT specifically expressed by HLA-G invades maternal tissues, thereby providing dNK cell tolerance and antiviral immune function between dNK and EVT due to HLA-G. For trophoblasts, HLA-G inhibits the cytotoxicity of dNK cells and protects trophoblasts from dNK cell-induced lysis.

[0004] In the prior art, maternal-fetal immune tolerance levels are assessed by measuring cytokine levels. However, there are many factors that affect cytokine levels, resulting in inaccurate assessment results. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the purpose of the present application is to provide a use of the HMGB3 gene to regulate HLA-G expression in maternal-fetal immune tolerance, aiming to solve the problem that the methods for evaluating the level of maternal-fetal immune tolerance in the prior art are relatively complex and the results are not accurate enough.

[0006] The technical solution of this application is as follows:

[0007] In a first aspect of the present application, there is provided a use of the HMGB3 gene to regulate HLA-G expression in maternal-fetal immune tolerance.

[0008] Optionally, the HMGB3 gene affects the level of maternal-fetal immune tolerance by regulating HLA-G expression.

[0009] The second aspect of the present application provides a use of the HMGB3 gene in preparing a kit for detecting maternal-fetal immune tolerance levels.

[0010] Optionally, the maternal-fetal immune tolerance level is assessed by detecting the expression level of the HMGB3 gene.

[0011] Optionally, the HMGB3 gene expression level is detected by real-time fluorescence quantitative PCR.

[0012] Optionally, the HMGB3 gene affects the maternal-fetal immune tolerance level by regulating the expression level of HLA-G.

[0013] In a third aspect of the present application, a kit for detecting maternal-fetal immune tolerance level is provided, wherein the product for assessing maternal-fetal immune tolerance level comprises a reagent for detecting the expression amount of HMGB3 gene.

[0014] Compared with the prior art, this application has the following advantages:

[0015] The present application conducts bioinformatics analysis on the differential gene expression of trophoblasts and extravillous trophoblasts, obtains that the HMGB3 gene can regulate the expression of HLA-G, and then proposes a use of the HMGB3 gene to regulate HLA-G expression in maternal-fetal immune tolerance. The maternal-fetal immune tolerance level is detected by detecting the expression of the HMGB3 gene. The operation is simple and quick, and the number of test samples required is small. It has certain theoretical significance for the study of the mechanism of maternal-fetal immune tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for describing the embodiments are briefly introduced below.

[0017] Figure 1 Gene differential expression analysis of trophoblasts and extravillous trophoblasts provided in the examples of this application:

[0018] A is a cluster analysis diagram of trophoblast stem cells and extravillous trophoblast cells; B is a distribution diagram of HMGB3 gene in trophoblast stem cells; C is a diagram of HLA-G and HMGB3 expression in primary human villous trophoblast cells and extravillous trophoblast cells; D is a diagram of HLA-G and HMGB3 expression in trophoblast stem cells and extravillous trophoblast cells; E is a gel electrophoresis analysis diagram of HMGB3 protein expression in trophoblast stem cells and extravillous trophoblast cells;

[0019] Figure 2 This is an analysis diagram of the effect of HMGB3 gene knockout and HMGB3 gene overexpression on HLA-G expression after TSC cells differentiated into EVTs provided in the examples of the present application:

[0020] A is an analysis graph of HLA-G expression in trophoblast stem cells overexpressing the HMGB3 gene and wild-type trophoblast stem cells; B is an analysis graph of HLA-G expression in trophoblast stem cells with HMGB3 knockout and wild-type trophoblast stem cells; C is a fluorescence analysis graph of HLA-G protein expression in trophoblast stem cells overexpressing the HMGB3 gene and wild-type trophoblast stem cells; D is a fluorescence analysis graph of HLA-G protein expression in trophoblast stem cells with HMGB3 knockout and wild-type trophoblast stem cells; E is a graph of DNA binding to the HLA-G promoter region in wild-type and overexpressed HMGB3 genes in EVT cells. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the drawings and embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the protection scope of the present application. In the absence of conflict, the following embodiments and features in the embodiments can be combined with each other.

[0022] It should be noted that if there are descriptions involving "first", "second", etc. in the implementation of this application, the descriptions of "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or suggesting their relative importance and implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the fact that ordinary technicians in the field can implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by this application.

[0023] The present application embodiment provides a use of HMGB3 gene to regulate HLA-G expression in maternal-fetal immune tolerance, wherein the HMGB3 gene regulates HLA-G expression, thereby affecting the level of maternal-fetal immune tolerance.

[0024] The present application example also provides an application of the HMGB3 gene in preparing a kit for detecting maternal-fetal immune tolerance levels.

[0025] In some embodiments, the maternal-fetal immune tolerance level is assessed by detecting the expression level of the HMGB3 gene.

[0026] In some embodiments, the expression level of the HMGB3 gene is detected by real-time fluorescence quantitative PCR.

[0027] In some embodiments, the HMGB3 gene affects the level of maternal-fetal immune tolerance by regulating the expression of HLA-G.

[0028] The HMGB3 gene is a member of the high mobility group protein B (HMGB) family. The HMGB family includes four subtypes, namely HMGB1, HMGB2, HMGB3 and HMGB4, which are mainly involved in regulating DNA replication, transcription, recombination and repair mechanisms. HMGB3 is a member of the X cluster (XQ28) of the chromatin binding protein family and is located in the nucleus, cytoplasm and chromosomes. The HMG family is the second most abundant chromatin protein family in eukaryotic cells after histones. It can interact with chromatin structures (nucleosomes, transcription factors, nucleosome remodeling complexes and histone H1) and adapt to the extracellular environment by regulating the transcription process. HMGB3 is closely related to embryo implantation. Through bioinformatics analysis of the differential gene expression of trophoblasts and extravillous trophoblasts, it was found that the HMGB3 gene can regulate the expression of HLA-G. HLA-G plays a core role in the interaction between EVT and iris natural killer cells (decidual Natural Killer cells, dNK) and is considered to be one of the key maternal-fetal immune tolerance molecules. The HMGB3 gene is then used as a marker to prepare a maternal-fetal immune tolerance level detection kit.

[0029] The present application also provides a kit for detecting maternal-fetal immune tolerance level, which includes a reagent for detecting the expression level of the HMGB3 gene.

[0030] The following is further described through specific implementation methods.

[0031] Example 1

[0032] Construction of trophoblast organoids and stem cell differentiation system. In this example, trophoblast organoids and stem cell differentiation systems were constructed to obtain trophoblast cells (CTB) and extravillous trophoblast cells (EVT) for subsequent analysis of differential gene expression.

[0033] (1) Chorionic villus tissues from normal early pregnant women undergoing voluntary abortion were collected to construct stably growing trophoblast organoids (TOs) and differentiate them into extravilloustrophoblast organoids (EVTOs), forming a systematic organoid identification technology. The specific steps include:

[0034] Wash the placental tissue in a culture dish to remove blood clots, scrape the villi with a scalpel, resuspend the digestion solution in matrix gel after secondary enzyme digestion, and then put it into a 48-well plate and add trophoblast organoid culture medium. Cell clusters can be seen on the 6th day, and a small amount of trophoblast organoids appear on the 11th day. When the diameter of the trophoblast organoids reaches 150-200μm, they are passaged, and an appropriate amount of organoids are wrapped in matrix gel and added to the culture dish. After culturing in EVT medium (EVT medium, EVTM) for 10 days, adherent EVT cells are obtained, which are trophoblast organoids. CTB can be further obtained from the trophoblast organoids for analysis of differential gene expression.

[0035] (2) Expanded potential stem cells (EPSCs) are cells of the pre-implantation embryonic stage, i.e., the 4-cell and 8-cell stage, that have the potential to develop into extraembryonic cells. Human expanded potential stem cells (hEPSCs) can directly generate human trophoblast stem cells (hTSCs) in vitro. hTSCs are induced into EVT cells using differentiation medium, which can be used to identify changes in known markers. The specific steps include:

[0036] hEPSCs were cultured and passaged on a mouse embryonic fibroblast feeder layer (SNL76 / 7). After two weeks of culture of hEPSCs in trophoblast stem cell culture medium, TSC-like cell clumps were collected and further cultured. hTSCs were obtained after about 10 passages. hTSCs were cultured in EVT induction medium (EVTM) for 8 days, and hTSCs were induced to generate mesenchymal EVTs. The obtained EVTs can be used for analysis of differential gene expression.

[0037] Example 2

[0038] Single-cell sequencing technology (scRNA-seq) was used to analyze the expression of differentially expressed genes in CTB and EVT.

[0039] (1) The cell suspension prepared in Example 1 (trophoblast organoids and mesenchymal morphology of EVT) was digested with Accutase enzyme, the cell suspension was added to the amplification reagent, and the library was constructed using the NEXT GEM Chip G Single Cell Kit. The oil-in-water structure was prepared by Chromium Controller, reverse transcription amplification was performed, and sequencing analysis was performed. The sequencing library was quality controlled by 2100 Bioanalyzer.

[0040] (2) Analyze the sequencing results obtained in (1). First, obtain the cell-gene expression matrix, use Seurat for cell quality control and gene filtering, then perform linear dimensionality reduction (RunPCA function), clustering (FindNeighbors and FindClusters functions), differential expression analysis (FindAllMarkers function) and nonlinear dimensionality reduction analysis, and finally perform cell trajectory analysis (monocle3) and cell-cell interaction analysis (cellPhoneDB). The results of differential expression analysis are shown in Figure 2. Figure 1 As shown in C, D and E, from Figure 1 As can be seen from Figure C, the expression levels of HLA-G gene and HMGB3 gene in EVT cells are significantly higher than those in CTB cells, indicating that the expression of HMGB3 gene in EVT cells is higher than that in CTB cells; Figure 1 As can be seen from D and E, in TSC-EVT cells induced by hTSCs, the expression levels of HMGB3 gene and HLA-G gene were also higher than those in TSC cells, indicating that the expression level of HMGB3 gene in EVT cells increased.

[0041] (3) Use GENIE3 to infer the co-expression modules between transcription factors and candidate target genes based on co-expression, and perform cis-regulatory motif analysis on each co-expression module. First, perform enrichment analysis on the transcription factor binding domain (TF-motif) to identify direct targets. Each TF and its potential target gene is called a regulator. The AUCell algorithm is then used to score the activity of each regulator in each cell. Comparing the AUCell scores of a regulator between different cells identifies which cells have significant regulatory activity. The final result generates a binary regulator activity matrix, as shown in the following figure. Figure 1 As shown in A and B. Figure 1 As can be seen from A and B, the HMGB3 gene can regulate the expression of HLA-G in extravillous trophoblasts.

[0042] Example 3

[0043] Trophoblast stem cells with HMGB3 gene knockout or overexpression were obtained, and the expression of HLA-G in extravillous trophoblast cells was analyzed after they differentiated into extravillous trophoblast cells.

[0044] (1) Small guide RNA (sgRNA) was designed to knock out the HMGB3 gene in TSC cells, and a stable knockout strain was constructed in trophoblast stem cells using CRUSPR-Cas9 technology. The knockout effect was identified by PCR and Western blot. Finally, trophoblast stem cells with HMGB3 gene knockout (HMGB3-KO) and trophoblast stem cells overexpressing HMGB3 (HMGB3-OE) were obtained.

[0045] (2) Wild-type trophoblast stem cells (HMGB3-WT), HMGB3-KO cells, and HMGB3-OE cells were subjected to EVT-induced differentiation, and the expression of HLA-G in HMGB3-WT cells, HMGB3-KO cells, and HMGB3-G cells was analyzed by Q-PCR, Western blot, and immunofluorescence staining. Figure 2 As shown in A and B, from Figure 2 As can be seen from Figure A, the expression level of HLA-G in trophoblast stem cells overexpressing the HMGB3 gene (HMGB3-OE) is significantly higher than that in wild-type trophoblast stem cells (HMGB3-WT cells). Figure 2 As can be seen from B, the expression level of HLA-G in HMGB3-knockout trophoblast stem cells (HMGB3-KO) is significantly lower than that in wild-type trophoblast stem cells (HMGB3-WT cells), which further illustrates the effect of HMGB3 gene on the expression of HLA-G.

[0046] (3) Chromatin immunoprecipitation (CHIP) was used to verify the binding of HMGB3 to HLA-G genomic DNA. The specific experimental steps were as follows: the cell samples were cross-linked, lysed, and ultrasonically disrupted before immunoprecipitation with HMGB3 antibodies. The cross-links were then reversed and the enriched DNA was purified and analyzed by agarose gel electrophoresis. Figure 2 As shown in E. Combined Figure 2 As can be seen in E, the overexpressed HMGB3 gene binds more strongly to the HLA-G genomic DNA.

[0047] Based on the results of Example 2 and Example 3, it can be seen that during the differentiation of CTB to EVT, the HMGB3 gene can regulate the expression of HLA-G in cells.

[0048] In summary, the present application experimentally detects the expression level of the HMGB3 gene during the differentiation of trophoblasts into extravillous trophoblasts, and combined with bioinformatics analysis, finds that the HMGB3 gene can regulate the expression of HLA-G, and then the expression of HLA-G can be analyzed by detecting the expression of the HMGB3 gene, and the maternal-fetal immune tolerance level can be further detected. The operation is simple and fast, and the required test samples are small and easy to obtain.

[0049] It should be understood that the application of the present application is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to this application.

Claims

1. A use of HMGB3 gene to regulate HLA-G expression in maternal-fetal immune tolerance.

2. The use of HMGB3 gene to regulate HLA-G expression in maternal-fetal immune tolerance according to claim 1, characterized in that: The HMGB3 gene affects the maternal-fetal immune tolerance level by regulating HLA-G expression.

3. Application of HMGB3 gene in the preparation of a kit for detecting maternal-fetal immune tolerance level.

4. The use according to claim 3, characterized in that: The maternal-fetal immune tolerance level is evaluated by detecting the expression level of the HMGB3 gene.

5. The use according to claim 4, characterized in that: The HMGB3 gene expression level was detected by real-time fluorescence quantitative PCR.

6. The use according to claim 3, characterized in that: The HMGB3 gene affects the maternal-fetal immune tolerance level by regulating the expression level of HLA-G.

7. A maternal-fetal immune tolerance level detection kit, characterized in that: The maternal-fetal immune tolerance level detection kit comprises a reagent for detecting the expression amount of the HMGB3 gene.

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