Innovative screening method for key genes of intrahepatic cholangiocarcinoma with vascular invasion and application thereof
By integrating single-cell and conventional transcriptome data, and combining them with cellular functional phenotype validation, key genes for vascular invasion in intrahepatic cholangiocarcinoma were screened, overcoming the shortcomings of existing screening strategies and enabling precise treatment of intrahepatic cholangiocarcinoma.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- THE AFFILIATED SIR RUN RUN SHAW HOSPITAL OF SCHOOL OF MEDICINE ZHEJIANG UNIV
- Filing Date
- 2026-05-12
- Publication Date
- 2026-06-12
AI Technical Summary
Existing technologies lack key gene screening strategies for vascular invasion in intrahepatic cholangiocarcinoma, making it difficult to systematically depict tumor microenvironment differences at single-cell resolution. Furthermore, the lack of in vitro validation procedures makes it impossible to accurately locate key molecules driving vascular invasion.
By integrating single-cell transcriptome and conventional transcriptome data, specific cell subpopulations enriched in intrahepatic cholangiocarcinoma tissues were screened through cell clustering and functional phenotype validation. Key genes were screened in vitro using inhibition or knockout strategies, and their functions were further validated in vivo using the budding ability test of human umbilical vein endothelial cells.
The key genes driving vascular invasion in intrahepatic cholangiocarcinoma were precisely identified, providing new targeted therapeutic targets that can effectively inhibit the growth and vascular invasion process of intrahepatic cholangiocarcinoma.
Smart Images
Figure CN122201425A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, specifically to an innovative screening method and application for key genes involved in vascular invasion of intrahepatic cholangiocarcinoma, based on multi-omics data integration and in vivo phenotypic verification. Background Technology
[0002] Intrahepatic cholangiocarcinoma (ICC) is the second most common primary malignant liver tumor after hepatocellular carcinoma. In recent years, its global incidence has shown a significant upward trend. ICC has an insidious onset and is highly aggressive; most patients are diagnosed at a locally advanced stage or with distant metastases, losing the opportunity for radical surgical resection. Although gemcitabine-based chemotherapy and immunotherapy have provided some survival benefits, the overall prognosis remains extremely poor for patients with unresectable or metastatic ICC, with a short median survival. Vascular invasion (VI), especially microvascular invasion, has been proven to be a key independent risk factor for assessing tumor invasiveness, predicting early postoperative recurrence, and poor prognosis. In ICC, vascular invasion mainly involves the portal vein and hepatic venous system and is closely associated with shortened overall survival and disease-free survival. Therefore, in-depth research into the specific cell populations and key molecular mechanisms of vascular invasion in ICC has significant clinical translational implications for finding new therapeutic targets and improving patient management strategies.
[0003] The tumor microenvironment plays a central driving role in the progression and vascular invasion of intracellular carcinoma (ICC). Cancer-associated fibroblasts (CAFs) are the most abundant cellular component of the stroma in solid tumors, profoundly influencing tumor metastasis and angiogenesis. In recent years, the application of single-cell transcriptome sequencing technology has greatly advanced research on ICC heterogeneity and microenvironmental composition. Existing studies have revealed the interaction between specific CAF subsets and tumor cells in ICCs, and some researchers have used specific biomarkers to molecularly subtype ICCs. However, a systematic description of the differences in the tumor microenvironment at single-cell resolution for ICC patients with and without vascular invasion is currently lacking, as are precise screening strategies for key functional genes driving vascular invasion. Summary of the Invention
[0004] The purpose of this invention is to address the problems in existing technologies where the screening of key molecules in ICC is often limited to simple transcriptome sequencing comparisons, making it difficult to locate specific key cellular subpopulations and their effector molecules that drive vascular invasion in the microenvironment; or relying solely on single-cell data to describe heterogeneity, lacking a systematic validation process for subsequent angiogenesis functional phenotypes. This invention provides an innovative method that combines single-cell resolution with tissue-level transcriptome differences and performs reverse screening validation through specific angiogenesis functional phenotypes. This method aims to accurately identify key genes involved in vascular invasion in ICC, providing new drug targets for targeted therapy of ICC.
[0005] This invention integrates multi-omics data from single-cell transcriptomics and general transcriptomics to precisely locate candidate genes enriched in specific cell subpopulations within the microenvironment of intrahepatic cholangiocarcinoma (ICC) with vascular invasion. It utilizes in vitro endothelial cell budding function assays as the core screening phenotype, thereby efficiently and accurately identifying key genes driving vascular invasion in ICC. Furthermore, this invention provides the application of key genes obtained based on this screening method in the precision treatment of ICC.
[0006] The technical solution adopted in this invention is as follows:
[0007] An innovative screening method for key genes involved in vascular invasion of intrahepatic cholangiocarcinoma includes:
[0008] Single-cell transcriptome data of intrahepatic cholangiocarcinoma tissues with and without vascular invasion were obtained. Through cell clustering and annotation analysis, specific cell subpopulations significantly enriched in intrahepatic cholangiocarcinoma tissues with vascular invasion were identified, and sets of genes specifically highly expressed in these specific cell subpopulations were screened.
[0009] We obtained conventional transcriptome data from intrahepatic cholangiocarcinoma tissues with and without vascular invasion, and through comparative analysis, screened out differentially expressed genes that were significantly upregulated in intrahepatic cholangiocarcinoma tissues with vascular invasion.
[0010] Intersection analysis was performed between the set of highly expressed genes from single-cell transcriptomes and the set of differentially expressed genes from ordinary transcriptomes to extract key common candidate genes.
[0011] A suppression or knockdown strategy was designed for the common gene, and cell phenotype screening experiments were conducted in vitro. The cell phenotype screening experiments included the detection of the budding ability of human umbilical vein endothelial cells (HUVECs). Based on the screening results, genes that can significantly reduce the budding ability of HUVECs were selected, which are the key genes for vascular invasion in intrahepatic cholangiocarcinoma.
[0012] Furthermore, single-cell transcriptome data analysis of intrahepatic cholangiocarcinoma tissues with and without vascular invasion was conducted, specifically including:
[0013] Unsupervised clustering and cell type annotation were performed based on single-cell transcriptome data.
[0014] By comparing the cell composition ratios between intrahepatic cholangiocarcinoma tissue samples with and without vascular invasion, specific cell subpopulations with significantly enriched cell abundance in tissues with vascular invasion were identified.
[0015] Analyze the differential gene expression characteristics and functions of this specific cell subpopulation, and screen out the highly expressed genes that are specifically upregulated in this subpopulation.
[0016] Furthermore, the specific procedures for designing a suppression or knockout strategy for the shared gene and conducting in vitro cell phenotype screening experiments include:
[0017] Based on the common gene sequences obtained through screening, specific small molecule inhibitors are selected or corresponding short hairpin RNAs (shRNAs) are designed and synthesized.
[0018] The specific cells enriched in intrahepatic cholangiocarcinoma tissue with vascular invasion, as identified by single-cell transcriptome analysis, were treated using the above-mentioned inhibition or knockout methods, and the conditioned medium of the treated specific cells was collected.
[0019] The conditioned medium was used to culture HUVECs, and changes in the budding ability of HUVECs were detected by in vitro angiogenesis experiments, thereby screening out function-related genes.
[0020] Furthermore, the method also includes:
[0021] The key gene that can reduce the budding ability of HUVECs, which was screened by in vitro angiogenesis experiments, was further functionally validated in an in vivo animal model. By inhibiting the expression of this key gene, its effect on the growth of intrahepatic cholangiocarcinoma tumors and intrahepatic metastasis was observed, so as to confirm that targeting this key gene can inhibit the progression and vascular invasion of intrahepatic cholangiocarcinoma.
[0022] Furthermore, the key gene for vascular invasion in the intrahepatic cholangiocarcinoma is SLC2A1.
[0023] An innovative screening system for key genes involved in vascular invasion of intrahepatic cholangiocarcinoma, used to implement the innovative screening method for key genes involved in vascular invasion of intrahepatic cholangiocarcinoma, comprising:
[0024] The single-cell transcriptome analysis module is used to acquire and analyze single-cell transcriptome data of intrahepatic cholangiocarcinoma tissues with and without vascular invasion, identify specific cell subpopulations enriched in tissues with vascular invasion, and screen for genes specifically highly expressed in these specific cell subpopulations.
[0025] The general transcriptome analysis module is used to acquire and analyze general transcriptome data of intrahepatic cholangiocarcinoma tissues with and without vascular invasion, and to screen differentially expressed genes that are upregulated in tissues with vascular invasion.
[0026] Shared Gene Extraction Module: This module performs an intersection operation on the outputs of the single-cell transcriptome analysis module and the general transcriptome analysis module to extract a list of key shared candidate genes.
[0027] Cell phenotype screening and validation module: used to design inhibition or knockdown strategies for common genes and perform endothelial cell budding ability detection experiments to identify key genes based on changes in budding ability.
[0028] An innovative screening method for key genes involved in vascular invasion of intrahepatic cholangiocarcinoma. Applications of the key genes obtained from this screening include:
[0029] To create a single-cell atlas of vascular invasion in intrahepatic cholangiocarcinoma;
[0030] Specifically, specific cell subpopulations enriched in tissues with vascular invasion were identified and mapped into a single-cell atlas of vascular invasion in intrahepatic cholangiocarcinoma.
[0031] Investigating the mechanisms of vascular invasion in intrahepatic cholangiocarcinoma;
[0032] To prepare drugs for the treatment of intrahepatic cholangiocarcinoma.
[0033] Furthermore, the drug for treating intrahepatic cholangiocarcinoma was prepared as BAY-876, a specific inhibitor of SLC2A1.
[0034] The beneficial effects of this invention are: this invention proposes an innovative screening strategy for the discovery of key genes in the process of vascular invasion in intrahepatic cholangiocarcinoma (ICC), in order to provide a useful idea for the precision treatment of ICC. At the same time, this invention provides a drug for the preparation of treatment for intrahepatic cholangiocarcinoma. Attached Figure Description
[0035] Figure 1 This is a flowchart illustrating the innovative screening method for key genes involved in vascular invasion of intrahepatic cholangiocarcinoma according to the present invention.
[0036] Figure 2 This is a UMAP diagram of cell clustering and the distribution and enrichment of specific cell subpopulations in the two groups of single-cell transcriptome analysis of 6 cases of intrahepatic cholangiocarcinoma tissue samples with vascular invasion and 19 cases without vascular invasion, as shown in the embodiments of the present invention. Figure 2 In this diagram, A represents the cell type atlas of single-cell clustering. Figure 2 In the diagram, B represents the cell proportion distribution in the positive and negative groups for vascular invasion. Figure 2 C in the figure represents the tissue enrichment index of the six major cell types. Figure 2 In the diagram, D represents the cell subpopulation map of fibroblast clustering. Figure 2 E in the figure represents the tissue enrichment index of fibroblast subsets;
[0037] Figure 3 It is a list of candidate genes that are significantly overexpressed in a specific enriched cell subpopulation compared to other cell subpopulations;
[0038] Figure 4 This is a differential gene volcano plot between intrahepatic cholangiocarcinoma tissues with and without vascular invasion in ordinary transcriptome data;
[0039] Figure 5 This is a Venn diagram showing the number of genes commonly expressed and the overlap between the two data sources: one from a single-cell transcriptome and the other from a general transcriptome.
[0040] Figure 6 This is a graph showing the effect of conditioned medium on the budding ability of HUVECs after treating specific intrahepatic cholangiocarcinoma cells with an inhibitor designed for a common gene in step three of this invention. Figure 6 Figure A in the image represents a representative microscopic result showing the effect of conditioned medium on the budding ability of HUVECs. Figure 6 B in the figure is a statistical graph showing the total length of all germinations after HUVECs were treated with conditioned medium in each group; Figure 6 C in the figure represents the statistical chart of the number of all germinations after HUVECs were treated with conditioned medium in each group;
[0041] Figure 7 This is a diagram showing the effect of conditioned medium on the budding ability of HUVECs after knocking down specific intrahepatic cholangiocarcinoma cells with shRNA designed for a common gene in step three of this invention. Figure 7 Figure A in the image represents a representative microscopic result showing the effect of conditioned medium on the budding ability of HUVECs. Figure 7 B in the figure represents the total length of all buds and the number of all buds after HUVECs were treated with conditioned medium in each group.
[0042] Figure 8 This is a flowchart of the animal experiment used in step four of this invention;
[0043] Figure 9 This is a comparison chart of liver and tumor size in two groups at the end of the animal experiment in step four of this invention (20 days after treatment);
[0044] Figure 10 This is a statistical graph showing the ratio of liver weight to body weight (A) and tumor weight (B) of the two groups of mice at the end of the animal experiment in step four of this invention.
[0045] Figure 11 The images shown are (A) actual photos of the liver metastases of the two groups of tumors at the end of the animal experiment in step four of this invention, and (B) a statistical chart of the number of liver metastases. Detailed Implementation
[0046] The present invention will be further described below with reference to specific embodiments.
[0047] This invention provides an innovative screening method for key genes involved in vascular invasion of intrahepatic cholangiocarcinoma, such as... Figure 1 As shown, it includes the following steps:
[0048] Step 1: Obtain single-cell transcriptome data of intrahepatic cholangiocarcinoma tissues with and without vascular invasion, identify specific cell subpopulations enriched in intrahepatic cholangiocarcinoma tissues with vascular invasion, and screen for genes specifically highly expressed in these cell subpopulations.
[0049] In one specific implementation, the present invention first obtained raw single-cell transcriptome sequencing data from tumor tissues of 6 patients with intrahepatic cholangiocarcinoma exhibiting vascular invasion and 19 patients without vascular invasion from public databases. The raw reads were aligned to the human reference genome GRCh38 using CellRanger (v.3.1) software, and downstream analysis was performed using the Seurat (v5.1.0) R package. The specific implementation steps are as follows:
[0050] 1) Quality Control: The total UMI of a single cell is less than 60,000; the number of detected genes is between 500 and 10,000; the percentage of mitochondrial genes is less than 25%, and the percentage of hemoglobin genes is less than 1%. After quality control, the data is normalized, principal component analysis is performed using the RunPCA() function for dimensionality reduction, and the RunUMAP() function is used for visualization. Cell clustering is performed using the FindClusters() function with a resolution parameter set to 0.4.
[0051] 2) Cell Annotation: First, six major cell types were identified based on classical marker genes: NK / T cells (KLRD1 / CD3E), epithelial cells (EPCAM), myeloid cells (LYZ), fibroblasts (ACTA2), B cells (CD79A), and endothelial cells (VWF). Subsequently, a second round of subpopulation subdivision was performed for each major cell type. For example, fibroblasts were divided into 9 CAF subpopulations and 1 pericyte subpopulation at a resolution of 0.3. Figure 2 ).
[0052] 3) Tissue bias analysis: The tissue enrichment index (Ro / e) was calculated using the STARTRAC R package. A Ro / e value greater than 1 indicates that the cell subpopulation is significantly enriched in a specific tissue. The analysis results showed that a specific fibroblast subpopulation (tumor-like fibroblasts) was significantly enriched in intrahepatic cholangiocarcinoma tissue with vascular invasion (Ro / e value > 1). Further analysis of differentially expressed genes in this specific CAF subpopulation using the FindAllMarkers() function identified 912 genes specifically highly expressed in this subpopulation, forming candidate high-expression gene set A. Figure 3 List the top 10 genes.
[0053] Step 2: Obtain ordinary transcriptome data of intrahepatic cholangiocarcinoma tissues with and without vascular invasion, screen for differentially expressed genes upregulated in tissues with vascular invasion, and take the intersection with the set of highly expressed genes from Step 1.
[0054] In one specific implementation, this invention obtained conventional transcriptome data from independent cohorts of intrahepatic cholangiocarcinoma tissues with and without vascular invasion. Differential expression analysis was performed using the DESeq2 R package, and 113 genes significantly upregulated in tissues with vascular invasion were selected as differentially expressed gene set B, with |log2FoldChange| > 1 and P < 0.05 as the criterion. Figure 4 Subsequently, the intersection analysis of set A and set B of specific cells with high expression of genes obtained in step one was performed to extract 15 common genes. Based on GO and KEGG functional enrichment analysis and literature screening, the final three candidate key genes PTGS2, SLC2A1 and PLOD2 were obtained. Figure 5 These shared genes are not only enriched and expressed in specific microenvironment cells of vascular invasion tissues, but also show an upregulated trend at the overall tissue level, thus possessing strong functional indicative significance.
[0055] Step 3: Design knockdown or repression strategies for common candidate genes, conduct cell phenotype screening experiments in vitro, and identify key genes using the budding ability of human umbilical vein endothelial cells (HUVECs) as the core detection indicator.
[0056] In one specific implementation, this invention targets the common genes screened in step two and treats ICC-derived CAF cells with small molecule inhibitors (Acetaminophen (Ace), BAY-876 (BAY), and Minoxidil (Min)) of three candidate genes PTGS2, SLC2A1, and PLOD2. The conditioned medium (supernatant from ICC-derived CAF cell culture, including culture medium, CAF secretions, etc.) is collected for subsequent functional experiments. This invention designs and synthesizes the corresponding shRNA sequences for the candidate genes. Table 1 shows the shRNA target sequence information for some of the candidate genes. The shRNA plasmid is transfected into HEK 293T cells using a lentiviral packaging system. The viral suspension is collected and used to infect the primary specific CAF cells of intrahepatic cholangiocarcinoma isolated and cultured in step one. After obtaining stable knockdown CAF cell lines through drug screening, their conditioned medium is collected for subsequent functional experiments.
[0057] Table 1. Specific sequences of shRNA (only 6 types are shown).
[0058]
[0059] The method for detecting the budding ability of HUVECs is as follows:
[0060] After digesting and resuspending HUVECs, 80,000 cells were added to 4 mL of fresh complete culture medium, followed by the addition of 1 mL of methylcellulose to prepare a cell suspension. 25 μL of the mixture was added dropwise to a 10 cm² culture dish and incubated for 24 hours to form spheroids. Once the spheroids were visible under a microscope, they were gently separated and centrifuged at 200 × g for 5 minutes to form a precipitate. The spheroid precipitate was resuspended in 2 mL of methylcellulose. Next, 4 mL of collagen stock solution was mixed with endothelial cell culture medium on ice. Then, 2 mL of the collagen solution was added to the spheroid suspension in methylcellulose. The mixture was added to 24-well plates at 1 mL per well and incubated at 37°C with 5% CO2 for 30 minutes. Subsequently, the cells were divided into four groups of 14 wells each. Three groups received 100 μL of the above-mentioned specific CAF cell conditioned medium treated with different inhibitors in each well, while one group received 100 μL of specific CAF cell conditioned medium treated with DMSO as a control. Incubation continued for 24 hours. The budding of HUVECs in each group was observed and photographed under an inverted microscope, and the number of buds and total bud length were counted. Based on the inhibitor screening results (excluding the PTGS2 gene), key candidate genes were further identified and divided into 3 groups of 9 wells each. Two groups had 100 μL of specific CAF cell conditioned medium treated with the aforementioned knockdown genes (SLC2A1 and PLOD2) added to each well, while the other group had 100 μL of negative control CAF cell conditioned medium added. The cells were cultured for another 24 hours. The budding of HUVECs in each group was observed and photographed under an inverted microscope, and the number of buds and total bud length were counted. The screening results are as follows: Figure 6 and Figure 7 As shown. By comprehensively evaluating the degree of inhibition of the budding ability of HUVECs after the suppression or knockdown of each candidate gene, the gene with the most significant inhibitory effect was finally identified as SLC2A1, a key gene for vascular invasion in intrahepatic cholangiocarcinoma.
[0061] Step 4: The selected key genes were verified in vivo through animal experiments to confirm that targeting these key genes can inhibit the progression and vascular invasion of intrahepatic cholangiocarcinoma.
[0062] To verify the effectiveness of the screening strategy and the in vivo function of key genes, in vivo animal experiments were conducted on the screened key gene SLC2A1, such as... Figure 8 As shown, the specific implementation steps are as follows:
[0063] Intrahepatic cholangiocarcinoma tumor tissue was collected from male C57BL / 6 mice confirmed by CK19 immunohistochemical staining. The tumor tissue was cut into small pieces of approximately 1 mm³ and implanted orally into the livers of recipient C57BL / 6 mice to establish an orthotopic intrahepatic cholangiocarcinoma mouse model. On the third day post-surgery, mice were randomly divided into a treatment group (BAY-876) and a control group, with eight mice in each group. The treatment group received a specific inhibitor of the key gene (SLC2A1 inhibitor BAY-876, 3 mg / kg) via gavage daily, while the control group received an equal volume of physiological saline. The experimental procedure is as follows: Figure 8 As shown. The experimental endpoint results showed a significant reduction in the liver-to-body weight ratio and absolute tumor weight in the treatment group mice (as shown). Figures 9-10 Furthermore, the number of metastatic nodules in the livers of mice in the treatment group was significantly reduced ( ). Figure 11 Intrahepatic metastatic nodules metastasize via the bloodstream, and vascular invasion is the first step in hematogenous metastasis, indicating that vascular invasion has been mitigated. These results demonstrate that the key gene identified by the innovative screening method of this invention can effectively inhibit the growth and vascular invasion process of intrahepatic cholangiocarcinoma in vivo, validating the feasibility and effectiveness of this screening strategy. This key gene can serve as a potential target for precision treatment of intrahepatic cholangiocarcinoma and can be used to prepare drugs against intrahepatic cholangiocarcinoma.
[0064] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.
Claims
1. An innovative screening method for key genes involved in vascular invasion of intrahepatic cholangiocarcinoma, characterized in that, include: Single-cell transcriptome data of intrahepatic cholangiocarcinoma tissues with and without vascular invasion were obtained, specific cell subpopulations enriched in intrahepatic cholangiocarcinoma tissues with vascular invasion were identified, and genes specifically highly expressed in these cell subpopulations were screened. We obtained ordinary transcriptome data from intrahepatic cholangiocarcinoma tissues with and without vascular invasion, compared and screened differentially regulated genes in intrahepatic cholangiocarcinoma tissues with vascular invasion. Common genes were extracted from specifically overexpressed genes screened from single-cell transcriptome data and upregulated differentially expressed genes screened from ordinary transcriptome data. For the common genes, corresponding inhibition or knockout methods are designed, and cell phenotype screening experiments are performed in vitro. The cell phenotype screening experiments include the detection of the budding ability of human umbilical vein endothelial cells. Based on the screening experiment results, genes that can reduce the budding ability of human umbilical vein endothelial cells are selected, which are the key genes for vascular invasion of intrahepatic cholangiocarcinoma.
2. The method according to claim 1, characterized in that, The identification of specific cells enriched in intrahepatic cholangiocarcinoma tissue with vascular invasion, and the screening of highly expressed genes in these specific cells, specifically includes: Cell clustering and annotation were performed based on single-cell transcriptome data. Intrahepatic cholangiocarcinoma tissues with and without vascular invasion were compared to identify specific cell subpopulations that were significantly enriched in tissues with vascular invasion. The gene expression characteristics and functions of the specific cell subpopulations were analyzed to screen for genes that were specifically highly expressed in the specific cell subpopulations.
3. The method according to claim 1, characterized in that, The specific steps for designing corresponding inhibition or knockout methods for the shared gene and conducting in vitro cell phenotype screening experiments are as follows: Based on the common genes identified through screening, corresponding inhibitors or knockdown methods were designed to treat specific cells enriched in intrahepatic cholangiocarcinoma tissues with vascular invasion. Conditioned culture medium was collected from specific cells after treatment, and human umbilical vein endothelial cells were treated to screen for budding ability.
4. The method according to claim 3, characterized in that, The knockdown method includes using shRNA targeting the shared gene.
5. The method according to claim 1, characterized in that, It also includes further in vitro and in vivo experiments on the key gene selected based on the screening results that can reduce the sprouting ability of human umbilical vein endothelial cells, to confirm that targeting the key gene can inhibit the progression and vascular invasion of intrahepatic cholangiocarcinoma.
6. The method according to claim 1, characterized in that, The key gene for vascular invasion in intrahepatic cholangiocarcinoma is SLC2A1.
7. An innovative screening system for key genes involved in vascular invasion of intrahepatic cholangiocarcinoma, characterized in that, An innovative screening method for achieving the key gene for vascular invasion in intrahepatic cholangiocarcinoma as described in any one of claims 1-6 includes: The single-cell transcriptome analysis module is used to obtain single-cell transcriptome data of intrahepatic cholangiocarcinoma tissues with and without vascular invasion, identify specific cell subpopulations enriched in tissues with vascular invasion, and screen for atopic high-expression genes in these specific cell subpopulations. The standard transcriptome analysis module is used to obtain standard transcriptome data of intrahepatic cholangiocarcinoma tissues with and without vascular invasion, and to compare and screen differentially regulated genes in intrahepatic cholangiocarcinoma tissues with and without vascular invasion. The shared gene extraction module is used to select shared genes obtained from the two data analysis modules mentioned above. The cell phenotype screening and verification module is used to perform cell phenotype screening and functional verification experiments on the common genes, and to identify key genes based on the results of the human umbilical vein endothelial cell budding ability test.
8. The application of a key gene obtained by an innovative screening method for key genes involved in vascular invasion of intrahepatic cholangiocarcinoma as described in any one of claims 1-6, characterized in that, include: To create a single-cell atlas of vascular invasion in intrahepatic cholangiocarcinoma; Investigating the mechanisms of vascular invasion in intrahepatic cholangiocarcinoma; To develop drugs that target this key gene for the treatment of intrahepatic cholangiocarcinoma.
9. The application according to claim 8, characterized in that, The drug for treating intrahepatic cholangiocarcinoma is a specific inhibitor targeting this key gene.
10. The application according to claim 9, characterized in that, The drug developed for treating intrahepatic cholangiocarcinoma is BAY-876, a specific inhibitor of the key gene SLC2A1.