Application of HOXA3 gene as target spot in screening and treating medicine for hepatocellular carcinoma

Abstract

The invention provides an application of an HOXA3 gene as a target spot in screening and treating drugs for hepatocellular carcinoma. Through single-cell multi-omics analysis, the HOXA3 is found to be remarkably and highly expressed in liver cancer tissues and is closely related to poor prognosis of patients. Mechanism research shows that HOXA3 promotes tumor proliferation and metastasis through transcriptional up-regulation of YAP1 and RHOA, and an FGF19-FGFR4 / beta-catenin signal channel positively regulates the expression of HOXA3. The invention verifies that the combined application of the YAP1 inhibitor Verteporfin and the RHOA inhibitor Rhosin can significantly inhibit the HOXA3-mediated liver cancer progress, in-vivo experiments show that the tumor volume is reduced by 68%, the lung metastasis inhibition rate reaches 83%, and an effective treatment strategy is provided for HOXA3 positive hepatocellular carcinoma.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of the HOXA3 gene as a target in the screening and treatment of drugs for hepatocellular carcinoma. Background Technology

[0002] Hepatocellular carcinoma (HCC) is a common malignant tumor worldwide with a high mortality rate. Current treatments such as surgery, chemotherapy, and immunotherapy have limited effectiveness, especially in advanced-stage patients who are prone to drug resistance and metastasis. Transcriptional dysregulation is a key driver of HCC progression, with transcription factors (TFs), such as the Homeobox (HOX) gene family, playing a crucial role in tumorigenesis. HOXA3, a member of the HOX gene family, is highly expressed in tumors such as glioblastoma and renal cell carcinoma and promotes malignant progression, but its specific mechanism in HCC remains unclear. Fibroblast growth factor 19 (FGF19) activates downstream signaling pathways through the FGFR4 receptor, is frequently amplified in HCC, and is associated with poor prognosis. However, the mechanism by which FGF19 regulates the transcription factor network to drive HCC requires further investigation.

[0003] Therefore, it is necessary to develop a new target for screening and treating drugs for hepatocellular carcinoma. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the HOXA3 gene as a target in the screening and treatment of drugs for hepatocellular carcinoma. This invention discovers that inhibiting HOXA3 expression can significantly inhibit the proliferation of liver cancer cells (inhibition rate >65%) and metastasis (lung metastasis reduced by 83%). It innovatively proposes a combination therapy of YAP1 inhibitor Verteporfin and RHOA inhibitor Rhosin, providing a new approach to solving the treatment problem of liver cancer with high HOXA3 expression.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect of the invention, the use of the HOXA3 gene as a target gene in screening for drugs to prevent, alleviate, and / or treat hepatocellular carcinoma is provided, wherein the screening method includes screening for substances that can inhibit the expression or function of the HOXA3 gene.

[0006] In a second aspect of the invention, the use of HOXA3 gene-inhibiting substances or functional substances in the preparation of medicaments for the prevention, relief and / or treatment of hepatocellular carcinoma is provided.

[0007] Furthermore, the substances that inhibit HOXA3 gene expression or function include small molecule inhibitors, shRNA, or siRNA.

[0008] Furthermore, the sequence of the shRNA is shown in SEQ ID NO.1-3.

[0009] Furthermore, the drug prevents, alleviates, and / or treats hepatocellular carcinoma by at least one of the following actions: inhibiting the proliferation or metastasis of liver cancer.

[0010] In a third aspect of the invention, a medicament for the prevention, relief and / or treatment of hepatocellular carcinoma is provided, the medicament comprising a substance that inhibits the expression or function of the HOXA3 gene, and a combination of a YAP1 inhibitor and / or a RHOA inhibitor.

[0011] Furthermore, the YAP1 inhibitor is Verteporfin, and the RHOA inhibitor is Rhosin.

[0012] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0013] Furthermore, the excipients are selected from one of the following: fillers, disintegrants, binders, diluents, lubricants, sweeteners, or colorants.

[0014] Furthermore, the dosage form of the drug includes at least one of granules, tablets, pills, capsules, injections, and dispersants.

[0015] In a fourth aspect of the invention, the use of HOXA3 as a molecular marker in the preparation of products for the diagnosis of hepatocellular carcinoma is provided.

[0016] In a fifth aspect of the invention, the use of a detection reagent for HOXA3 in the preparation of products for diagnosing hepatocellular carcinoma is provided.

[0017] Furthermore, the detection reagents include ELISA kits, PCR primers, or antibodies.

[0018] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: 1. This invention discovers a novel and highly efficient diagnostic biomarker: Through single-cell multi-omics analysis, HOXA3 was systematically identified for the first time as a key transcriptional regulator in hepatocellular carcinoma. Clinical validation showed that HOXA3 expression was significantly upregulated in hepatocellular carcinoma tissues, with a diagnostic sensitivity of 92.5% and a specificity of 88.3%. Compared with traditional biomarkers, the AUC value of HOXA3 alone was 0.84, and the AUC increased to 0.91 when combined with YAP1.

[0019] 2. This invention identifies a novel therapeutic target: In vitro and in vivo experiments confirm that inhibiting HOXA3 expression significantly suppresses liver cancer cell proliferation (inhibition rate >65%) and metastasis (lung metastasis reduced by 83%). It elucidates for the first time the molecular mechanism by which HOXA3 promotes liver cancer progression through transcriptional regulation of YAP1 and RHOA. It reveals the complete regulatory network of the FGF19-β-catenin / YAP1-HOXA3-YAP1 / RHOA signaling pathway.

[0020] 3. A novel combination therapy regimen of the YAP1 inhibitor Verteporfin and the RHOA inhibitor Rhosin was proposed. In animal models, this combination therapy reduced tumor volume by 68% and significantly prolonged survival. This provides a new approach to addressing the treatment challenges of HOXA3-overexpressing liver cancer. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 Single-cell analysis revealed the expression and accessibility of HOXA3 in liver cancer. Figure 1 A: The diagram illustrates the integrated analysis workflow of scRNA-seq and scATAC-seq, including sample processing (24 HCC tumor tissues and 6 adjacent normal tissues), data integration, and cell type identification. Figure 1 B: Distribution map of major cell types in scRNA-seq and scATAC datasets, including seven cell types such as hepatocytes, T cells, and endothelial cells; bar charts compare the differences in cell proportions between tumors and adjacent normal tissues, showing a decrease in T cells and an increase in hepatocytes in HCC samples. Figure 1 C: The heatmap shows the distribution of differentially accessible regions (DARs) in different cell types in scATAC-seq; the pie chart summarizes the genomic characteristics of DARs (such as promoter regions and intron regions). Figure 1 D: The bubble chart shows pathways enriched by upregulated differentially expressed genes (DEGs) in HCC hepatocytes, such as metabolic-related pathways. Figure 1 E: Bubble diagram shows the signaling pathways enriched by DARs in HCC hepatocytes. Figure 1 F: The heatmap shows the differences in the activity of transcription factor (TF) motifs in different cell types, with hepatocytes having the highest number of motifs specific to them. Figure 1 G: The heatmap shows the copy number variation (CNV) signal of malignant cells, used to distinguish tumor cells. Figure 1H: UMAP plots show the clustering results of malignant cells, reflecting tumor heterogeneity. Figure 1 I: The hierarchical clustering graph shows the similarity of the four metaprograms (MPs) identified by nonnegative matrix factorization (NMF). Figure 1 J: The UMAP plot is colored according to MP feature scores, showing the distribution of the MP1 subgroup. Figure 1 K: Heatmap shows the accessibility of DARs in MPs; pie chart shows the genome annotation of DARs. Figure 1 L: The heatmap shows the activity differences of TF motifs in MPs. Figure 1 M: The pathway enrichment analysis diagram shows the unique pathways of each MP. MP1 is enriched in oncogenic pathways such as FGF and ERK / MAPK. Figure 1 N: Venn diagram shows the intersection of MP1 characteristic genes and active TFs, highlighting the candidate role of HOXA3. Figure 1 O: Violin plot shows the difference in HOXA3 expression between HCC hepatocytes and adjacent hepatocytes, with significantly increased expression in HCC. Figure 1 P: Chromatin accessibility trajectory diagrams show the open state of the HOXA3 promoter region in different cell types, with the highest accessibility in hepatocytes.

[0023] Figure 2 Effects of HOXA3 overexpression / knockdown on the proliferation and invasion of liver cancer cells. Figure 2 A: The qPCR results show the expression levels of HOXA3 mRNA in normal liver tissue, adjacent normal tissue, and HCC tissue (n=20 normal, n=50 adjacent normal, n=50 HCC); the subplots compare HOXA3 expression in patients with / without recurrence or metastasis. Figure 2 B: Representative immunohistochemical (IHC) images show HOXA3 protein expression in HCC and adjacent normal tissues; semi-quantitative scoring plots compare HOXA3 levels.

[0024] Figure 2 C: Kaplan-Meier survival curves show that patients with high HOXA3 expression in both independent cohorts had poorer relapse and overall survival rates. Figure 2 D: Comparison of qPCR and Western Blot results on the expression of HOXA3 in normal liver tissue and various HCC cell lines (such as MHCC97H), with higher expression in highly metastatic cell lines. Figure 2 E: Western Blot images show the effects of HOXA3 overexpression in PLC / PRF / 5 cells and HOXA3 knockdown in MHCC97H cells. Figure 2 F: Transwell experimental images and quantification plots show that HOXA3 overexpression enhances PLC / PRF / 5 cell migration and invasion, while knockdown inhibits these phenotypes in MHCC97H cells. Figure 2 G: In vivo imaging of the orthotopic transplantation model shows enhanced tumor growth in the HOXA3 overexpression group and growth inhibition in the knockdown group. Figure 2 H: Survival curves showed that the survival time of mice in the HOXA3 overexpression group was shortened, while that in the knockdown group was prolonged. Figure 2 I: Representative H&E staining images of lung metastases show an increase in metastatic nodules in the HOXA3 overexpression group and a decrease in the knockdown group. Figure 2 J: Statistical chart of lung metastatic nodules to compare the degree of metastasis in each group.

[0025] Figure 3 : Validate the function of HOXA3 using an in vivo model. Figure 3 A: The experimental flowchart shows the dosing regimen for DEN / CCl4-induced HCC (DEN 25 mg / kg, CCl4 0.5 μl / g weekly). Figure 3 B: Representative images of liver gross morphology and H&E staining show the tumor burden in different groups; H&E images of lung metastases show the metastasis status. Figure 3 C: The Western Blot results show the time-series changes in HOXA3 protein expression during HCC. Figure 3 D: Schematic diagram illustrating the process of achieving hepatocyte-specific Hoxa3 knockout using the AAV8-TBG-Cre vector. Figure 3 E: Experimental timeline plot combining DEN / CCl4 treatment and AAV8 injection. Figure 3 F: Western blotting images verify the efficiency of AAV8-mediated Hoxa3 knockout. Figure 3 G: Gross images of the liver and H&E staining plots comparing the number of tumors, liver weight / body weight ratio (LW / BW), and maximum tumor size between the control and Hoxa3 knockout groups. Figure 3 H: Survival curves show that the survival time of mice in the Hoxa3 knockout group is prolonged. Figure 3 I: H&E images and nodule count statistics of lung metastases show reduced metastasis in the knockout group. Figure 3 J: A schematic diagram illustrating the construction of genetically engineered mice, showing the Hoxa3flox / flox strain. Figure 3 K: Validation plot shows the success of hepatocyte-specific Hoxa3 knockout (Hoxa3Δhep).

[0026] Figure 3 L: Liver images and statistical graphs show reduced tumor burden in the Hoxa3Δhep group. Figure 3 M: Survival curves comparing the survival rates of the Hoxa3flox / flox and Hoxa3Δhep groups. Figure 3 N: Statistical graph of lung metastases to verify the knockout effect. Figure 3 Schematic diagram of the O:Hoxa3 knock-in (Hoxa3hepOE) mouse construction. Figure 3 P: The verification chart shows the Hoxa3hepOE strain. Figure 3 Q: The tumor burden graph shows an increase in tumors in the Hoxa3hepOE group. Figure 3 R: The survival curve shows a shortened survival in the Hoxa3hepOE group. Figure 3 S: The lung metastasis statistics graph shows enhanced metastasis in the knock-in group.

[0027] Figure 4 The mechanism by which HOXA3 regulates YAP1 and RHOA. Figure 4 A: The RNA sequencing volcano plot shows the differentially expressed genes (upregulated and downregulated genes) caused by HOXA3 overexpression. Figure 4 B: Bubble charts show pathways enriched by HOXA3 overexpression, such as the Hippo signaling pathway. Figure 4 C: The CUT & TAG signal heatmap and density map show the distribution of HOXA3 binding sites near the transcription start site (TSS); the bottom right corner shows the HOXA3 binding motif. Figure 4 D: Venn diagram showing 43 overlapping genes identified by both RNA sequencing and CUT&TAG. Figure 4 E: The heatmap shows the genes with the most significant expression changes among the overlapping genes, highlighting YAP1 and RHOA. Figure 4 F: Gene trajectory map showing the binding signal of HOXA3 in the YAP1 and RHOA promoter regions of CUT&TAG. Figure 4 G: The qPCR and Western Blot results show that HOXA3 overexpression upregulates YAP1 and RHOA expression, while knockdown downregulates them. Figure 4 H: The statistical graph of the dual-luciferase reporter assay shows that HOXA3 overexpression activates the YAP1 and RHOA promoter activities. Figure 4 I: The luciferase activity graph shows the YAP1 promoter truncation and mutant experiments, which determined the key roles of binding sites 1 and 2. Figure 4 J: Similar statistical graphs illustrate RHOA promoter truncation and mutation experiments to determine the necessity of binding site 1. Figure 4 K: The ChIP experimental results confirm that HOXA3 directly binds to the YAP1 and RHOA promoters in HCC cells and patient samples. Figure 4 L: Western blotting images validate the expression changes of YAP1 and RHOA under HOXA3 regulation. Figure 4 M: Transwell experimental images and quantization plots show that HOXA3 knockdown still inhibits invasion in the context of YAP1 overexpression. Figure 4 N: Representative in vivo imaging images of the orthotopic transplantation model show that HOXA3 promotes tumor growth through YAP1 / RHOA. Figure 4O: Survival curves show the differences in survival among mice in the relevant groups.

[0028] Figure 4 P: H&E images of lung metastases show the extent of metastasis. Figure 4 Q: Quantitative effect of lung metastatic nodule count statistics.

[0029] Figure 5 Clinical relevance of HOXA3 to YAP1 / RHOA. Figure 5 A: Representative IHC images show the expression patterns of HOXA3, YAP1, and RHOA in HCC and adjacent normal tissues. Figure 5 B: The correlation plot shows a positive correlation between HOXA3 and YAP1 and RHOA expression in the two independent cohorts (P<0.001). Figure 5 C: Kaplan-Meier curves show that patients with high YAP1 expression have poorer relapse and survival rates. Figure 5 D: A similar graph illustrates the prognostic significance of high RHOA expression. Figure 5 E: Subgroup analysis curves show that patients with high expression of both HOXA3 and YAP1 have the worst prognosis. Figure 5 F: A similar graph illustrates the poor prognosis of high HOXA3 / RHOA dual expression. Figure 5 G: qPCR results comparing the mRNA levels of HOXA3, YAP1, and RHOA in adjacent normal tissues, primary HCC, and metastatic HCC. Figure 5 H: Representative images of the IHC show the expression of the three in different tissues. Figure 5 I: IHC score statistical graph quantifies the difference in expression, with the highest expression in the transfer sample.

[0030] Figure 6 The mechanism by which the FGF19 pathway upregulates HOXA3. Figure 6 A: The qPCR and Western Blot results show that FGF19 treatment of PLC / PRF / 5 cells resulted in a dose-dependent increase in HOXA3 expression. Figure 6 B: The luciferase activity graph shows that FGF19 activates the HOXA3 promoter. Figure 6 C: Western blot images show that FGF19 induces upregulation of FGFR4, p-FRS2, p-GSK3β, β-catenin, and HOXA3. Figure 6 D: Similar images show FGFR4 knockdown or the inhibitor BLU9931 blocking this pathway. Figure 6 E: The luciferase activity graph shows the HOXA3 promoter truncation and mutation experiments, which determined the key role of β-catenin / TCF4 binding site 2. Figure 6F: Western blot images showed that ERK inhibitors blocked FGF19-induced HOXA3 expression, while other pathway inhibitors had no effect. Figure 6 G: Protein expression diagrams confirm that ERK inhibitors reduce p-GSK3β and β-catenin activation. Figure 6 H: The ChIP results show that FGF19 enhances the binding of TCF4 to the HOXA3 promoter through the ERK pathway. Figure 6 I: Co-IP experimental images show that FGF19 stimulation enhances the interaction between endogenous β-catenin and YAP1, and the complex contains TCF4 and TEADs. Figure 6 J: Confocal immunofluorescence images show the colocalization of β-catenin and YAP1 in the nucleus and cytoplasm, with FGF19 treatment enhancing the colocalization. Figure 6 K: The Co-IP results diagram identifies the ARM domain of β-catenin as the key interaction region. Figure 6 L: Similar experiments confirm the necessity of the TAD / PDZ domain in YAP1. Figure 6 M: Summary of findings from the interaction domain diagram. Figure 6 N: The molecular docking model diagram shows the interaction interface and key amino acids between β-catenin and YAP1 proteins. Figure 6 O: The qPCR results show that β-catenin or YAP1 knockdown inhibits FGF19-induced HOXA3 expression. Figure 6 P: Western blotting images validate changes in protein levels. Figure 6 Q: The luciferase activity graph shows that β-catenin knockdown completely blocks FGF19 activation of the HOXA3 promoter. Figure 6 R: The ChIP results confirm that YAP1 binding to the HOXA3 promoter depends on β-catenin.

[0031] Figure 7 The role of HOXA3 in FGF19-mediated liver cancer. Figure 7 A: Western blot images show the protein levels of FGF19 overexpression and HOXA3 knockdown in PLC / PRF / 5 cells. Figure 7 B: Representative in vivo imaging images of the orthotopic transplantation model show that FGF19 overexpression enhances tumor growth, and HOXA3 knockdown reverses this effect. Figure 7 C: Bioluminescence signal statistical graph quantifies tumor burden. Figure 7 D: Survival curves show the survival rates of mice in each group. Figure 7 E: Statistical graph of lung metastatic nodules to compare the degree of metastasis. Figure 7 F: Representative H&E images of lung tissue showing metastatic morphology. Figure 7G: Schematic diagram showing the construction of hepatocyte-specific Fgf15 knock-in (Fgf15hepOE) mice. Figure 7 H: Gross images of the liver and H&E staining show that the liver of Fgf15hepOE mice is normal without treatment. Figure 7 I: Schematic diagram showing the construction of Fgf15hepOE; Hoxa3Δhep double transgenic mice. Figure 7 J: Liver images and statistical plots after DEN / CCl4 treatment showed increased tumor burden in the Fgf15hepOE group and reduced tumor burden in the dual transgenome group. Figure 7 K: Survival curves compare the survival rates of different groups. Figure 7 L: Quantitative results of tumor number, LW / BW ratio, and maximum tumor size. Figure 7 M: Western blotting images showed that β-catenin, HOXA3, YAP1 and RHOA proteins were upregulated in Fgf15hepOE mice, and YAP1 / RHOA ratio decreased after HOXA3 knockdown. Figure 7 N: Representative IHC images show the co-expression of FGF19 and HOXA3 in HCC tissues. Figure 7 O: The correlation plot shows that FGF19 and HOXA3 expression are positively correlated in both cohorts. Figure 7 P: Kaplan-Meier curves show that patients with double high expression of FGF19 / HOXA3 have the worst prognosis.

[0032] Figure 8 : Verification of the efficacy of combined treatment. Figure 8 A: The experimental diagram shows the grouping and dosing regimens of the orthotopic transplantation model (Vehicle, Verteporfin monotherapy, Rhosin monotherapy, combination therapy). Figure 8 B: Representative in vivo imaging images show that the combined treatment group exhibits the strongest tumor suppression. Figure 8 C: Bioluminescence signal statistical graph quantifies tumor growth inhibition. Figure 8 D: Survival curves show that the survival time of mice in the combined treatment group was significantly prolonged. Figure 8 E: The lung metastatic nodule count graph shows the suppression of metastasis by combined treatment. Figure 8 F: Representative H&E images of lung tissue confirm reduced metastasis. Figure 8 G: The experimental flowchart shows the dosing regimen in the DEN / CCl4-induced Hoxa3hepOE mouse model. Figure 8 H: Gross images of the liver and H&E staining showed that the tumor burden was the lightest in the combination therapy group. Figure 8 I: Survival curves compare the survival rates of each group. Figure 8 J: The tumor count chart shows the advantages of combination therapy. Figure 8 K: The LW / BW ratio is used to quantify changes in liver weight. Figure 8 L: The maximum tumor size statistical chart shows the treatment effect. Figure 8 M: A schematic diagram summarizing the role of the FGF19-HOXA3-YAP1 / RHOA axis and combined therapy. Detailed Implementation

[0033] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0034] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0035] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

[0036] The relevant terms in this invention embodiment are explained as follows: 1. Vehicle (carrier / control group) Definition: In experiments, "Vehicle" refers to a carrier solution without active ingredients, typically used to dissolve active drugs or as a control group to eliminate the influence of the solvent itself on the experimental results. In this invention, the Vehicle group serves as a negative control to compare and verify the therapeutic effects of Verteporfin and Rhosin.

[0037] 2. Verteporfin is a specific YAP1 inhibitor. CAS#: 129497-78-5.

[0038] 3. Rhosin (a RhoA inhibitor) is a highly effective and specific RHOA inhibitor. CAS#: 1173671-63-0.

[0039] To solve the above-mentioned technical problems, the overall concept of the present invention is as follows: This invention, through integrated single-cell multi-omics analysis (scRNA-seq and scATAC-seq), discovered that HOXA3 is a key transcription factor in the malignant cell subset (MP1) of hepatocellular carcinoma. High expression of HOXA3 in hepatocellular carcinoma tissues is significantly associated with patient recurrence, metastasis, and reduced survival.

[0040] Functional experiments showed that overexpression of HOXA3 promoted the proliferation and metastasis of liver cancer cells, while knockdown of HOXA3 inhibited these phenotypes.

[0041] Mechanistically, FGF19 upregulates HOXA3 expression by activating the FGFR4 / p-FRS2 / p-GSK3β / β-catenin pathway, and β-catenin forms a complex with YAP1 to synergistically enhance HOXA3 transcription. HOXA3 further directly binds to the YAP1 and RHOA promoters, upregulating these two oncogenes and forming a positive feedback loop that promotes liver cancer progression.

[0042] In treatment, the combined use of the YAP1 inhibitor Verteporfin and the RHOA inhibitor Rhosin can effectively inhibit HOXA3-mediated liver cancer growth and metastasis. Animal models (such as DEN / CCl4-induced mouse liver cancer models and orthotopic transplantation models) have validated the efficacy of this strategy.

[0043] This invention reveals for the first time the core role of HOXA3 in liver cancer, providing new diagnostic biomarkers and therapeutic targets.

[0044] The present application will now be described in detail with reference to embodiments and experimental data.

[0045] Example 1: Expression and clinical significance of HOXA3 in hepatocellular carcinoma 1. Methods: Samples (n=110) from the TCGA database and clinical samples were collected. HOXA3 expression was detected by qPCR, Western blotting, and immunohistochemistry (IHC). Statistical analysis and prognostic parameters (such as tumor size, metastasis, and survival rate) were performed.

[0046] 2. Results: HOXA3 mRNA and protein expression in liver cancer tissues were significantly higher than in adjacent normal tissues (P<0.001), and patients with high expression had a lower 5-year survival rate (HR=2.1). Single-cell analysis showed that HOXA3 was active in the MP1 subset. Figure 1 O, 1P).

[0047] In summary, HOXA3 can be used as an independent prognostic biomarker with a diagnostic AUC of 0.84, and the AUC increases to 0.91 when combined with YAP1.

[0048] Example 2: In vitro experiment to verify the function of HOXA3 1. In PLC / PRF / 5 (low HOXA3 expression) and MHCC97H (high HOXA3 expression) cell lines, HOXA3 overexpression and knockdown models were constructed by lentiviral transfection.

[0049] HOXA3 shRNA sequence: The shRNA sequence used was designed targeting the HOXA3 coding region. The specific sequence is shown in Table 1. Table 1

[0050] In this embodiment, HOXA3 shRNA-2 is preferably used for knockdown experiments.

[0051] MHCC97H-shHOXA3 cell construction process: Stable cell line construction was performed using a lentiviral system, and the procedure is as follows: HEK-293T cells were co-transfected with the lentiviral vector (PLKO.1-TRC, Addgene) encoding shHOXA3 (or control shControl) and the packaging plasmids (pMD2.G and psPAX2), and cultured in DMEM medium for 72 hours using PEI transfection reagent.

[0052] Collect the viral supernatant, filter it through a 0.45-μm filter, and store it at -80°C.

[0053] MHCC97H cells were infected with the virus. 10 μg / ml polybrene (Sigma H9268) was added during infection to enhance efficiency. The infection time was 24 hours.

[0054] Forty-eight hours after infection, cells were selected for one week with 3 μg / ml puromycin (OriGene) to obtain a stable HOXA3 knockdown MHCC97H-shHOXA3 cell line. Control cells were constructed using the same method with non-targeting shRNA (shControl, Sigma SHC016).

[0055] Functional experiments: CCK-8 proliferation assay, Transwell invasion assay and scratch healing assay were performed to evaluate cell phenotype.

[0056] 2. Results: Overexpression of HOXA3 increased cell proliferation by 58% and the number of cells that migrated through the cell membrane by 2.1 times; knockdown of HOXA3 inhibited proliferation by 65% ​​and reduced metastasis by 70%. Figure 2 F, 2G, 2H, 2I).

[0057] In summary, HOXA3 directly promotes the malignant phenotype of liver cancer.

[0058] Example 3: In vivo experiment to verify the function of HOXA3 1. Methods: A mouse orthotopic transplantation model was established (injection of PLC / PRF / 5-HOXA3 or MHCC97H-shHOXA3 cells), and tumor growth and lung metastasis were monitored by in vivo imaging. Hepatocyte-specific Hoxa3 knockout (Hoxa3Δhep) or knock-in (Hoxa3hepOE) was achieved using the AAV8 vector.

[0059] 2. Results: In the HOXA3 overexpression group, tumor volume increased by 2.3-fold, and lung metastatic nodules reached 18.3 per mouse; in the knockdown group, tumor volume decreased by 76%, and lung metastases decreased by 88%. Figure 3 G, 3H, 3I, 3J. Hoxa3 knockout prolongs mouse survival ( Figure 3 H).

[0060] In summary, targeting HOXA3 effectively inhibits the progression of liver cancer.

[0061] Example 4: Study on the HOXA3 mechanism 1. Methods: HOXA3 target genes were analyzed by RNA sequencing and cut & tagging. Dual-luciferase reporter assays and ChIP were used to verify the binding of HOXA3 to the YAP1 and RHOA promoters. Cells were treated with FGF19 stimulation and pathway inhibitors (such as ERK inhibitors).

[0062] 2. Results: HOXA3 directly binds to the YAP1 promoter (sites -897 to -170 bp) and the RHOA promoter (sites -587 to -126 bp). FGF19 upregulates HOXA3 via the ERK / β-catenin axis. Figure 4 C-4K, Figure 6 A-6R).

[0063] In summary, the FGF19-β-catenin / YAP1-HOXA3-YAP1 / RHOA axis forms a positive feedback loop.

[0064] Example 5: Validation of Therapeutic Application 1. Methods: In the orthotopic transplantation model, patients were divided into groups and treated with Vehicle, Verteporfin (50 mg / kg), Rhosin (30 mg / kg) or combination therapy. Tumor volume, lung metastasis and survival were assessed.

[0065] 2. Results: The combined treatment group showed a 68% reduction in tumor volume, the strongest inhibition of lung metastasis, and a significantly prolonged survival time in mice. Figure 8 B-8F).

[0066] In conclusion, the combined use of YAP1 and RHOA inhibitors is a potential treatment for HOXA3-positive liver cancer.

[0067] Example 6: Validation of the clinical relevance and diagnostic value of HOXA3 and YAP1 / RHOA To verify the correlation between HOXA3 expression and downstream effectors YAP1 and RHOA in clinical samples of hepatocellular carcinoma and their diagnostic and prognostic value.

[0068] I. Experimental Methods: 1. Clinical sample collection: Two independent HCC cohorts were collected (cohort 1: n=260; cohort 2: n=240), including cancerous tissue, paired adjacent normal tissue, and metastatic lesion samples.

[0069] Record the patient's clinicopathological parameters (tumor size, differentiation degree, TNM stage, survival time).

[0070] 2. Immunohistochemical (IHC) detection: Tissue microarrays were stained with anti-HOXA3, YAP1, and RHOA antibodies and scored using the H-score method (0-12 points).

[0071] The staining results were evaluated in a double-blind manner, with the positive threshold set at H-score ≥ 6.

[0072] 3. Statistical analysis: Spearman correlation analysis was used to study the expression association between HOXA3 and YAP1 and RHOA.

[0073] The Kaplan-Meier method and Log-rank test were used to analyze survival differences, and Cox regression models were used to assess independent prognostic factors.

[0074] ROC curve analysis demonstrates diagnostic efficacy (AUC value calculation).

[0075] II. Results: 1. HOXA3 expression is positively correlated with YAP1 / RHOA expression: In cohort 1, the YAP1 positivity rate in the HOXA3 positive group (n=168) was 72.0% (121 / 168), which was significantly higher than that in the HOXA3 negative group (P<0.001).

[0076] Similarly, HOXA3 and RHOA expression were significantly positively correlated (P<0.001), and the results were consistent between the two cohorts.

[0077] 2. Association between co-expression and prognosis: The 5-year survival rate of patients with double HOXA3 / YAP1 positivity was only 15%, significantly lower than that of the double negative group (85%) (HR=5.2, P<0.001).

[0078] The prognosis was equally poor in the HOXA3 / RHOA double-positive group. Figure 5 F).

[0079] 3. Diagnostic efficacy verification: The AUC of HOXA3 alone for diagnosing HCC is 0.84, and the AUC increases to 0.91 when combined with YAP1 (sensitivity 92.5%, specificity 88.3%).

[0080] Conclusion: HOXA3 is closely associated with the expression of YAP1 and RHOA, and the combination of the three can serve as a powerful biomarker combination for the diagnosis and prognostic assessment of HCC.

[0081] Example 7: The core role of HOXA3 in the FGF19 / FGF15 signaling pathway and validation of its therapeutic target This embodiment uses a genetically engineered mouse model and molecular experiments to elucidate the function of HOXA3 as a key downstream effector of FGF19 and the targeted intervention strategy.

[0082] I. Experimental Methods: 1. Cell model construction: FGF19 was stably overexpressed in PLC / PRF / 5 cells and then introduced into HOXA3 shRNA (sequence shown in SEQ ID NO:X).

[0083] The levels of FGF19 and HOXA3 proteins were verified by Western blotting.

[0084] 2. Animal model design: Hepatocyte-specific Fgf15 knock-in (Fgf15hepOE) mice were constructed (FGF15 is a homolog of mouse FGF19).

[0085] Further, Fgf15hepOE; Hoxa3Δhep double transgenic mice were constructed.

[0086] We used a DEN / CCl4-induced spontaneous HCC model to monitor tumor growth and lung metastasis.

[0087] 3. Treatment intervention: In the orthotopic transplantation model (PLC / PRF / 5-HOXA3 cells), patients were divided into groups and given Vehicle, Verteporfin (50 mg / kg), Rhosin (30 mg / kg), or combination therapy to evaluate efficacy.

[0088] II. Results: 1. HOXA3 mediates the pro-cancer effect of FGF19: FGF19 overexpression increased cell invasion capacity by 2.1 times, while HOXA3 knockdown completely reversed this effect (P<0.01).

[0089] In vivo experiments showed that the tumor burden in Fgf15hepOE mice was significantly increased (tumor number: 15.3±2.1 vs. control group 5.2±1.1), while the tumor number in double transgenic mice (Fgf15hepOE; Hoxa3Δhep) decreased to 6.8±1.4 (P<0.05).

[0090] 2. Molecular mechanism verification: In Fgf15hepOE mice, the expression of β-catenin, HOXA3, YAP1, and RHOA proteins was upregulated in liver tissue, while the levels of YAP1 and RHOA were significantly reduced after Hoxa3 knockout. Figure 7 M).

[0091] 3. Enhanced clinical relevance: In HCC patients, FGF19 and HOXA3 expression were positively correlated (r=0.75, P<0.001), and patients with double positivity had a very poor prognosis (5-year survival rate <20%).

[0092] In summary, HOXA3 is a core mediator driving the progression of liver cancer through the FGF19 / FGF15 signaling pathway, and targeting the HOXA3-YAP1 / RHOA axis (such as in combination with Verteporfin and Rhosin) has significant therapeutic potential.

[0093] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0094] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0095] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of the HOXA3 gene as a target gene in screening drugs for the prevention, alleviation, and / or treatment of hepatocellular carcinoma, characterized in that, The screening methods include screening for substances that can inhibit HOXA3 gene expression or functional substances.

2. Application of substances that inhibit HOXA3 gene expression or functional substances in the preparation of drugs for the prevention, relief and / or treatment of hepatocellular carcinoma.

3. The application according to claim 2, characterized in that, The substances that inhibit HOXA3 gene expression or function include small molecule inhibitors, shRNA, or siRNA.

4. The application according to claim 3, characterized in that, The sequence of the shRNA is shown in SEQ ID NO: 1-3.

5. The application according to any one of claims 1-4, characterized in that, The drug prevents, alleviates, and / or treats hepatocellular carcinoma by at least one of the following actions: inhibiting the proliferation and metastasis of hepatocellular carcinoma cells or inducing apoptosis.

6. A drug for preventing, alleviating, and / or treating hepatocellular carcinoma, characterized in that, The drug contains a substance that inhibits the expression or function of the HOXA3 gene, as well as a combination of a YAP1 inhibitor and / or a RHOA inhibitor.

7. The drug according to claim 6, characterized in that, The YAP1 inhibitor is Verteporfin, and the RHOA inhibitor is Rhosin.

8. The drug according to claim 6, characterized in that, The drug also includes pharmaceutically acceptable excipients, and the dosage form includes injections, tablets, or capsules.

9. Application of HOXA3 as a molecular marker in the preparation of products for the diagnosis of hepatocellular carcinoma.

10. The application of the HOXA3 detection reagent in the preparation of products for diagnosing hepatocellular carcinoma, characterized in that, The detection reagents include ELISA kits, PCR primers, or antibodies.

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