Application of ITGA6 inhibitor in preparation of medicine for treating uveal melanoma

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of an ITGA6 inhibitor in preparation of a medicine for treating uveal melanoma. By integrating bioinformatics analysis of databases of TCGA, GTEx, CPTAC and the like, the invention systematically reveals that the ITGA6 has high specific expression in uveal melanoma and is significantly related to poor prognosis of a patient for the first time. In-vitro experiments prove that by knocking down ITGA6, proliferation, migration, invasion and colony forming ability of UVM cells can be effectively inhibited. According to the invention, small molecular compounds such as 4.5-diphenyl-1H-imidazole, MS-275, W-13 and the like and siRNA (small interfering Ribonucleic Acid) targeting ITGA6 are screened out to be used as effective inhibitors, and specific drug dosage forms such as intraocular injection and the like are provided. In addition, the ITGA6 inhibitor can be used in combination with a PD-1 / PD-L1 inhibitor, a CTLA-4 inhibitor or a chemotherapeutic drug to generate a synergistic anti-tumor effect. Mechanism research shows that ITGA6 promotes UVM progress by regulating a signal channel and a tumor immune microenvironment. The invention provides a new targeted treatment strategy for uveal melanoma, and has important clinical application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more particularly to the use of an ITGA6 inhibitor in the preparation of a drug for uveal melanoma. Background Technology

[0002] Uveal melanoma (UVM) is the most common primary malignant tumor of the eye in adults, with the highest incidence rate among intraocular malignancies. This disease has unique biological characteristics; although local treatments such as radiation therapy or enucleation can effectively control the primary tumor, nearly half of patients eventually develop distant metastases, with the liver being the most common site of metastasis. Once metastasis occurs, the median survival is only about 10 to 12 months, and the five-year survival rate is extremely low.

[0003] Currently, systemic treatment options for metastatic uveal melanoma are extremely limited. Traditional chemotherapy drugs such as dacarbazine and formustine are ineffective, with objective response rates generally below 10%. While immune checkpoint inhibitors have made breakthroughs in the treatment of cutaneous melanoma, their response rates in uveal melanoma are unsatisfactory, which is closely related to its unique immune microenvironment. Furthermore, regarding targeted therapy, although research on common driver gene mutations such as GNAQ / GNA11 has been conducted for many years, no significantly effective targeted drugs have yet been approved for clinical use.

[0004] Integrin α6 (ITGA6), an important member of the integrin family, is known to participate in the regulation of cell adhesion, migration, and proliferation by interacting with extracellular matrix components such as laminin. Existing studies have shown that ITGA6 is abnormally highly expressed in various epithelial-derived malignancies such as hepatocellular carcinoma and lung cancer, and is associated with tumor progression and poor prognosis. However, in uveal melanoma, a specific type of tumor, there are currently no systematic research reports on the expression characteristics, clinical significance, and specific mechanisms of action of ITGA6 in disease development and progression. In particular, significant knowledge gaps exist in the following aspects: First, the expression level of ITGA6 in uveal melanoma tissue and its correlation with the clinicopathological characteristics of patients are not yet clear; second, there is insufficient evidence to suggest that ITGA6 expression can serve as a prognostic predictor for uveal melanoma patients; third, the specific function and molecular mechanism of ITGA6 in regulating the malignant phenotype of uveal melanoma cells urgently need to be elucidated; fourth, whether targeted inhibition of ITGA6 can be an effective strategy for treating uveal melanoma remains to be experimentally verified.

[0005] Therefore, in-depth exploration of the biological function of ITGA6 in uveal melanoma and the development of novel treatment strategies based on ITGA6 inhibition are of great theoretical significance and clinical value for improving the clinical prognosis of this refractory disease. Summary of the Invention

[0006] The purpose of this invention is to provide the use of an ITGA6 inhibitor in the preparation of a drug for uveal melanoma. Through bioinformatics analysis integrating databases such as TCGA, GTEx, and CPTAC, this invention is the first to systematically reveal that ITGA6 is specifically highly expressed in uveal melanoma and is significantly associated with poor patient prognosis. In vitro experiments have confirmed that knocking down ITGA6 can effectively inhibit the proliferation, migration, invasion, and colony formation of UVM cells. This invention screened small molecule compounds such as 4,5-diphenyl-1H-imidazole, MS-275, and W-13, as well as siRNAs targeting ITGA6, as effective inhibitors, and provided specific drug formulations such as intraocular injections. Furthermore, ITGA6 inhibitors can be used in combination with PD-1 / PD-L1 inhibitors, CTLA-4 inhibitors, or chemotherapeutic drugs to produce a synergistic anti-tumor effect. Mechanistic studies have shown that ITGA6 promotes UVM progression by regulating signaling pathways and the tumor immune microenvironment. This invention provides a new targeted therapy strategy for uveal melanoma and has significant clinical application value.

[0007] To achieve the above objectives, the present invention provides the following technical solution: Use of an ITGA6 inhibitor in the preparation of a medicament for the treatment of uveal melanoma, wherein the ITGA6 inhibitor is selected from 4,5-diphenyl-1H-imidazole, MS-275, W-13, or a small interfering RNA (siRNA) that specifically silences the expression of the human ITGA6 gene.

[0008] Furthermore, the ITGA6 inhibitor is siRNA, and the sequence of the siRNA is selected from any of the following: 5'-GCAUCUACUACAACCUACA-3', 5'-CCAGAAGAUCAUCAAGAAA-3', 5'-GGACAUCAUCUUCUACAAA-3'.

[0009] Furthermore, the dosage form of the drug is an injection.

[0010] Furthermore, the injection is an intraocular injection.

[0011] The use of an ITGA6 inhibitor in the preparation of a medicament for the treatment of uveal melanoma in combination with a second active ingredient, wherein the ITGA6 inhibitor is selected from 4,5-diphenyl-1H-imidazole, MS-275, W-13, or a small interfering RNA (siRNA) that specifically silences the expression of the human ITGA6 gene, and the second active ingredient is selected from chemotherapeutic drugs or immune checkpoint inhibitors.

[0012] Furthermore, the immune checkpoint inhibitor is a PD-1 / PD-L1 inhibitor or a CTLA-4 inhibitor; the chemotherapy drug is selected from dacarbazine or formustine.

[0013] Use of an ITGA6 inhibitor in the preparation of a medicament for treating patients with uveal melanoma characterized by high ITGA6 expression, wherein the ITGA6 inhibitor is selected from 4,5-diphenyl-1H-imidazole, MS-275, W-13, or a small interfering RNA (siRNA) that specifically silences human ITGA6 gene expression.

[0014] Furthermore, the high expression characteristic of ITGA6 is determined by immunohistochemistry or RT-qPCR, wherein high expression is defined as the ITGA expression level of the detected sample being higher than the median value of the sample set.

[0015] A method for in vitro non-therapeutic inhibition of uveal melanoma cell migration or invasion, the method comprising contacting uveal melanoma cells with an effective amount of an ITGA6 inhibitor, wherein the ITGA6 inhibitor is an siRNA, and the sequence of the siRNA is selected from any of the following: 5'-GCAUCUACUACAACCUACA-3', 5'-CCAGAAGAUCAUCAAGAAA-3', 5'-GGACAUCAUCUUCUACAAA-3'.

[0016] A method for screening candidate drugs for the treatment of uveal melanoma, the method comprising the following steps: (a) Uveal melanoma cells expressing ITGA6 were cultured in the presence of the test compound; (b) Detect the expression level or activity of ITGA6 in the cells; (c) Select compounds that can reduce the expression level or activity of ITGA6 relative to control cells not exposed to the test compound as candidate drugs.

[0017] This invention, through systematic bioinformatics analysis and rigorous in vitro experimental verification, comprehensively reveals for the first time the crucial role of ITGA6 in uveal melanoma, and based on this, provides an effective treatment strategy, producing the following significant beneficial effects: 1. This invention, for the first time, confirms through large-scale bioinformatics data analysis that ITGA6 exhibits specific high expression characteristics in uveal melanoma tissues, and that its high expression is closely related to poor patient prognosis. Specifically, patients with high ITGA6 expression had significantly shorter overall survival (OS), progression-free survival (PFS), and disease-specific survival (DSS), making ITGA6 a reliable prognostic biomarker and providing a new reference indicator for clinical prognostic assessment. In the TCGA-UVM cohort, the median OS was 14.2 months in the high ITGA6 expression group and 28.7 months in the low expression group (HR=2.45, 95%CI 1.56-3.85, p<0.001). Figure 5 B.

[0018] 2. This invention demonstrates through in vitro cell experiments that reducing ITGA6 expression can effectively inhibit the malignant biological behavior of uveal melanoma cells. In two different uveal melanoma cell lines (92-1 and MUM2B), knocking down ITGA6 expression with specific siRNA significantly reduced cell proliferation activity (48-hour inhibition rate 42.6%, 72-hour inhibition rate 39.2%, p<0.01), migration and invasion abilities were significantly weakened (number of migrating cells reduced by more than 60%, number of invasive cells reduced by more than 55%, p<0.001), and the number of colonies formed was also greatly reduced (more than 70%, p<0.001). These results directly demonstrate the key role of ITGA6 in maintaining the malignant phenotype of uveal melanoma cells, providing solid experimental evidence for ITGA6-targeted therapeutic strategies.

[0019] 3. This invention, through integrated analysis of multiple drug sensitivity databases (such as PRISM and GDSC), successfully screened several specific small molecule compounds with potential inhibitory effects on ITGA6, including 4,5-diphenyl-1H-imidazole (IC50 = 2.3 μM), MS-275 (IC50 = 1.8 μM), and W-13 (IC50 = 3.1 μM). These compounds showed sensitivity related to ITGA6 expression levels in various tumor models, providing clear candidate molecules for the development of targeted therapies against uveal melanoma.

[0020] 4. This invention reveals that ITGA6 expression is closely associated with multiple malignant characteristics of uveal melanoma. Gene set enrichment analysis (GSEA) showed that high ITGA6 expression was significantly associated with the activation of pro-cancer pathways such as epithelial-mesenchymal transition (EMT, NES=2.13, FDR<0.01), angiogenesis (z=2.31, p<0.001), metastasis, proliferation, and stem cell characteristics. This finding not only reveals the potential mechanism by which ITGA6 promotes tumor progression but also provides a new perspective for understanding the highly aggressive nature of uveal melanoma.

[0021] 5. This invention confirms a significant association between ITGA6 expression and the characteristics of immune cell infiltration in the tumor microenvironment of uveal melanoma. High ITGA6 expression is associated with the infiltration levels of immune cells such as B cells (r=0.52, p<0.001), CD8+ T cells (r=0.62, p<0.001), and macrophages. This suggests that targeting ITGA6 may affect the tumor immune microenvironment, providing a theoretical basis for the combined use of ITGA6 inhibitors and immunotherapy.

[0022] 6. Based on the above findings, this invention provides multiple treatment strategies for uveal melanoma, including the use of specific ITGA6 small molecule inhibitors, gene silencing techniques, and combination therapy with other anti-tumor drugs. These strategies all have clear mechanisms of action and experimental support, providing new directions and options for the clinical treatment of this refractory disease. For example, the combination of ITGA6 inhibitors and PD-1 / PD-L1 inhibitors can produce a synergistic effect (combination index CI=0.65), significantly increasing the apoptosis rate (25.3% with monotherapy vs. 48.7% with combination therapy, p<0.01).

[0023] In summary, this invention not only systematically elucidates the carcinogenic effect and mechanism of ITGA6 in uveal melanoma for the first time, but also provides a validated and effective treatment strategy, which has important practical application value for improving the clinical prognosis of patients with uveal melanoma. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 The expression profile analysis results of ITGA6 in pan-cancer and UVM provided in Example 1 of this invention are as follows: (A) Difference in ITGA6 mRNA expression between UVM tumor tissue and normal control (TCGA database). (B) Difference in ITGA6 mRNA expression between tumor and normal tissue in different cancer types. (C) Organ-specific expression distribution of ITGA6 in tumor and normal tissue. (D) Validation results of ITGA6 protein expression level in the CPTAC database. (E) Immunohistochemical analysis of ITGA6 cytoplasmic immunoreactivity in tumor tissue encoded by staining intensity.

[0026] Figure 2The following are pan-cancer mutation maps of ITGA6 provided in Examples 1 and 9 of this invention: (A) Distribution and number statistics of ITGA6 gene variants in various cancers. (B) Comparison of mutation rates of ITGA6 and its related pathways. (C) A bubble chart showing the differential methylation level of the ITGA6 locus in pan-cancer analysis; the horizontal axis represents cancer type, the vertical axis represents methylation level, the bubble size represents the degree of methylation, and the color coding uses red / blue to represent the high / low methylation state of tumor tissue and normal tissue, respectively. (D) Mutation frequency and type of ITGA6 gene variants in top-tier cancers. (E) Spatial distribution of ITGA6 gene mutation hotspot regions.

[0027] Figure 3 The correlation between ITGA6 expression and pan-cancer clinical characteristics provided in Example 1 of this invention. (A) Correlation between ITGA6 expression and pan-cancer T stage: In both clear cell renal cell carcinoma (KIRC) and the whole renal cell carcinoma cohort (KIPAN), ITGA6 expression showed a gradual decreasing trend with increasing tumor stage. (B) Correlation between ITGA6 expression and pan-cancer N stage: Significant differences in ITGA6 expression were observed between bladder urothelial carcinoma (BLCA) and KIPAN N stages. (C) Correlation between ITGA6 expression and pan-cancer M stage: In both KIRC and KIPAN, ITGA6 expression levels in M1 stage tumors were significantly lower than in other stages. (D) Correlation between ITGA6 expression and pan-cancer tumor stage: ITGA6 expression gradually decreased with tumor stage progression in both KIRC and KIPAN. (E) Correlation between gender and pan-cancer ITGA6 expression: Female KIPAN patients showed significantly higher ITGA6 expression than male KIPAN patients.

[0028] Figure 4 The diagnostic value of ITGA6 in pan-cancer studies provided in Example 1 of this invention. (A) Evaluation of the diagnostic value of ITGA6 in pan-cancer studies; (B) Receiver operating characteristic (ROC) curves of ITGA6 in basal cell carcinoma, paraganglioma, and head and neck squamous cell carcinoma.

[0029] Figure 5 The prognostic value of ITGA6 in pan-cancer studies provided in Example 1 of this invention. (A) Hazard ratios (HRs) of ITGA6 expression for OS, DSS, DFI, and PFI. (B) Relationship between ITGA6 expression levels and OS survival curves in patients with different cancer types.

[0030] Figure 6The following are examples of ITGA6 functional enrichment analyses based on GSEA and GSVA provided in Examples 1 and 9 of this invention. (A) Differences in ITGA6 enrichment in 50 Hallmarks and 83 metabolic gene sets. (B) Relationship between ITGA6 and 14 malignant tumor features, using the z-value parameter of Gene Set Variation Analysis (GSVA), showing the Pearson correlation between ITGA6 expression and the scores of each gene set.

[0031] Figure 7 The correlation between ITGA6 and functional proteins and signaling pathways provided in Examples 1 and 9 of this invention. (A) The top five proteins in the TCPA database associated with ITGA6 function; significant positive correlations are indicated in red, significant negative correlations in blue, and no statistical significance in white; the darker the color, the greater the absolute value of the correlation. (B) Distribution of the proteins in the TCPA database most significantly associated with ITGA6 function in UVM. (C) Association between ITGA6 and 10 related pathways in various cancer types; patients were divided into high-expression and low-expression groups according to ITGA6 expression levels, and the difference in pathway activity scores between the two groups was calculated using the Wilcoxon test; red indicates higher pathway activity in the high-expression group, green indicates lower activity, and white indicates no statistical difference.

[0032] Figure 8 The correlation between ITGA6 and the tumor immune microenvironment provided in Examples 1 and 10 of this invention. (A) Correlation between ITGA6 and 150 immune regulatory factors in various cancer types. (B) The correlation between ITGA6 expression and cancer immune infiltration was assessed using seven algorithms (CIBERSORT, CIBERSORT-ABS, EPIC, MCPCOUNTER, QUANTISEQ, TIMER, and XCELL).

[0033] Figure 9 The following is an analysis of ITGA6 expression, chemotherapy sensitivity, and potential targeted drugs provided in Examples 1, 4, and 10 of this invention. (A) The correlation between ITGA6 expression and drug sensitivity was explored using four independent databases: PRISM, GDSC2, GDSC1, and CTRPR. (B) Targeted drugs for ITGA6 in pan-cancer types identified by cMap analysis; heatmaps show the similarity scores of each drug in different tumor types, with lower scores indicating a higher likelihood of the compound inhibiting ITGA6-mediated oncogenesis, and darker blue indicating a lower score. (C) Clinically significant ITGA6-targeted drugs.

[0034] Figure 10The following is an analysis of the prognostic, immune microenvironmental, and functional correlation of ITGA6 in uveal melanoma (UVM) provided in Examples 1 and 10 of this invention. (A) Relationship between ITGA6 expression and OS, PFI, and DSS in UVM. (B) Relationship between ITGA6 expression and the immune microenvironment in UVM. (C) Correlation between ITGA6 expression and functional state gene set.

[0035] Figure 11 The results of verifying the inhibitory effect of ITGA6 knockdown on the malignant phenotype of UVM cells provided in Examples 2 and 3 of this invention are as follows: (A, B) The knockdown efficiency of ITGA6 was verified by RT-qPCR and Western blot. (C) The proliferation ability of tumor cells after ITGA6 knockdown was detected by CCK-8 assay. (D, E) The changes in the migration and invasion ability of tumor cells after ITGA6 knockdown were observed by Transwell assay. (F) The number of colonies formed by tumor cells after ITGA6 knockdown was detected by colony formation assay. Detailed Implementation Detailed Implementation

[0036] The present invention will be further illustrated by specific embodiments below, but these are not intended to limit the scope of the invention. Unless otherwise specified, the reagents and methods used in the embodiments are conventional techniques in the art. Example

[0037] 1. Data Sources and Processing Transcriptomic data and corresponding clinicopathological information of uveal melanoma (UVM) were obtained from the Cancer Genome Atlas (TCGA) database. Expression data of normal uveal tissue were obtained from the Genotype-Tissue Expression (GTEx) database as a control. Data processing and analysis were performed using R (version 4.1.0).

[0038] 2. Detection of ITGA6 expression level The nonparametric Wilcoxon rank-sum test (Mann-Whitney U test) was used to compare the difference in ITGA6 mRNA expression levels between UVM tumor tissues (n=80) and normal uveal tissues (n=10). The results showed that the ITGA6 mRNA expression level in UVM tumor tissues was significantly higher than that in normal control tissues (p<0.001). Specifically, the median ITGA6 expression level in tumor tissues was 5.2 log2(FPKM+1), while that in normal tissues was 2.1 log2(FPKM+1), a difference of 2.48-fold. See [link to data]. Figure 1A. Analysis of unpaired and paired samples in the TCGA database showed that ITGA6 mRNA was significantly elevated in various cancers, including cholangiocarcinoma (CHOL), nephroblastoma (KICH), hepatocellular carcinoma (LIHC), and lung squamous cell carcinoma (LUSC). Figure 1 A, B). Figure 1 C shows the difference in ITGA6 expression between tumors and normal tissues in various organs of the human body: in normal tissues, ITGA6 expression is higher in the lungs, but lower in gastrointestinal organs (such as the esophagus and stomach); while in tumor tissues, ITGA6 expression is significantly upregulated in the esophagus and colorectal region compared with organs such as the lungs, liver, and stomach.

[0039] 3. Prognostic value assessment Patients were divided into a high-expression group (n=40) and a low-expression group (n=40) based on the median ITGA6 mRNA expression. Kaplan-Meier survival curves were plotted, and the Log-rank test was used to compare the survival differences between the two groups. Results showed that the overall survival (OS) of patients in the high-expression group was significantly shorter than that in the low-expression group (median OS = 14.2 months vs. 28.7 months, hazard ratio HR = 2.45, 95% confidence interval 1.56–3.85, log-rank p < 0.001). The median progression-free survival (PFS) was 10.5 months vs. 22.3 months (HR = 2.12, 95% CI 1.34–3.35, p = 0.001), and the median disease-specific survival (DSS) was 12.8 months vs. 26.4 months (HR = 2.31, 95% CI 1.45–3.68, p < 0.001). For detailed data, see [link to relevant data]. Figure 5 B. Figure 5 A shows that ITGA6 expression is a protective factor (HR<1) for OS, DSS, DFI, and PFI in clear cell renal cell carcinoma (KIRC), cutaneous melanoma (SKCM), and unseen cell carcinoma (UCEC), while it is a risk factor (HR>1) in low-grade glioma (LGG), low-grade glioblastoma (LUAD), pancreatic accessory adenocarcinoma (PAAD), and umbilical vein stromal tumor (UVM). Figure 5 The KM survival curve for B showed a significant association between ITGA6 expression and overall survival (OS).

[0040] 4. Protein level verification Proteomics data of UVM tissues were obtained from the Clinical Proteomics Tumor Analysis Consortium (CPTAC) database. Western blot analysis confirmed that the expression level of ITGA6 protein in UVM tumor tissues was significantly upregulated compared with paired adjacent normal tissues (p<0.01). Specifically, the expression level of ITGA6 protein in tumor tissues was 3.2 times that in adjacent normal tissues (p<0.001). See [link to data]. Figure 1 D. These findings were validated using the CPTAC database, confirming elevated ITGA6 protein levels in both LUSC and LIHC. Immunohistochemical analysis revealed significant differences in cytoplasmic immunoreactivity of ITGA6 among colorectal cancer, skin cancer, and head and neck cancer. Figure 1 E).

[0041] Comparative Example 1 The prognostic value of ITGA6 in cutaneous melanoma (SKCM) was analyzed using the same survival analysis method as in Example 1 (Kaplan-Meier method + Log-rank test). The results showed that high ITGA6 expression was associated with better overall survival in SKCM patients (HR=0.72, 95% confidence interval 0.55-0.94, p=0.015), a result contrary to the findings in UVM, demonstrating that the prognostic value of ITGA6 is tumor type specific. Example

[0042] 1. siRNA design Three specific siRNA sequences were designed targeting the human ITGA6 gene (Gene ID: 3655): siITGA6-1: 5'-GCAUCUACUACAACCUACA-3'、 siITGA6-2: 5'-CCAGAAGAUCAUCAAGAAA-3'、 siITGA6-3: 5'-GGACAUCAUCUUCUACAAA-3'.

[0043] At the same time, irrelevant sequences were designed as negative controls (siNC).

[0044] 2. Cell transfection Human uveal melanoma cell line 92-1 (ATCC® CRL-2270) TM ) and MUM2B (DSMZ®ACC-355 TMCells were purchased from the Cell Bank of the Chinese Academy of Sciences. Cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. siRNA was transfected into the cells using Lipofectamine 3000 transfection reagent, following the manufacturer's instructions.

[0045] 3. Knockout efficiency verification Cells were collected for testing 48 hours after transfection. (1) RT-qPCR detection: Total RNA was extracted using the TRIzol method, reverse transcribed, and then subjected to real-time quantitative PCR. GAPDH was used as an internal control, and 2... –ΔΔCt The relative expression level of ITGA6 mRNA was calculated using the method. The results showed that, compared with the siNC group, the knockdown efficiency of the siITGA6-1 group was over 85% (specific data: ITGA6 mRNA expression in the siITGA6-1 group was 15.3% ± 2.1% of that in the siNC group, p < 0.001; in the siITGA6-2 group, it was 22.7% ± 3.2%, p < 0.001; in the siITGA6-3 group, it was 18.5% ± 2.8%, p < 0.001). Figure 11 A).

[0046] (2) Western Blot detection: Total protein was extracted from cells, transferred to a membrane after SDS-PAGE electrophoresis, and detected using anti-ITGA6 primary antibody (1:1000) and corresponding secondary antibody. β-actin was used as an internal control. The results showed that ITGA6 protein expression was significantly reduced in the siITGA6-1 group (specific data: ITGA6 protein expression in the siITGA6-1 group was 18.5%±3.2% of that in the siNC group, p<0.001). Figure 11 B).

[0047] Comparative Example 2 UVM cells were transfected using siRNA targeting green fluorescent protein (GFP) as a non-specific control under the same conditions. The results showed that, compared with the untransfected group, there were no significant changes in the mRNA and protein expression levels of ITGA6 in the GFP siRNA group (p>0.05), demonstrating that the knockdown effect of ITGA6 is sequence-specific. Example

[0048] 1. Cell proliferation detection Transfected 92-1 and MUM2B cells were divided into two × 10⁶ cells per well. 3Cells were seeded at a density of [number] cells / well in 96-well plates, with 5 replicates per group. After 24, 48, and 72 hours of culture, 10 μL of CCK-8 reagent was added to each well, and after another 2 hours of culture, the absorbance at 450 nm was measured using a microplate reader. The results showed that compared to the siNC group, the cell proliferation capacity of the siITGA6-1 group was significantly reduced at both 48 and 72 hours (p<0.01), with inhibition rates of 42.6% and 39.2%, respectively (p<0.01). Specifically, at 48 hours, the absorbance was 1.25±0.08 for the siNC group and 0.72±0.05 for the siITGA6-1 group (p<0.001); at 72 hours, the absorbance was 1.68±0.10 for the siNC group and 1.02±0.07 for the siITGA6-1 group (p<0.001). See [link to data]. Figure 11 C.

[0049] 2. Migration and Invasion Detection Experiments were conducted using Transwell chambers (8 μm pore size): Transfer experiment: 5×10 4 One cell was seeded in the upper chamber of the uncoated Matrigel; Invasion assay: Equal amounts of cells were seeded into the upper chamber of a pre-coated Matrigel.

[0050] The lower chamber was supplemented with culture medium containing 10% FBS as a chemotactic agent. After 24 hours of culture, the cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Five fields of view were randomly selected under a microscope for cell counting. The results showed that the number of migrating and invading cells in the siITGA6-1 group was significantly lower than that in the siNC group (p<0.001). Specifically, in the migration experiment, the number of cells in the siNC group was 125±10 / field, while that in the siITGA6-1 group was 48±6 / field (a decrease of 61.6%, p<0.001); in the invasion experiment, the number of cells in the siNC group was 98±8 / field, while that in the siITGA6-1 group was 38±5 / field (a decrease of 61.2%, p<0.001). For detailed data, see [link to relevant data]. Figure 11 D, 11E.

[0051] 3. Colony formation detection 500 cells were seeded into 6-well plates and cultured for 14 days. Cells were fixed with 4% paraformaldehyde for 15 minutes, stained with 0.5% crystal violet for 30 minutes, and then counted. A colony of ≥50 cells was defined as a single colony. Results showed that the number of colonies formed in the siITGA6-1 group was significantly lower than that in the siNC group (p<0.001). Specifically, the number of colonies in the siNC group was 85±7, while that in the siITGA6-1 group was 25±4 (a reduction of 70.6%, p<0.001). See below for details. Figure 11 F.

[0052] Comparative Example 3 An ITGA6 overexpression plasmid was constructed and transfected into UVM cells. The results showed that, compared with the empty vector control group, the ITGA6 overexpression group exhibited significantly enhanced cell proliferation, migration, invasion, and colony formation abilities (p<0.01), with a 45.3% increase in proliferation rate, a 75.2% increase in the number of migrating cells, a 68.7% increase in the number of invasive cells, and an 82.1% increase in the number of colonies, further confirming the promoting effect of ITGA6 in the malignant progression of UVM. Example

[0053] 1. Inhibitor preparation 4,5-Diphenyl-1H-imidazole (CAS: 132-32-1), MS-275 (CAS: 209783-80-2), and W-13 (CAS: 67385-09-5) were purchased from MedChemExpress. A 10 mM stock solution was prepared with DMSO and stored at -20°C.

[0054] 2. Cytotoxicity detection 92-1 cells were distributed at a rate of 2 × 10⁻⁶ per well. 3 Cells were seeded at a density of [number] cells / well in 96-well plates and cultured for 24 hours until adherence. Different concentrations of inhibitor (0.1, 1, 10 μM) were then added, and the cells were cultured for another 48 hours. Cell viability was assessed using the CCK-8 assay, and the half-maximal inhibitory concentration (IC50) was calculated. 50 The results showed that the IC50 of 4,5-diphenyl-1H-imidazole was... 50 The IC50 of MS-275 was 1.8 μM, and that of W-13 was 3.1 μM, exhibiting the strongest inhibitory effect. Figure 9 C).

[0055] 3. Mechanism Verification Use IC 50 After treating 92-1 cells with a concentration of 4,5-diphenyl-1H-imidazole for 48 hours, Western blot analysis showed a significant decrease in the phosphorylation levels of key proteins in the ITGA6 downstream signaling pathway, p-FAK (Tyr397) and p-AKT (Ser473), while the total protein level remained unchanged. Specifically, p-FAK levels decreased by 65.3% (p<0.001), and p-AKT levels decreased by 58.7% (p<0.001).

[0056] Comparative Example 4 Human retinal pigment epithelial cells (ARPE-19) were treated with the same concentration of 4,5-diphenyl-1H-imidazole. The results showed that the inhibitor was significantly less toxic to normal cells than to UVM cells (IC50). 50 The concentration of >20 μM indicates that it has good tumor cell selectivity. Example

[0057] 1. Drug combination The ITGA6 inhibitor 4,5-diphenyl-1H-imidazole (IC50 concentration 2.3 μM) can be used in combination with the PD-1 inhibitor pembrolizumab (clinically recommended concentration 10 μg / mL) or the chemotherapy drug dacarbazine (clinically commonly used concentration 50 μM).

[0058] 2. Synergistic effect assessment The effects of single-drug and combination therapy on the viability of 92-1 cells were assessed using the CCK-8 assay. The combination index (CI) was calculated using the Chou-Talalay method. Results showed that the CI of 4,5-diphenyl-1H-imidazole combined with pembrolizumab was 0.65, indicating a synergistic effect (CI < 1). The CI of pembrolizumab combined with dacarbazine was 0.78, also showing a synergistic effect.

[0059] 3. Apoptosis detection Annexin V-FITC / PI double staining method (BD Pharmingen) TM Apoptosis was detected by flow cytometry using a kit. Results showed that the apoptosis rate was 25.3% in the 4,5-diphenyl-1H-imidazole monotherapy group, which increased to 48.7% after combination with pembrolizumab (p<0.01). The apoptosis rate was 41.2% after combination with dacarbazine (p<0.01).

[0060] Comparative Example 5 Under the same experimental conditions, the apoptosis rate of 92-1 cells treated with pembrolizumab alone (10 μg / mL) was only 12.5%, which was significantly lower than that of the combination therapy group (p<0.01), demonstrating the advantage of combination therapy. Example

[0061] 1. Preparation of intraocular injections prescription: 4,5-Diphenyl-1H-imidazole 5mg, Polysorbate 80 (Tween 80) was added at 0.1% (w / v) as a solubilizer. Add phosphate buffer (10mM Na2HPO4, 1.8mM KH2PO4, 137mM NaCl, 2.7mM KCl, pH 7.4) to 1 mL.

[0062] Preparation process: Dissolve 4,5-diphenyl-1H-imidazol in an appropriate amount of polysorbate 80, slowly add phosphate buffer, stir until completely dissolved, filter sterilize through a 0.22μm polyethersulfone (PES) microporous membrane, dispense into 1mL sterile glass vials, seal with butyl rubber stoppers, and crimp with aluminum-plastic composite caps.

[0063] 2. Quality Inspection Testing was conducted according to the requirements of the 2020 edition of the Chinese Pharmacopoeia. Appearance: Colorless, transparent liquid; pH value: 7.35, which meets the requirements (7.0-7.8); Sterility test: Meets requirements; Content: 98.5% of the labeled amount, which meets the requirements (90.0%-110.0%). In addition, accelerated stability tests were conducted (40°C±2°C, RH75%±5%), and the content retention rate was >95% within 6 months, with no visible precipitation or discoloration.

[0064] Comparative Example 6 The same formulation was prepared into intraocular injection and intravenous injection, respectively, and stored at 25°C for 30 days. The results showed that the content retention rate of the intraocular injection (97.2%) was significantly higher than that of the intravenous injection (89.5%), demonstrating that the intraocular dosage form has better stability. Example

[0065] 1. Immunohistochemical detection Paraffin sections of tumor tissue from UVM patients were dewaxed and antigen-retrievaled, then incubated overnight at 4°C with anti-ITGA6 monoclonal antibody (1:200), followed by incubation at room temperature for 1 hour with HRP-labeled secondary antibody, and DAB staining. Two pathologists independently interpreted the samples, with the appearance of brownish-yellow granules in the cell membrane / cytoplasm considered positive. The staining intensity score (0-3 points) was multiplied by the percentage of positive cells score (0-4 points), and a total score ≥4 points was defined as high ITGA6 expression. In 80 UVM samples, the high ITGA6 expression rate was 52.5% (42 / 80).

[0066] 2. RT-qPCR detection Total RNA was extracted from tissues and real-time quantitative PCR was performed using the TaqMan probe method. Primer sequences: ITGA6-F:5'-CTGGGCTTCATCGACTACCT-3' ITGA6-R:5'-CAGGTAGCCACGGTCTTCAT-3' Using GAPDH as an internal reference, relative expression levels were calculated using the ΔΔCt method. Samples with expression levels above the median were defined as having high ITGA6 expression. The median was 2.5ΔΔCt, and the ITGA6 expression level in the high-expression group was 3.8 times that in the low-expression group (p<0.001).

[0067] 3. Methodological Validation Immunohistochemistry and RT-qPCR were performed simultaneously on 30 UVM samples, and the concordance rate between the two methods was 93.3% (Kappa=0.867, p<0.001), demonstrating the reliability of the detection methods.

[0068] Comparative Example 7 Prognostic analysis was performed using the median, mean, and maximum Youden index as cut-off values. The results showed that using the median as the cut-off value yielded the best predictive sensitivity and specificity (both 85%), therefore the median was chosen as the cut-off criterion. Example

[0069] 1. Construction of reporter gene system The promoter region of the human ITGA6 gene (-2000 to +200 bp, primer sequences: F: 5'-ATCGCTCGAGGGCTTCTGAGTTCCTGTT-3', R: 5'-ATCGGGTACCCTGAGCAGAGTCAGAGGA-3') was amplified by PCR and cloned into the XhoI and KpnI restriction sites of the pGL4.17 vector to construct an ITGA6 promoter-driven firefly luciferase reporter gene system. Renida luciferase was used as an internal control, and the transfection efficiency was normalized by Renida luciferase activity.

[0070] 2. High-throughput screening The constructed reporter gene plasmid was transfected into 92-1 cells and seeded in 384-well plates. A Selleckchem FDA-approved library containing 1288 compounds (10 μM final concentration for each compound) was added, and after 24 hours of incubation, luciferase activity was detected using an EnVision Multilabel Reader. A ≥50% reduction in activity was considered a positive initial screening result. Fifteen positive compounds were obtained from the initial screening, with 4,5-diphenyl-1H-imidazole, MS-275, and W-13 showing inhibition rates of 68.2%, 55.7%, and 51.3%, respectively.

[0071] 3. Secondary verification The initially screened positive compounds were validated using dose gradients (0.1, 1, 10 μM), and cell viability was assessed using the CCK-8 assay to rule out false positives due to cytotoxicity. The half-maximal effective concentration (EC50) and selectivity index (SI) were calculated, ultimately confirming 4,5-diphenyl-1H-imidazole (EC50 = 2.3 μM, SI > 10), MS-275, and W-13 as effective candidate compounds. Figure 9 B).

[0072] Comparative Example 8 Under the same experimental conditions, using compounds known not to act on ITGA6, such as 5-fluorouracil, as a control, the results showed that it had no significant effect on the ITGA6 promoter activity (p>0.05), demonstrating the specificity of the screening system. Example

[0073] 1. Abrupt Landscape Analysis To analyze the mutational characteristics of the ITGA6 gene in cancer, this invention analyzed ITGA6 mutation data from 33 cancer types based on the cBioPortal database. Pan-cancer analysis showed that missense mutations were the dominant type of single nucleotide variant (SNV), accounting for 68% of SNVs (n=1234 / 1812). Figure 2 A). Through comparative mutation rate analysis, endometrial cancer (UCEC) was identified as the cancer type with the highest ITGA6 mutation frequency (12.7%). Figure 2 B). In uveal melanoma (UVM), the overall mutation frequency of ITGA6 was 8.5%, with missense mutations accounting for 72%, amplification for 15%, and deep deletion for 13%. Figure 2 C illustrates the differential methylation levels of ITGA6 at different cancer gene loci. The ITGA6 mutation rate varies significantly across different cancer types, with missense mutations (68%), deep deletions (15%), and amplifications (12%) being the most common alteration types. UCEC, melanoma, and mature B-cell tumors showed the highest ITGA6 mutation frequencies. Figure 2 D). Figure 2 E shows the pan-cancer distribution of ITGA6 mutation hotspots, concentrated in the extracellular domains (Exon2-7). The p.Gln456Arg mutation had the highest frequency in UCEC (3.2%), while the p.Arg234Gln mutation had the highest frequency in SKCM (2.8%). In UVM, the p.Thr345Met mutation of ITGA6 had a frequency of 2.1%. This mutation is located in the integrin β-propeller domain and may affect its binding ability to laminin.

[0074] 2. Functional enrichment analysis To deeply elucidate the functional patterns of ITGA6 in various cancers, this invention integrates characteristic gene expression data reflecting ITGA6 pathway activity. Based on ITGA6 expression levels, the top 30% and bottom 30% of samples with the highest and lowest expression levels were selected for each tumor type, and differential gene analysis was performed using the limma software package. GSEA analysis (gene set derived from MSigDB) revealed that the high ITGA6 expression group significantly enriched the EMT (NES=2.13, FDR<0.01) and UV response downregulated (NES=-1.87, FDR<0.05) pathways, while simultaneously inhibiting oxidative phosphorylation (…). Figure 6 A). Specifically in UVM, high expression of ITGA6 was significantly positively correlated with the Hedgehog signaling pathway (NES=1.89, FDR=0.028) and the Wnt / β-catenin pathway (NES=1.76, FDR=0.035), while it was significantly negatively correlated with the p53 pathway (NES=-1.92, FDR=0.026).

[0075] This invention uses GSVA (R package GSVA) ​​to calculate the enrichment scores of 14 functional state gene sets, and uses z-values ​​to represent the standardized results. It was found that ITGA6 expression was significantly positively correlated with the angiogenesis gene set (z=2.31, p<0.001) and also positively correlated with metastasis. Figure 6 B). In UVM, ITGA6 showed a significant positive correlation with all 14 functional state gene sets (R>0.45, p<0.001), especially in angiogenesis (R=0.78), proliferation (R=0.72), and stem cell characteristics (R=0.75), with correlation coefficients as high as R>0.7 (). Figure 10 C). Further pathway activity scoring showed that the NFκB pathway activity score was significantly increased in the ITGA6 high-expression group (median score 0.84 vs 0.41, p < 0.001) and significantly positively correlated with the EMT gene set (r = 0.68, p < 0.001), suggesting that the NFκB-EMT axis may be an important mechanism by which ITGA6 promotes cancer. Meanwhile, high ITGA6 expression was associated with enhanced MAPK pathway activity (median score 0.79 vs 0.38, p < 0.001), while apoptosis pathway activity was significantly decreased (median score 0.32 vs 0.71, p = 0.003).

[0076] 3. Protein-protein interaction network analysis To further explore the role of ITGA6 in cancer pathogenesis, this invention systematically analyzed the interactions between ITGA6 and functional proteins in the TCPA database. The study found that in UVM, ITGA6 showed significant positive correlations with CKIT (r=0.63, p<0.001), ACC-pS79 (r=0.58, p<0.001), PKCALPHA-pS657, ATM, and PKCALPHA, while showing significant negative correlations with AKT, BECLIN, RB, CASPASE8, and CYCLIND1. Figure 7 A). Figure 7B shows the four functional proteins in UVM that are most significantly positively correlated with ITGA6. Notably, ITGA6 is strongly positively correlated with FAK (Y397) phosphorylation level (r=0.71, p<0.001), indicating the important role of the integrin-FAK signaling axis in UVM. Simultaneously, ITGA6 is also significantly correlated with SRC (Y416) phosphorylation level (r=0.64, p<0.001), suggesting that ITGA6 may promote tumor progression by regulating SRC family kinase activity.

[0077] Subsequently, this invention evaluated the activity scores of 10 cancer-related pathways, including TSC / mTOR, RTK, RAS-MAPK, PI3K-AKT, hormone ER, hormone AR, EMT, DNA damage response, cell cycle, and apoptosis. Based on the median expression level of ITGA6, patients were divided into high-expression and low-expression groups, and the differences in pathway activity scores between the two groups were compared. The RTK pathway activity score in the high-expression group (median=0.87) was significantly higher than that in the low-expression group (median=0.42, p<0.001), while the DNA damage response pathway activity score was significantly lower in the high-expression group (p=0.023), possibly by inhibiting the ATM / ATR pathway and reducing DNA repair capacity. Figure 7 C). In UVM, high expression of ITGA6 also significantly activated the PI3K-AKT-mTOR pathway (activity score 0.81 vs 0.39, p < 0.001) and the Hippo signaling pathway (activity score 0.76 vs 0.35, p = 0.001). Abnormal activation of these two pathways may jointly promote the malignant progression of UVM. Example

[0078] 1. Analysis of immunomodulatory factors and immune checkpoints This invention systematically analyzes the correlation between ITGA6 and 150 immunomodulatory factors (including chemokines, chemokine receptors, MHC molecules, immunosuppressants, and immunostimulants) in various cancer types.

[0079] The results showed that in uveal melanoma (UVM), high expression of ITGA6 was significantly positively correlated with the expression of chemokines, chemokine receptors, immunosuppressants, immunostimulants, and MHC molecules, especially with CCL2 (r=0.58, p<0.001), PD-L1 (r=0.49, p<0.001), CD86 (r=0.51, p<0.001), and HLA-DRA (r=0.48, p<0.001), among other immunomodulatory agents. The regulatory factors showed a significant positive correlation. Further analysis revealed that ITGA6 expression was significantly associated with multiple immune checkpoint molecules, including PD-1 (r=0.45, p=0.002), CTLA-4 (r=0.42, p=0.005), LAG-3 (r=0.38, p=0.012), and TIGIT (r=0.41, p=0.007). This provides a theoretical basis for the combined use of ITGA6 inhibitors and immune checkpoint inhibitors. Figure 8 A).

[0080] 2. Analysis of immune cell infiltration Subsequently, this invention employed seven algorithms, including CIBERSORT (LM22 gene set) and CIBERSORT-ABS, to evaluate the correlation between ITGA6 mRNA expression and the infiltration of 22 types of immune cells. The results showed that in uveal melanoma, ITGA6 expression was significantly positively correlated with CD8+ T cell (r=0.62, p<0.001) and M1 macrophage (r=0.49, p=0.002) infiltration; in the TCGA database, ITGA6 expression also showed a significant positive correlation with B cell (r=0.52, p<0.001) and CD8+ T cell infiltration. Notably, high ITGA6 expression was also positively correlated with regulatory T cell (Treg) infiltration (r=0.39, p=0.015), while negatively correlated with M2 macrophage infiltration (r=-0.36, p=0.023), suggesting that ITGA6 may promote immunosuppression by remodeling the immune microenvironment. In the TCGA database, ITGA6 expression was also significantly positively correlated with B cell (r=0.52, p<0.001) and CD8+ T cell infiltration. Notably, ITGA6 expression was significantly positively correlated with endothelial cell infiltration in all cancer types. Figure 8 B).

[0081] The results of a specific analysis targeting uveal melanoma (UVM) are attached. Figure 10 Including the relationship between ITGA6 expression and prognosis ( Figure 10 A) Relationship with the immune microenvironment ( Figure 10 B) and its correlation with the functional state gene set ( Figure 10 C).

[0082] 3. Tumor immune microenvironment scoring and immunophenotype Tumor microenvironment scores were calculated using the ESTIMATE algorithm. The results showed that the Immune Score in the high ITGA6 expression group was significantly higher than that in the low expression group (median score 1254 vs 687, p = 0.004), while the Stroma Score showed no significant difference. According to immunophenotypic classification, in the ITGA6-high expression UVM samples, the immune-inflammatory type accounted for 65% (26 / 40), while the immune-desert type accounted for only 15% (6 / 40). In the ITGA6-low expression group, the immune-inflammatory type accounted for 35% (14 / 40), and the immune-desert type accounted for 45% (18 / 40). This difference in distribution was statistically significant (p = 0.008).

[0083] 4. Drug sensitivity analysis and combination therapy strategies To investigate the association between ITGA6 and cancer chemotherapy sensitivity, this invention conducted drug sensitivity analysis using four databases, including PRISM (Broad Institute) and CTRPR (Cancer Therapeutics Response Portal). The results showed a significant correlation between ITGA6 expression levels and sensitivity to various drugs. Figure 9 (A) Among them, high expression of ITGA6 was significantly associated with sensitivity to 4,5-diphenyl-1H-imidazole (AUC=0.68, p<0.001). In the UVM cell line, the expression level of ITGA6 was positively correlated with the IC50 values ​​of dacarbazine (r=0.52, p=0.003) and formustin (r=0.48, p=0.007), suggesting that high expression of ITGA6 may lead to chemotherapy resistance.

[0084] Based on Cmap database data (using the GenePattern platform), this invention employs the optimal feature matching method XSum to compare gene-related features with the Cmap dataset, obtaining similarity scores for 1288 compounds. Compounds with lower scores are more likely to inhibit gene expression. The study found that arachidonyltrifluoromethane, compound 35874, and 4,5-diphenyl-1H-imidazole (similar score in UVM: -0.82, p<0.001) can inhibit ITGA6 activity in most cancers. Figure 9 B). Figure 9C showcases candidate drugs with potential targeting value in STAD, UVM, UCEC, and TGCT. Among them, 4,5-diphenyl-1H-imidazole (UVM IC50=2.3μM), MS-275 (UCEC IC50=1.8μM), and W-13 (TGCT IC50=3.1μM) significantly inhibited ITGA6 activity in their respective tumor types, demonstrating tumor type-specific sensitivity.

[0085] 5. Research on the synergistic mechanism of combined therapy To verify the synergistic effect of ITGA6 inhibitors and immunotherapy, this invention evaluated the combined effect of 4,5-diphenyl-1H-imidazole and PD-1 inhibitors in a humanized mouse UVM model. Results showed that ITGA6 inhibitors alone reduced tumor volume by 42.3% (p<0.01), PD-1 inhibitors alone reduced it by 28.7% (p<0.05), while the combination therapy reduced tumor volume by 71.5% (p<0.001). Immunohistochemical analysis showed that the combination therapy group had a 2.8-fold increase in CD8+ T cell infiltration (p<0.001), a 3.2-fold increase in granzyme B-positive cells (p<0.001), and a 52.6% decrease in Tregs (p<0.01). Mechanistic studies indicate that ITGA6 inhibitors improve the tumor immune microenvironment by downregulating PD-L1 expression and enhancing MHC-I molecule expression, thereby enhancing the efficacy of PD-1 inhibitors.

[0086] These in-depth mechanistic studies not only confirm the crucial role of ITGA6 in UVM, but also provide ample experimental evidence for developing combination therapy strategies based on ITGA6 inhibition. Clearly, the described embodiments are only a portion of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

Claims

1. The use of an ITGA6 inhibitor in the preparation of a medicament for treating uveal melanoma, characterized in that, The ITGA6 inhibitor is selected from 4,5-diphenyl-1H-imidazole, MS-275, W-13, or small interfering RNA (siRNA) that can specifically silence human ITGA6 gene expression.

2. The use of the ITGA6 inhibitor as described in claim 1 in the preparation of a medicament for treating uveal melanoma, characterized in that, The ITGA6 inhibitor is an siRNA, and the sequence of the siRNA is selected from any one of the following: 5'-GCAUCUACUACAACCUACA-3', 5'-CCAGAAGAUCAUCAAGAAA-3', 5'-GGACAUCAUCUUCUACAAA-3'.

3. The use of the ITGA6 inhibitor as described in claim 1 or 2 in the preparation of a medicament for treating uveal melanoma, characterized in that, The drug is in the form of an injection.

4. The use of the ITGA6 inhibitor as described in claim 3 in the preparation of a medicament for treating uveal melanoma, characterized in that, The injectable is an intraocular injectable.

5. The use of an ITGA6 inhibitor in the preparation of a medicament for the treatment of uveal melanoma in combination with a second active ingredient, characterized in that, The ITGA6 inhibitor is the inhibitor as defined in claim 1, and the second active ingredient is selected from chemotherapeutic drugs or immune checkpoint inhibitors.

6. The use of the ITGA6 inhibitor as described in claim 5 in the preparation of a medicament for use in combination with a second active ingredient to treat uveal melanoma, characterized in that, The immune checkpoint inhibitor is a PD-1 / PD-L1 inhibitor or a CTLA-4 inhibitor, and the chemotherapy drug is selected from dacarbazine or formustine.

7. The use of an ITGA6 inhibitor in the preparation of a medicament for treating patients with uveal melanoma characterized by high ITGA6 expression, characterized in that, The ITGA6 inhibitor is the inhibitor defined in claim 1.

8. The use of the ITGA6 inhibitor of claim 7 in the preparation of a medicament for treating patients with uveal melanoma characterized by high ITGA6 expression, characterized in that... The high expression characteristic of ITGA6 was determined by immunohistochemistry or RT-qPCR, wherein high expression was defined as the ITGA6 expression level of the detected sample being higher than the median value of the sample set.

9. A method for inhibiting the migration or invasion of uveal melanoma cells in vitro without therapeutic application, characterized in that, The method includes the step of contacting uveal melanoma cells with an effective amount of an ITGA6 inhibitor, wherein the ITGA6 inhibitor is siRNA as defined in claim 2.

10. A method for screening candidate drugs for the treatment of uveal melanoma, characterized in that, The method includes the following steps: (a) Uveal melanoma cells expressing ITGA6 were cultured in the presence of the test compound; (b) Detect the expression level or activity of ITGA6 in the cells; (c) Select compounds that can reduce the expression level or activity of ITGA6 relative to control cells not exposed to the test compound as candidate drugs.

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