Up-regulation of thrombospondin-1 inhibits the progression of gallbladder cancer

Abstract

Gallbladder cancer, the most prevalent malignant neoplasm of the biliary tract, has garnered significant attention due to its dismal prognosis and high degree of malignancy. Identifying key regulatory genes is crucial for the development of effective therapeutic strategies. The differential gene expression in biliary tract malignancies was identified using the Gene Expression Omnibus (GEO) database. Subsequently, the interactions among these differentially expressed genes were analyzed employing the STRING database, and the resultant regulatory network was visualized using Cytoscape software. Utilizing the Cytoscape plugin CytoHubba, the core genes within the network were identified, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Ultimately, the overexpression of THBS1 in the gallbladder cancer cell line (NOZ) was achieved through lentiviral transfection and both in vivo and in vitro experiments were conducted to evaluate its effects. We found that thrombospondin-1 (THBS1) was the core gene of gallbladder cancer and its expression was low in gallbladder cancer. Experimental data, both in vivo and in vitro, indicate that the up-regulation of THBS1 exerts an inhibitory effect on the proliferation, migration, and invasion of gallbladder cancer cells. Furthermore, it facilitates the process of apoptosis and suppresses tumor growth and angiogenesis. The expression of THBS1 is low in gallbladder cancer. Up-regulation of THBS1 can effectively inhibit the occurrence and development of gallbladder cancer and can be used as a biomarker for the diagnosis and treatment of gallbladder cancer.

Keywords: Biomarker; Gallbladder cancer; THBS1.

Fig. 1

A Heat map depicting differential gene expression in GSE132305. B Volcano plot illustrating differential gene expression in GSE132305. C Heat map depicting differential gene expression in GSE127897. D Volcano plot illustrating differential gene expression in GSE127897. E Intersection genes from GSE132305 and GSE127897 (up-regulated). F Intersection genes from GSE132305 and GSE127897 (down-regulated)

Fig. 2

A Gene interaction network diagram for the intersection of GSE132305 and GSE132305, with 19 down-regulated and 4 up-regulated genes (red indicates up-regulation, green indicates downregulation). B Core genes within the network regulation, and the results indicate that VCAN, EMP1, GPX3, ADRB2, THBD, CCL2, NFKBIZ, EGR1, THBS1, and CXCL2 are the key HUB genes, with a darker color signifying a stronger correlation. C GO enrichment analysis (BP, CC, and MF showing the top three enriched functions), E KEGG enrichment analysis (displaying the top ten enriched pathways), D GO circle diagram, F KEGG circle diagram

Fig. 3

A Immunohistochemical analysis of THBS1 in gallbladder cancer and normal gallbladder tissue reveals that THBS1 staining was light in gallbladder cancer and deep in cholecystitis tissue. The AOD values were higher in cholecystitis tissue than in gallbladder cancer tissue. B Fluorescence diagram of gallbladder cancer cell line (NOZ) after lentiviral transformation (from left to right: Normal/Fluorescence/Merged), RT-PCR analysis detected elevated THBS1 expression in the THBS1-OE group compared to the THBS1-NC group. C Colony formation experiments demonstrated a reduced number of colonies in the THBS1-OE group relative to both the THBS1-NC and Control groups. D Wound healing assays indicated a slower wound closure rate in the THBS1-OE group compared to the THBS1-NC and Control groups. E, F Transwell migration and invasion experiments showed that the number of cells passing through the gel chamber with or without matrix in THBS1-OE group was lower than that in THBS1-NC and Control groups (E without matrix glue, F with matrix glue). G Western blot experiments indicated that the pro-apoptotic proteins Bax and Caspase-3 were activated, whereas the anti-apoptotic protein Bcl-2 was down-regulated in gallbladder cancer cells upon THBS1 up-regulation. H The results of flow cytometry apoptosis detection showed that the apoptosis rate of THBS1-OE group was higher than that of THBS1-NC group and control group

Fig. 4

A In the nude mouse xenograft model, tumors were excised on day 30 for mass determination, showing that the THBS1-OE group had a lower tumor weight than the THBS1-NC and Control groups. B Immunohistochemical analysis of the excised tumors from nude mice exhibited markedly increased THBS1 staining in the THBS1-OE group compared to the THBS1-NC and Control groups. The AOD values were higher in the THBS 1-OE group than in the THBS 1-NC and Control groups. C Hematoxylin and Eosin (H&E) staining indicated a reduced number of newly formed vessels in the THBS1-OE group compared to the corresponding THBS1-NC and Control groups (× 40 zoom level)