GLO1 regulates hepatocellular carcinoma proliferation and migration through the cell cycle pathway

Abstract

Background: Hepatocellular carcinoma (HCC) is a malignant tumor characterized by a high mortality rate. The occurrence and progression of HCC are linked to oxidative stress. Glyoxalase-1 (GLO1) plays an important role in regulating oxidative stress, yet the underlying mechanism remains unclear. GLO1 may serve as a prognostic biomarker and therapeutic target for HCC.

Methods: Based on TCGA database hepatocellular carcinoma samples, we conducted a bioinformatics analysis to explore the correlation between GLO1 expression and HCC cell proliferation and viability. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that differentially expressed genes (DEGs) were mainly enriched in the cell cycle pathway. We analyzed the relationships between GLO1 and 24 genes enriched in the cell cycle pathway using a protein-protein interaction (PPI) network. Finally, experimental validation was performed to assess GLO1's impact on the distribution of cells at different cell cycle stages and on the proliferation and migration of HCC cells.

Results: Our study demonstrated that GLO1 was overexpressed in HCC tissues and was associated with a poor prognosis. Data analysis indicated that overexpression of GLO1 activated the cell cycle pathway and positively correlated with expression of the majority of key cell cycle genes. Experimental validation showed that GLO1 expression affects the number of HCC cells in G2 and S phases and regulates HCC cell proliferation and migration.

Conclusions: GLO1 represents a promising therapeutic target for HCC, providing valuable insights into its role in the viability and proliferation of HCC cells.

Keywords: GLO1; Biomarker; Cell cycle; Hepatocellular carcinoma; Therapeutic target.

Fig. 1

GLO1 expression levels and survival analysis in HCC. (A) Comparison of GLO1 expression levels in HCC tissues and normal tissues was investigated by TCGA database. *p < 0.5, **p < 0.01, ***p < 0.001 between the two groups. (B) Protein levels from the Human Protein Atlas (HPA) assay GLO1 in normal and HCC tumor tissues. (C) GLO1 expression levels in HCC tissues compared to normal tissues verified by the GEO database. Between the two groups *p < 0.5, **p < 0.01, ***p < 0.001. (D) Kaplan-Meier survival analysis based on high and low GLO1 expression. overall survival (OS), disease-free survival (DFS), disease-specific survival (DSS), and progression-free survival (PFS)

Fig. 2

Analysis of GLO1 expression and immune cell infiltration in HCC. (A) Association of GLO1 expression levels with differences in immune and stromal scores in HCC. Between the two groups *p < 0.5, **p < 0.01, ***p < 0.001. (B) Correlation analysis of GLO1 mRNA expression levels with immune score and stromal score in HCC. (C) The relationship between GLO1 gene expression and the level of infiltration of six types of immune cells in HCC was investigated using the Tumor Immune Estimation Resource (TIMER) database

Fig. 3

Identification of DEGs in HCC and GO and KEGG enrichment analysis. (A) Volcano plots show the overall distribution of DEGs between the high and low GLO1 expression groups. (B) Heatmap of DEGs generated by comparison between the high and low GLO1 expression groups. (C) GO enrichment analysis of 458 DEGs was conducted via DAVID. GO terms are classified as biological process, cellular component, or molecular function terms. (D) KEGG pathway analysis revealed the signaling pathways in which the DEGs were enriched. Each point represents the enrichment level. The color corresponds to -log10 (adjusted p-value), and the size corresponds to the number of enriched genes

Fig. 4

Correlation analysis and PPI network analysis. (A) Correlation analysis of GLO1 and 24 genes enriched in the cell cycle pathway. (B) The PPI network of GLO1 and 24 cell cycle–related genes was constructed using the NetworkAnalysis tool

Fig. 5

GLO1 knockout and overexpression in Hep3B and Huh-7 cells. (A) mRNA expression after GLO1 knockout and overexpression in Hep3B and Huh-7 cell lines was detected by qRT-PCR. (B, C) Protein expression after GLO1 knockout and overexpression in Hep3B and Huh-7 cell lines was determined by Western blot analysis (Original version in Supplementary Fig. 3). Statistical significance was determined by t-test: * p < 0.05; ** p < 0.01; *** p < 0.001

Fig. 6

Effect of GLO1 knockout and overexpression of HCC cells in each cell cycle stage. GLO1 knockout and overexpression mainly affected the number of Hep3B and Huh-7 cells in G2 and S phases. Statistical significance was determined by t-test: * p < 0.05; ** p < 0.01; *** p < 0.001

Fig. 7

GLO1 affects HCC cell proliferation, migration, and invasion by regulating the cell cycle pathway. (A, B) Proliferative capacity of GLO1-knockout and GLO1-overexpressing HCC cells was determined by CCK-8 assay. (C-F) Migration ability of GLO1-knockout and GLO1-overexpressing HCC cells was determined by wound healing assay. (G, H) Invasive ability of GLO1-knockout and GLO1-overexpressing HCC cells was determined by invasion assay. Statistical significance was determined by t-test: * p < 0.05; ** p < 0.01; *** p < 0.001

Fig. 8

GLO1 affects the growth of HCC cells in zebrafish. (A) Schematic depicting the procedure for constructing a zebrafish HCC xenograft model. (B) Fluorescence microscopy images of DiI stained HCC cells injected into the yolk region of zebrafish. Bright-field microscopy of whole zebrafish (AB line) 24 h after injection of human HCC cells (upper panel). Fluorescence microscopy of DiI stained HCC cells under red fluorescent protein (RFP) channel (middle). Merged bright-field and RFP field microscopy images (bottom). (C) Growth of DiI stained HCC cell xenografts in zebrafish