Abstract: Objective To investigate the expression of the ubiquitination enzyme UBE2S in different cell types in hepatocellular carcinoma (HCC) microenvironment and its impact on proliferation and stemness of HCC cells. Methods TCGA and CPTAC database were used to analyze the transcriptional and promoter methylation levels and protein expressions of UBE2S in HCC. Specific expression patterns of UBE2S, intercellular communication and key transcription factors in different cell types were analyzed based on single-cell sequencing data from TISCH website. We further examined UBE2S expressions in clinical samples of HCC tissues, HCC cells and T cells using immunohistochemistry and immunofluorescence staining. We also tested the effects of UBE2S knockdown on stemness of HCC-LM3 and HepG2 cells using clone formation experiments and sphere formation assay. Results Analysis based on TCGA database suggested significant overexpression of UBE2S in both paired and non-paired tumor tissues (P<0.001), and its transcriptional level increased with tumor grades. The methylation level of UBE2S promoter was significantly decreased in HCC (P<0.001), and its transcription level increased obviously in HCC with TP53 mutation (P<0.001). Analysis of CPTAC database also demonstrated overexpression of UBE2S protein in HCC tissues (P<0.001). Three prognostic models suggested that HCC patients with high UBE2S expression had poorer prognosis (P<0.001). Single-cell sequencing data analysis revealed high expressions of UBE2S in T cells and high intensities of interaction between endothelial cells, epithelial cells and fibroblasts in HCC microenvironment. Immunohistochemistry and immunofluorescence staining demonstrated high UBE2S expressions in clinical samples of HCC tissues, HCC cells and T cells. In HCC-LM3 and HepG2 cells, UBE2S knockdown significantly inhibited cell clone formation and tumor sphere formation (P<0.05). Conclusion UBE2S is highly expressed in T cells in HCC microenvironment in close correlation with a poor prognosis. High UBE2S expression promotes the stemness of HCC cells.

Key words: single-cell sequencing; UBE2S; hepatocellular carcinoma; T cells