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.
目的: 探究UBE2S在肝癌微环境不同细胞类型的特异性表达及对肝癌细胞干性的影响。
方法: 使用TCGA数据库分析肝癌中UBE2S转录水平及其启动子甲基化水平差异，使用CPTAC数据库分析UBE2S蛋白水平差异。使用TISCH单细胞测序数据挖掘UBE2S在不同细胞类型中的表达，并分析细胞通讯的强度以及细胞群各自的关键转录因子。免疫组化验证UBE2S在肝癌组织中的表达；多色免疫荧光检测UBE2S在肝癌细胞以及T细胞的表达情况。利用shRNA敲低肝癌细胞HCC-LM3中的UBE2S，分为shCtrl（对照组）、shUBE2S-1（敲低组1）、shUBE2S-2（敲低组2）3个组，利用克隆形成实验、悬浮成球实验检测UBE2S敲低对HCC-LM3干性影响。
结果: UBE2S在TCGA数据库中非配对肿瘤组织中高表达（P < 0.001），配对肿瘤组织中也高表达（P < 0.001），随着肿瘤分级增高，转录水平逐渐增高（P < 0.001）；在CPTAC数据库中肝癌组织UBE2S蛋白水平表达也增高（P < 0.001）；肝癌中UBE2S启动子甲基化水平降低（P < 0.001）；TP53突变组UBE2S转录水平更高（P < 0.001）。单细胞测序结果显示，UBE2S在T细胞等细胞群高表达；内皮与上皮及成纤维细胞之间的细胞交互强度较高，SMAD1等转录因子可能是Tprolif细胞关键转录因子。与正常组织相比，UBE2S在肝癌组织高表达；且在肝癌细胞和T细胞中均有较高表达。克隆形成实验显示：与shCtrl组相比，敲低UBE2S能减少肝癌细胞克隆形成数目（shUBE2S-1，P < 0.05、P < 0.01；shUBE2S-2，P < 0.01、P < 0.05）；悬浮成球实验显示：与shCtrl组相比，敲低UBE2S能减少肝癌细胞肿瘤球形成数目（shUBE2S-1，P < 0.05、P < 0.01；shUBE2S-2，P < 0.01、P < 0.01）。
结论: UBE2S在肝癌组织T细胞中高表达，与预后不良相关，且其能促进肝癌细胞干细胞特性。
Keywords: T cells; UBE2S; hepatocellular carcinoma; single-cell sequencing.