Upregulation of ubiquitin-conjugating enzyme E2T (UBE2T) predicts poor prognosis and promotes hepatocellular carcinoma progression

Abstract

Reportedly, ubiquitin-conjugating enzyme E2T (UBE2T) is closely related to the progression of several malignancies. This work is aimed to probe the role of UBE2T in the progression of hepatocellular carcinoma (HCC) patients. The microarray analysis was executed to screen the differentially expressed genes (DEGs) in HCC tissues. The Cancer Genome Atlas (TCGA) and Gene Expression Profiling Interactive Analysis (GEPIA2) databases, PCR and immunohistochemistry were utilized to validate the dysregulation of UBE2T in HCC. Kaplan-Meier analysis was employed to determine the relationship between UBE2T expression and the prognosis of HCC patients. PCR was carried out to detect UBE2T protein expression in HCC cell lines. Cell Counting Kit-8 (CCK-8) assay and 5-bromo-2'-deoxyuridine (BrdU) experiments were conducted to examine the proliferation of HCC cells. Scratch healing and Transwell experiments were conducted to examine the migration of HCC cells. Bioinformatics analysis and dual-luciferase reporter gene experiments predicted and validated the targeting relationship with miR-212-5p and UBE2T. We found that UBE2T expression was remarkably up-modulated in HCC tissues and cell lines, and its high expression was linked to a worse prognosis in HCC patients. UBE2T overexpression enhanced HCC cell proliferation and migration. Additionally, UBE2T was verified as a downstream target of miR-212-5p. In conclusion, UBE2T overexpression is markedly linked to unfavorable prognosis in HCC patients. UBE2T, regulated by miR-212-5p, significantly enhances the malignant phenotypes of HCC cells, which can be used as a target for HCC diagnosis and prognosis.

Keywords: HCC; UBE2T; miR-212-5p; migration.

Graphical abstract

Figure 1.

Identification of DEGs based on bioinformatics analysis. (a-c) Volcano plots of DEGs in the GSE64041, GSE74656, and GSE76427 datasets. (d) Venn diagrams illustrated the number of up-modulated DEGs in the three gene expression datasets. (e and f) TCGA and GEPIA2 databases were utilized to analyze UBE2T expression in HCC. (g and h). GEPIA2 database was adopted to analyze the relationship between UBE2T expression and overall survival and disease-free survival of HCC patients

Figure 2.

UBE2T overexpression was remarkably linked to adverse prognosis of HCC patients. (a and b) UBE2T expression in HCC tissues and cell lines was detected by qRT-PCR. (c) IHC was executed to detect UBE2T protein expression in HCC tissues and paracancerous tissues. (d) Survival curves were plotted to analyze the relationship between UBE2T expression and overall survival of HCC patients. (e) Correlation between UBE2T expression and TNM stage in HCC patients. (f) Correlation between UBE2T expression and tumor tissue differentiation grade in HCC patients

Figure 3.

UBE2T enhances the proliferation and migration of HCC cells. (a) pcDNA3.1-UBE2T and si-UBE2T were transfected into Hep3B and HepG2 cell lines, respectively, and the transfection efficiency was detected by qRT-PCR. (b and c) CCK-8 and BrdU experiments are employed to detect the proliferation of HCC cells after transfection. (d and e) Scratch healing and Transwell experiments were executed to detect the migration of HCC cells after transfection

Figure 4.

UBE2T was a downstream target of miR-212-5p. (a) StarBase database and miRDB database were applied to predict miRNAs that could interact with UBE2T. (b) The binding site between miR-212-5p and UBE2T 3ʹUTR. (c) Relative luciferase activity was determined 48 h after HEK293T cells were transfected with miR-212-5p mimics/mimics NC and reporter vectors carrying UBE2T sequence. (d) qRT-PCR and Western blot were utilized to detect the effects of miR-212-5p mimics and inhibitors on UBE2T expression in HCC cells. (e) qRT-PCR was performed to detect miR-212-5p expression in HCC tissues. (f) Pearson’s correlation coefficient analysis was used to measure the correlation between miR-212-5p expression and UBE2T expression in HCC tissues

Figure 5.

miR-212-5p impeded the malignant phenotype of HCC cells dependent on UBE2T. (a and b) HepG2 cells were transfected with mimics NC, miR-212-5p mimics, and miR-212-5p mimics+pcDNA3.1-UBE2T, respectively, and UBE2T expression in HepG2 cells was detected by qRT-PCR and Western blot. (c and d) CCK-8 and BrdU assay were executed to detect the viability of HCC cells after the transfection. (e and f) Wound-healing assay and Transwell assay were conducted to detect the migration of HCC cells after the transfection