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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.

SUBMITTER: Ren X

PROVIDER: S-EPMC8806210 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Ubiquitin-conjugating enzyme E2T (UBE2T) plays a significant role in carcinogenesis. Previous studies have demonstrated that UBE2T promotes the development and progression of numerous types of cancer. However, the association between UBE2T expression and colorectal cancer (CRC) remains unclear. In the present study, UBE2T protein expression was examined in the tissues of patients with CRC via immunohistochemistry. In addition, UBE2T expression data and corresponding clinical information were obtained from The Cancer Genome Atlas (TCGA). In the clinical samples, the associations between UBE2T expression and clinicopathological factors were evaluated by the ?2 or Fisher's exact tests. In TCGA data, associations between UBE2T expression and clinical characteristics were evaluated using a logistic regression model. Overall survival was analyzed using Kaplan-Meier and Cox regression analyses. Reverse transcription-quantitative PCR (RT-qPCR) and western blotting assays were used to examine UBE2T expression in normal and CRC cell lines. Gene set enrichment analysis (GSEA) was performed on the dataset from TCGA. UBE2T protein was highly expressed in the cytoplasm of tumor cells in 29/50 clinical samples, whereas in the adjacent normal tissues, it was only highly expressed in 2/50 samples. Furthermore, UBE2T expression was associated with the N classification (P<0.001), clinical TNM stage (P<0.001) and histological grade of tumors (P=0.010). Survival analysis showed an association between high UBE2T expression and poor survival rate in patients with CRC (P=0.002). Cox regression analysis also revealed that UBE2T expression was an independent prognostic factor for these patients (P=0.006). RT-qPCR and western blotting showed that UBE2T was expressed in CRC cell lines at higher levels than that in a normal colon cell line. Analysis of TCGA data revealed that UBE2T was highly expressed in tumor samples compared with normal samples, but was not associated with prognosis. GSEA showed that high expression of UBE2T was associated with the Kyoto Encyclopedia of Genes and Genomes pathways ‘cell cycle’, ‘oxidative phosphorylation’, ‘DNA replication’, ‘p53 signaling pathway’, ‘ubiquitin mediated proteolysis’ and ‘pentose phosphate pathway’. These results indicate that UBE2T may play an important role in the progression of CRC and serve as a potential prognostic factor during the treatment of cancer.

Project description:Background - Hepatocellular carcinomas (HCCs) are heterogeneous tumors with respect to etiology, cell of origin and biology. The course of the disease is unpredictable and is in part dependent on the tumor microenvironment. One of the microenvironmental factors is hypoxia, which is known to promote aggressiveness in other malignant tumors. We hypothesized that certain regions in HCC exist with chronic hypoxia and a characteristic gene expression pattern. Moreover, during the development of HCC there is an important contribution of this chronic hypoxia on prognosis via this gene expression. Until now, most research has been performed in acute hypoxic models (< 24 hours). Methods – Human hepatoblastoma cells HepG2 were cultured in either normoxic (20% O2) or hypoxic (2% O2) conditions for 72 hrs, the time it takes to adapt to chronic hypoxia. After 3 days the cells were harvested and analyzed by microarray technology. The highly significant differentially expressed genes were selected and used to assess the clinical value of our in vitro chronic hypoxia gene signature in four published patient studies. Three of these independent microarray studies on HCC patients were used as training sets to determine a minimal prognostic gene set and one study was used for validation. Gene expression analysis and correlation with clinical outcome was assessed with the bioinfomatic method of Goeman et al (). Results – In the HepG2 cells, 3592 genes were differentially expressed in cells cultured at 2% oxygen for 72 hrs. Out of these, 265 showed a high significant change (2-fold change and p=0.0001). The level of gene expression after 72 hrs was different from the acute hypoxic response (during the first 24 hours) and represented chronicity. Using computational methods we identified 7 out of the 265 highly significant genes that showed correlation with prognosis in all three different training sets and this was independently validated in a 4th dataset. With our approach we could include the largest number of HCC patients in one single study. Conclusion – We identified a 7-gene signature, which is associated with chronic hypoxia and predicts prognosis in patients with HCC. In the future this signature could be used as a diagnostic tool. In addition, chronic hypoxia gene expression information can be used in the search for new therapeutic targets. Two conditions were compared and each sample has a biological replicate. Samples are hybridized in dye-swap, resulting in 4 hybridizations.

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

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