The Role And Mechanism Of E3 Ubiquitin Ligase UBE4B In The Occurrence And Development Of Gastric Cancer

Objective:Gastric cancer(GC)is one of the common malignancies of the digestive system.According to the Global Cancer Statistics 2020,there are approximately 1 million new cases and 769,000 deaths annually,making it the fifth most common cancer and the third most common cause of cancer death worldwide.UBE4 B is an E3 ubiquitin ligase containing a U-box domain,and increasing evidence suggests that UBE4 B is involved in the occurrence and development of various tumors and affects the prognosis of cancer patients.However,no study has elucidated the role and mechanism of UBE4 B in GC.Therefore,this study aims to preliminarily explore the role and possible mechanisms of UBE4 B in the development of GC.Method:Firstly,the expression of UBE4 B in GC tissues and normal gastric mucosa tissues was analyzed by bioinformatics.The correlation between UBE4 B expression and the prognosis and clinicopathological features of GC patients was evaluated.In addition,UBE4 B expression was validated in human specimens using immunohistochemistry(IHC).GC cells with UBE4 B knockdown or overexpression were constructed,and the effects of UBE4 B on cell proliferation and migration were investigated using CCK8,colony formation and scratch assays.Western blot was performed to assess the impact of UBE4 B on the expression of proliferation and apoptosis-related proteins in GC cells.Finally,the potential mechanisms of UBE4 B in GC initiation and progression were explored through transcriptome analysis.Result:1.UBE4 B expression was elevated in GC tissue and correlates with unfavorable prognosis and clinicopathological features in patients with GC.(1)Analysis of the GEO and TCGA-GETx databases showed that the expression of UBE4 B in GC tissues was significantly higher than that in normal gastric mucosal tissues(p<0.001).IHC results showed that the expression of UBE4 B in 20 human GC tissues was significantly higher than that in adjacent normal gastric mucosal tissues(p<0.001).(2)Kaplan-Meier Plotter analysis revealed that GC patients with high UBE4 B expression had a significantly worse prognosis than those with low UBE4 B expression(p<0.001).(3)Analysis of the GSE54129 dataset showed that the expression of UBE4 B was correlated with the age,N stage,M stage,and Stage of GC patients(p<0.05).2.Effect of UBE4 B on the biological behavior of GC cells.(1)We successfully constructed the HGC-27 and BGC-823 GC cells with UBE4 B knockdown,and AGS GC cells with UBE4 B overexpressing;(2)Knockdown of UBE4 B could significantly inhibit the proliferation and migration of GC cells,reduce the expression of p-Akt,Bcl-2 and increase the expression of Bax;(3)Overexpression of UBE4 B could significantly promote the proliferation and migration of GC cells,increase the expression of p-Akt and Bcl-2,and decrease the expression of Bax.3.Effect of UBE4 B on transcription levels in GC cells.(1)There were significant differences in gene expression between the UBE4 B knockdown and the control group.A total of 833 differentially expressed genes,of which 456 were significantly up-regulated and 377 were significantly down-regulated(FC≥1.5,FDR<0.05);(2)GO enrichment analysis results indicated that differentially expressed genes were mainly located in extracellular matrix,extracellular region,cell surface,plasma membrane,and are associated with biological processes such as extracellular matrix organization,cell-matrix adhesion,cell adhesion,angiogenesis,and cell differentiation(p<0.05);(3)KEGG enrichment analysis showed that the differentially expressed genes were significantly associated with MAPK signaling pathway,PI3K-Akt signaling pathway,Ras signaling pathway,Rap1 signaling pathway,and Notch signaling pathway(p<0.05).4.UBE4 B was associated with Notch signaling pathway(1)Expression of m RNA levels of Notch signaling pathway genes DLL1,DLL4,HES1 and HEY1 were downregulated after knockdown of UBE4 B in HGC-27 GC cells(p<0.05);(2)Expression of m RNA levels of Notch signaling pathway genes DLL1,DLL4,HES1 and HEY1 genes were upregulated after overexpression of UBE4 B in AGS GC cells(p<0.05).Conclusions:1.High expression of UBE4 B in GC was associated with poor prognosis and clinicopathological features of GC patients;2.UBE4 B knockdown could inhibit the proliferation and migration of GC cells,and overexpression of UBE4 B could promote the proliferation and migration of GC cells;3.UBE4 B might be involved in the occurrence and development of GC through multiple different mechanisms,UBE4 B was associated with the Notch signaling pathway.