Overexpression of negative regulator of ubiquitin-like proteins 1 (NUB1) inhibits proliferation and invasion of gastric cancer cells through upregulation of p27Kip1 and inhibition of epithelial-mesenchymal transition

Abstract

Background: Gastric cancer (GC), one of the most common causes of malignant tumors, is characterized by a high degree of heterogeneity, which represents a bottleneck in gaining comprehensive insights into its pathogenesis. Negative regulator of ubiquitin-like proteins 1 (NUB1) is a transcription factor that negatively regulates ubiquitylation system. Although the abnormal expression of NUB1 has been reported in many types of cancer, its expression pattern and functions in GC are poorly understood.

Materials and methods: The link between NUB1 expression and clinicopathological characteristics was analyzed by immunohistochemical staining, and the suitability of NUB1 as a prognostic marker was explored using a public database on mRNA expression levels. NUB1 overexpression was performed by lentiviral transfection. Cell proliferation was estimated using the cell counting kit-8 (CCK-8) assay. The effect on NUB1 on cell cycle was analyzed by fluorescence-activated cell sorting (FACS). Real-time PCR (RT-PCR) and western blotting experiments were used to explore the mechanism of p27Kip1 regulation by NUB1. Cell migration and invasion were determined by wound healing and transwell assays, respectively. Expression levels of epithelial-mesenchymal transition (EMT) indicator proteins were determined by western blotting.

Results: In this study, based on a comparative analysis of cancer tissues from 116 post-operative GC patients with the respective paracancerous healthy tissues, we found that NUB1 was downregulated in GC tissues. At the same time, a low expression level of NUB1 was closely related to poor prognosis. Results from In vitro cancer cell experiments verified that overexpressed NUB1 inhibited GC proliferation, migration, and invasion. In addition, NUB1 upregulated the expression of p27Kip1 and blocked the G1/S phase transition in cell cycle. Finally, NUB1 inhibited the process of EMT by upregulating E-cadherin and downregulating N-cadherin, vimentin, and matrix metalloproteinase-2 (MMP-2).

Conclusion: Reduced NUB1 levels were positively associated to poor prognosis of GC, whereas NUB1 overexpression inhibited the proliferation and blocked the G1/S phase transition in GC cells. This may be strongly coupled to the post-translational modification mechanism (PTM), which could, in turn, reduce the level of ubiquitinylated p27Kip1 and upregulate its expression. In addition, NUB1 overexpression inhibited GC migration and invasion by regulating EMT. In view of the positive tumor-suppressive effect of NUB1 on GC occurrence and progression reported here, this study enhances our understanding of the molecular mechanism of NUB1-mediated GC regulation, and may provide insights into novel drug targets or anti-tumor strategies with better accuracy and efficacy.

Keywords: Epithelial-mesenchymal transition; Gastric cancer; NUB1; Ubiquitylation; p27Kip1.