The Mechanism Of E3 Ubiquitin Ligase FBXO11 Ubiquitination Degrading ZEB1 Regulates Invasion And Metastasis Of Lung Cancer Cells

According to the GLOBOCAN global cancer statistics in 2020,lung cancer is the malignant tumor with the second highest incidence and the first fatality rate^[1].Tumor metastasis is a main cause of death in patients with lung cancer,but the mechanism of invasion and metastasis of lung cancer is still unclear.The Epithelia to Mesenchymal Transition（EMT）cells are closely related to tumor invasion and metastasis,and expression of the epithelial cell marker E-cadherin is downregulated as an essential symbol of EMT.Snail family,Twist family and ZEB family are important driving factors of EMT.ZEB1 directly represses E-cadherin expression and initiates EMT by binding its zinc finger structure to the E-box,the promoter region of the E-cadherin gene.Ubiquitin-proteasome system（UPS）uses E3 ubiquitin ligase to recognize the substrate protein and transfer ubiquitin to the substrate for degradation by the proteasome,which is a common degradation method in living organisms.FBXO11 is a widely studied E3 ubiquitin ligase.Therefore,this study attempted to establish a connection between FBXO11 and transcription factor ZEB1 in lung cancer cells to reveal the epigenetic mechanism by which FBXO11 ubiquitination degrades ZEB1 and thus regulates lung cancer metastesis.In this paper,human lung cancer cell lines,lung cancer database and mice were used to carry out in-depth research.By inducing EMT in human non-small cell lung cancer cell line A549 through Ni Cl2,we found that the transcription factor that plays a major regulatory role in lung cancer cells is ZEB1,and the correlation between FBXO11 and ZEB1 was further confirmed by the analysis of lung cancer data using GEPIA.Through Co-IP and GST pull-down experiments,the interaction between FBXO11 and ZEB1 was confirmed.At the same time,HEK293T cells were co-transfected with FBXO11 and ZEB1,and degradation,ubiquitination and half-life experiments proved that FBXO11 could degrade ZEB1 through ubiquitination proteasome pathway.sh RNA was used to knock down FBXO11 in A549 cells,and Western Blot,Real-time PCR and immunofluorescence experiments were used to clearly knock down FBXO11,and the expression of ZEB1 was increased.Meanwhile,the expression of EMT epithelial marker E-cadherin decreased and that of mesenchymal marker N-cadherin increased.Combined with morphological changes,Wound healing and Transwell experiments,it was found that after FBXO11 knockdown,A549 cells became more elongated,dispersed,and had enhanced migration and invasion ability.Moreover,after FBXO11 and ZEB1 knockdown,the changes of A549caused by FBXO11 knockdown basically disappeared.All these indicated that FBXO11could regulate the migration and invasion of lung cancer cells through ZEB1.Finally,we reconfirmed that FBXO11 knockdown can promote tumor invasion and metastasis through subcutaneous tumor formation in mice,and Kaplan-Meier survival curve analysis showed that high expression of FBXO11 is associated with good prognosis of lung cancer.According to the above results,a following conclusion can be drawn:FBXO11regulates EMT of lung cancer cells by ubiquitination and degradation of ZEB1 through ubiquitin-proteasome pathway,which provides a theoretical basis for elucidating the invasion and metastasis mechanism of lung cancer and a potential target for exploring new therapy of lung cancer.