The Promoting Effect And Mechanism Of Nrf2 On Cell Metastasis In Cervical Cancer

Background and objective:Cervical cancer is the fourth most common malignancy among women around the world,and its metastasis pathway mainly involves direct spread and lymphatic metastasis.However,due to the high recurrence and metastasis rate of advanced cervical cancer,the prognosis and survival of patients are unsatisfactory.Therefore,metastasis,as the major problem limits the survival of patients with advanced cervical cancer,its mechanism exploring is still an important issue.The premise of metastasis is that cancer cells leave the primary lesion,undergo epithelial-mesenchymal transition（EMT）to acquire strong ability of movement and invasion,and then "walk" in the blood vessels and lymph vessels to resist cell death caused by anoikis,in an appropriate site undergo mesenchymal-epithelial transformation（MET）,invade and proliferate to form metastatic lesions,and the distant metastasis of cancer is completed.Previous studies have confirmed that Nrf2 can promote the EMT in liver cancer,esophageal squamous cell carcinoma and pancreatic cancer through various mechanisms,thereby permitting the distant metastasis of malignant cells,and Nrf2 also can enhance liver cancer cells anoikis resistance to facilitate the metastasis.However,in cervical cancer,the role of Nrf2 in the regulation of EMT and anoikis resistance have not been elucidated.Here,we used cervical cancer HeLa and SiHa cells as the research objects to explore the function of Nrf2 in EMT and anoikis resistance and uncover the novel molecular mechanism.This study confirmed that Nrf2 signaling pathway promoting cervical cancer cell metastasis,and inhibiting the expression of Nrf2 maybe a novel target.Methods:1.We generated Nrf2 stable knockout HeLa cells using sg RNA targeting Nrf2,and transient over-expression of Nrf2 in HeLa and SiHa cells through transfection Nrf2 plasmid.By observing cellular morphology,wound healing assays and Transwell assays,the effects of Nrf2 on the migration of HeLa and SiHa cells were determined.2.Western blot analyses were used to measure the effects of Nrf2 on the expression levels of EMT-associated proteins.3.We constructed the model of anoikis in cervical cancer cells,cell counting and Western blot analyses were used to exam the influence of Nrf2 on cells anoikis.4.We generated Snail transient over-expression in HeLa,SiHa and HeLa-sg Nrf2 cells through transfecting Snail plasmid,and determined the role of Snail in Nrf2-mediated anoikis.Results:1.Nrf2 knockout significantly inhibited the migration ability of cervical cancer HeLa cells,however,Nrf2 over-expression could promote the migration in HeLa and SiHa cells.2.Nrf2 knockout increased the expression level of E-cadherin and decreased the expression level of N-cadherin,Snail,Slug,Smad and vimentin.But in the situation of Nrf2 over-expression,the level of E-cadherin decreased significantly and N-cadherin,Snail,Slug,Smad and vimentin increased significantly.3.In suspension condition,the expression level of Bax was observably increased and Bcl2,FAK,pro-Caspase-3 were decreased markedly in Nrf2 knockout HeLa cells.However,Nrf2 over-expression could change these changes.4.In suspension condition,the over-expression of Snail in Nrf2 knockout HeLa cells increased the expression of FAK,pro-PARP and pro-Caspase-3 observably.Conclusion:Nrf2 enhanced the EMT and the resistance to anoikis by promoting the expression of Snail,and finally accelerated the metastasis of cervical cancer HeLa and SiHa cells.