The Effects And Interaction Between Flotillin-1 And Flotillin-2 In Hepatocellular Carcinoma Cells

ObjectiveThe present study confirmed that both Flotillin-1 and Flotillin-2 promoted the metastasis and invasion of HCC cells,and Flotillin-1 interacts with Flotillin-2。Methods1.The role of Flotillin-1 and Flotillin-2 in HCC was analyzed by IHC and Kaplan-Meier and log-rank test analyses。2.After the Flotillin-1 and Flotillin-2 knockdown HCC cells were constructed,the ability for Flotillin-1 and Flotillin-2 knockdown HCC cells to migration and invasion in HCC cells was detected by Transwell assay and the expression of EMT-related proteins was detected by Western blot.3.The expression of Flotillin-1 and Flotillin-2 in HCC tissues and cells was detected to analyze their correlation。4.In order to determine the interaction between Flotillin-1 and Flotillin-2,the expression levels of Flotillin-1 and Flotillin-2 was detected after knock-down and overexpression experiments。5.It is determined whether Flotillin-1 and Flotillin-2 combined to form heteromerers in HCC cells by Co-IP assay.6.After CHX inhibitor treatment,the stability of Flotillin-1 or Flotillin-2 protein knockdown was detected.Under the action of MG132,the ubiquitination level was detected by immunoblotting in knockdown Flotillin-1 and Flotillin-2 HCC cells so as to define the way of interaction between them.Results1.Flotillin-1 and Flotillin-2 were highly expressed in HCC tissues,especially with lymph node metastasis。2.Down-regulation of Flotillin-1 and Flotillin-2 repressed HCC cell invasion and migration.meanwhile Flotillin-1 and Flotillin-2 similarly regulated the expression level of EMT-related proteins.3.Correlation analysis showed that Flotillin-1 was positively correlated with Flotillin-2 in HCC tissue and cells4.In HCC cells,Flotillin-1 and Flotillin-2 bind to each other.5.The protein expression of Flotillin-2 was decreased in Flotillin-1 knockdown HCC cells,while the mRNA expression level did not significantly change.However,after overexpression of Flotillin-1,both the protein and mRNA level of Flotillin-2 in HCC cells did not significantly change.And vice versa.6.In HCC cells,knockdown of Flotillin-2 accelerated degradation of Flotillin-1 and damaged the stability of Flotillin-1.The ubiquitination level of Flotillin-1 was also increased.Similarly,the degradation rate of Flotillin-2 was increased by Flotillin-1 silencing,and the ubiquitination level was increased.Conclusions1.Flotillin-1 and Flotillin-2 were significantly increased in HCC patients with lymph node metastasis.2.Down-regulation Flotillin-1 and Flotillin-2 suppressed the HCC cells migration and invasion.3.The expression levels of Flotillin-1 and Flotillin-2 were positively correlated in HCC cells and tissues.4.Flotillin-1 and Flotillin-2 were combined to form a complex.5.Only when Flotillin-1 expression was down-regulated,could Flotillin-2 protein level be reduced,and vice versa.6.Flotillin-1 and Flotillin-2 regulated the expression of the other by increasing the level of ubiquitination.