Biological Functional And Molecular Mechanistic Study Of CacyBP/SIP In Hepatocelluar Carcinoma

Aims:Hepatocellular carcinoma(HCC)is a leading cause of cancer-related death in our country.Up until now,HCC remains a challenge to early diagnosse,treat effecively,prevent recurrence after resection and it imposes a substantial economic burden on patients,families and the society in China.Hence,it is of great importace to identify potentail biomarkers sensitive for accurate diagnosis and molecular target effective for precise treatment of HCC.CacyBP/SIP is initially described as a binding parter of calcyclin and has been shown to be involved in a wide range of cellular processes,including cell differentiation,proliferation,protein ubiquitination,cytoskeletal dynamics,tumorigenesis.However,the expression profile,biological functions and molecular mechanisms of CacyBP/SIP in HCC are still poorly understood and remain to be further explored.Methods:Firstly,we detect CacyBP/SIP mRNA and protein expression in hepatocellular carcinoma patients who underwent surgical resection by qRT-PCR,western blotting and immunohistochemistry.Then statistical analyses are performed to compare the correlation between CacyBP/SIP expression and clinicopathological parameters.Secondly,shRNA and overexpression lentiviral vectors are constructed and employed to knockdown and overexpress CacyBP/SIP in liver cancer cells.The role of CacyBP/SIP in HCC cell proliferation,migration and invasion were established via CCK-8,colony formation assay and transwell assay in vitro and nude mice xenograft assay,tail vein metastatic assay,liver orthotopic implantion assay in vivo.Moreover,western blotting and ELISA were also performed to validate the changes of epithelial-mesenchymal transition markers and TGF-?/Smad2/Smad3 signaling pathway.Thirdly,we employed CHX to treat cells and analyzed ZEB1 protein half-life.Results:Firstly,we found that CacyBP/SIP expression was significantly upregulated in HCC cancer tissues compared with adjacent normal tissues.And it is positively correlated with tumor size,vascular invasion,cirrhosis,early recurrence.Moreover,its expression is negatively correlated with overall survival time.Secondly,knockdown of CacyBP/SIP inhibited HCC cell proliferation,migration and invasion by CCK-8 assay,colony formation assay,transwell assay,subcutaneous xenograft assay and liver orthotopic implantion assay.Overexpression of CacyBP/SIP could promote proliferation,migration and invasion in vitro and in vivo.Further studies revealed that CacyBP/SIP affected epithelial-mesenchymal transition,secretion of TGF-?1 and therefore TGF-?/Smad2/Smad3 signaling pathway.Thirdly,reduction of CacyBP/SIP inhibited nuclear translocation of ZEB-1 and overexpression of CacyBP/SIP increased the steady-state level of ZEB-1 protein through a decrease in ZEB-1 degradation.Conclusions:Our study defined that CacyBP/SIP expression was significantly upregulated in HCC and revealed that it was closely related to the tumor size,vascular invasion,cirrhosis,early recurrence and overall survival time.Functional study demondtrated that CacyBP/SIP exerted promotional effect on HCC cell proliferarion,migration and invasion,which was attributed to its effect on epithelial-mesenchymal transition,secretion of TGF-?1,TGF-?/Smad2/Smad3 signaling pathway and ZEB-1 degradation.These data elucidate that CacyBP/SIP is an important oncogene contributing to malignant behavior of HCC and provide a potentially molecular target for treatment of HCC.