The Molecular Mechanism Of Six1 Promoting Colorectal Cancer Cell Proliferation And Migration

BackgroundThe sine oculis homeobox 1(Six1),a member of Six family protein,has been reported to be an essential transcription factor involved in the development of many tissues and organs,including kidney and muscle tissue,as well as the auditory system and certain sensory organs.Six1 was also found to be highly expressed and implicated in both tumor initiation and tumor progression in a variety of human cancers,including breast cancer,Hodgkin’s lymphoma,cervical cancer,osteosarcoma,hepatocellular carcinoma,colorectal cancer(CRC),ovarian cancer,rhabdomyosarcoma,Wilms tumors.Six1 has been reported to play a key role in the proliferation and migration of CRC cells but the underlying molecular mechanisms is still poorly characterized.Objectives1.To analyze the function of Six1 in colorectal cancer cell proliferation and migration;2.To explore the molecular mechanism underlying Six1-promoted proliferation and migration of colorectal cancer cells.Method1.The effect of Six1 on proliferation of CRC cellsThe pXJ-40-Six1 vectors and Six1-siRNA were transfected into CRC HCT116 and LoVo cells,respectively.The cells were digested after 48 hours of continuous culture and inoculated into 96-well plates.The effects of Six1 overexpression on the proliferation of colorectal cancer cell line HCT116 and Six1 gene silencing on the proliferation of colorectal cancer cell line LoVo were measured by MTT assay.2.The effect of Six1 on migration of CRC cellsHCT116 cells trasnsfected with pXJ-40-Six1 vectors and LoVo cells transfected with Six1 siRNA were seeded into 6-well plates and cultured for 1 or 2 days.Then the cell culture medium was replaced with fresh serum-free but double-antibiotic-containing medium and starved for a suitable time.At the bottom of the 6-well plate,200 ul pipette tip was used to create a wound.The healing of the wounds was recorded by inverted microscope at different time points(0h,12 h,24h,36 h.).The migration ability of CRC cells was estimated by calculating the change of the area of the wound healing.The cell migration was also examined by transwell assay.The starved cells in medium without serum were seeded into the upper chamber of the transwell,with complete medium containing serum as chemoattractant in lower chamber.After culturing for 24 h,the noninvasive cells in the upper surface of the chamber were removed and the invasive cells migrating to the lower surface were fixed and stained with 0.5% crystal violet.Migrating cells were photographed and counted under a microscope.3.The molecular mechanisms underlying Six1-promoted proliferation and migration of CRC cellsHCT116 cells transfected with pXJ-40-Six1 vectors and LoVo cells transfected with Six1 siRNA were cultured for 2 days.Total protein of the cells was extracted according to the protein extraction procedure,and the related signaling pathways,such asβ-catenin,JNK,p53,Wnt,AKT,p38 and AMPKα were examined by western blot.Subsequent experiments were carried out with β-catenin specific siRNA and of Wnt/β-catenin signaling pathways specific activator and inhibitor to evaluate the contribution of Wnt/β-catenin signaling pathway in Six1-promoted proliferation and migration of CRC cells.Results1.Six1 overexpression promotes CRC cells proliferationThe results showed that the number of living cells a in the overexpression group of Six1 were significantly higher than those in the control group.Compared with the blank control group,the viability of colorectal cancer cells transfected with pXJ-40-Six1 was about two times higher.On the contrary,the number of living cells and the total cell viability of the interference group of Six1 were significantly lower than that of the control group.These results suggested that Six1 promotes the proliferation of CRC cells.2.Six1 overexpression enhances the migration potential of CRC cellsThe results of wound healing showed that overexpression of Six1 in HCT116 cells resulted in a longer healing distance than that in HCT116 cell control group at the same time.Transwell results showed that over-expression of Six1 in HCT116 cell line was associated with a higher migration rate than that in control group.On the contrary,in LoVo cells,the migration of colorectal cancer cells was significantly inhibited in the interfering Six1 group compared with thecontrol group.These results indicate that Six1 can promote the migration of CRC cells.3.Six1 promotes CRC proliferation and migration through activating Wnt/β-catenin signaling pathway.The results of western blot showed that the Six1 overexpression and gene silencing have no effects on phosphorylation of JNK,p53,AKT,p38 and AMPKα.However,Six1 overexpression in colorectal cancer cells increased the nuclear translocation of β-catenin.,Consistently,Six1 gene silencing suppressed the nuclear translocation of β-catenin.These results indicates that Six1 is an upstream effector of Wnt/β-catenin pathway.Further study with β-catenin specific siRNA or Wnt pathway inhibitor revealed that reduced β-catenin nuclear translocation could reverse Six1 overexpression-promoted proliferation and migration of CRC.In contrary,Wnt pathway activator could block Six1 gene silencedmediated inhibition of proliferation and migration of CRC.These results suggested that Six1 promoted the proliferation and migration of CRC by activating Wnt/β-catenin signaling pathway.ConclusionIn this study,we found that Six1 could significantly promote the proliferation and migration of colorectal cancer cells.Further investigation revealed that Wnt/β-catenin signaling pathway is involved in Six1-promoted proliferation and migration of CRC cells.Our study provides a new target for the development of drugs to inhibit the migration of colorectal cancer cells.