LYAR Expression And Functional Studies In Colorectal Cancer

Ly-1 antibody reactive clone(LYAR),identified initially from a mouse T-cell leukemia line,encodes a 45 kD nucleolar protein that consists of 380 amino acid residues,and is confirmed as a transcription factor with a DNA-binding motif(GGTTAT/G).In order to study the role of LYAR in CRC,immunohistochemistry(IHC)analysis was performed to detect the expression levels of LYAR on three tissue arrays harbored 77 paraffin-embedded normal colorectal mucosa and CRC tissues.Comparing with the adjacent non-tumorous tissues,we found that there were about 49.4%(38/77)of total CRC tissues with significant higher expression levels of LYAR.In order to examine the function of LYAR in colorectal cancer,we performed experiments determining cell phenotype associated closely with cancer progression.Our results showed that cell proliferation,cell cycle,apoptosis,colony formation,adhesion were not significantly affected in HCT8 and HCT15 colon cells when LYAR was knocked down using specific siRNA.However,compared with Non-silencing control(HCT8-NC and HCT15-NC)cells,cell migration and invasion capability were markedly impaired in HCT8 LYAR-siRNA(LYAR Knock-down by siRNA)and HCT15 LYAR-siRNA cells.These results suggest that LYAR might promote cell migration and invasion in colorectal cells.To characterize the potential target affected by LYAR in HCT8 cells,we analyzed the global gene expression profile by microarray analysis using HCT8-NC cells and LYAR knock-down(LYAR-siRNA)cells.We found that both LGALS1 mRNA and protein levels were significantly reduced accordingly when LYAR gene expression was silenced in HCT8,HCT116,LoVo and RKO cells.This suggested that LYAR may regulate the expression of LGALS1 in colorectal cancer.Subsequently,we carried out bioinformatics assay on the promoter of the LGALS1 gene and found a specific LYAR-binding sequence(CTAACC)located at-1359 bp on the upstream LGALS1 promoter.To confirm that LYAR binds to this specific sequence,we performed chromatin immunoprecipitation(ChIP)assay and luciferase reporter assay.Our results indicated that LYAR promotes galectin-1 expression by binding the CTAACC sequence of the promoter of LGALS1.In colon cancer,galectin-1 played an important role in the regulation of cell migration.To investigate whether galectin-1 plays a role in LYAR-promoted cancer cell migration and invasion,we performed a rescue experiment.Ectopic expression of galectin-1 could partially restore the potential of cell migration and invasion of HCT8 LYAR-KD(LYAR Knock-down by shRNA)and HCT116 LYAR-KD cells.Thus galectin-1 contributed to the LYAR-promoted cell migration and invasion of CRC cells.In summary,we showed that LYAR promotes cell migration and invasion through upregulating LGALS1 in CRC.This study may help reationalize LYAR as a potential therapeutic target for treatment of CRC.In addition,this research also studied the relationship between the changes of the expression of Suv420H1 and cell proliferation,apoptosis and cell cycle of K562 cells.Our results found that Suv420H1 promots K562 cell proliferation and cell cycle progression by upregulating the expression of cyclin D1.