SphK1 Regulates EMT Through CLCA1 To Promote The Proliferation, Invasion And Migration Of Colorectal Cancer

Object: Studies have shown that SphK1 plays an important role in regulating EMT in colorectal cancer metastasis.Until now,the regulation of EMT by SphK1 has not been clarified.CLCA1 is the first protein number of the calcium-activated chloride channel regulator(CLCA)family to be identified.It participates in the chloride ion conduction of the epithelial cell membrane,and participate in the regulation of various biological processes such as tumorigenesis and metastasis,cell adhesion,and apoptosis.Recent study has found that CLCA1 was involved in regulated the EMT.This study aims to explore the effect and mechanism of SphK1 on the growth of colorectal cancer cells HT29 in vitro,and give a theoretical basis for the prevention and treatment of colorectal cancer.Methods: Immunohistochemical assay was used to analysis of the expression of CLCA1 in colorectal cancer and normal tissues adjacent to the cancer.Using genetic engineering technology to up-regulate the expression of SphK1 in HT29,one of the human colon cancer cell lines.The expression of SphK1 m RNA and protein were used RT-PCR and western blot to detect.The proliferation ability of colon cancer cells in each group were used colony formation assay and CCK8 assay to detect,wound healing assay were used to observe the migration ability of cells,using Transwell assay to observe the migration and invasion capacity of each group of cells,using western blot to detect the expression of CLCA1 and EMT-related proteins.Results:(1)The results of immunohistochemical staining showed that CLCA1 expression was reduced in colon cancer compared with normal tissues adjacent to cancer.(2)The Colony formation assay showed that compared with blank control group(CON group)and negative transfection control group(NC group),the number of colonys was significantly increased in SphK1 overexpression group.(3)The CCK8 assay showed that compared with CON group and NC group,the OD 450 nm value of the cells was significantly increasing in overexpression SphK1 group.(4)Wound healing assay showed that compared with CON group and NC group,in overexpressing SphK1 group,the wound healing rate of colon cancer cell HT29 was significantly improved.(5)The Transwell assay showed that compared with CON group and NC group,the migration and invasion cell numbers of colon cancer cells HT29 was increased significantly in overexpression SphK1 group.(6)Western blot assay showed that in overexpression SphK1 group,the expression of CLCA1 and E-cadherin protein were decreased,whereas the protein expression of vimentin were increased.Conclusions: SphK1 may promote the proliferation,invasion and migration of colorectal cancer cells by promoting EMT through CLCA1.