The Function And Molecular Mechanisms Of SSH3 In The Invasion And Metastasis Of Coloretal Cancer

Colorectal cancer(CRC)is one of the most common malignant tumors in digestive tract,and the incidence of CRC has increased recent years.Thus,it is very important to investigate the mechanisms of carcinogenesis and metastasis of CRC deeply.SSH3 gene encodes SSH3 protein which belongs to the SSH family.SSH3 protein is a phosphotase that contains the A,B and the PTP domains that are highly conserved among SSH family.It can dephosphorylate and activate ADF/Cofilin.However,there are few investigations about SSH3,and there are no studies about SSH3 in tumor so far.Thus,we will test the expression of SSH3,analyze the correlation between SSH3 expression and clinicopathological parameters,illustrate the functions and the associated molecular mechanisms of SSH3 in CRC,and eventually explore the upstream regulating mechanisms of SSH3 in our study.The results from Oncomine database analysis,RT-PCR,Westernblot,and Immunohistochemistry revealed showed that SSH3 was up-regulated in CRC.Interestingly,the expression of SSH3 protein was higher in the tumor thrombosis than that in the primary tumor.And there was a positive correlation between SSH3 and the clinicopathological parameters such as differentiation,TNM stage,metastasis and prognosis in CRC.The results from transwell migration assays,scratch wound healing assays and 3-D cell culture assays showed that,the invasion and metastasis ability increased in the SSH3 overexpression CRC cells compared with the control group.However,the invasion and metastasis ability decreased in the SSH3 knockdown CRC cells compared with the control group.To determine the effect of SSH3 on the lung localization of CRC in vivo,indicated cells were injected into the tail vein.The results revealed that the tumor nodules in the SSH3 overexpression group were more than that in the control group,and the tumor nodules were less in the SSH3 knockdown group than that in the control group.The results of the surgical orthotopic transplataion assay revealed that the number of liver metastatic nodules was more in the SSH3 overexpression group than that in the control group.These results indicated that overexpression of SSH3 promoted the invasion and metastasis of CRC,and vice versa.The results of GSEA(Gene Set Enrichment Analysis)revealed that the cytoskeletal signaling pathway and the cytoskeletal related Racl signaling pathway were up-regulated and the pathway-related genes were enriched significantly in the SSH3 high group.The results of the analysis from the public database and mass spectrometry revealed that the protein most likely interacted with SSH3 was LIMK1 and Rac1.The co-localization of SSH3 and F-actin in CRC cells was tested using confocal laser scanning technique,and the results revealed that SSH3 and F-actin was co-localized in CRC cell lines.We also observed the changes in cytoskeleton in the SSH3 overexpression or knockdown CRC cells.The results showed that the cytoskeleton was reorganized when SSH3 was up-regulated in CRC cells.However,the relative regular cytoskeleton nearly disappeared when SSH3 was down-regulated in CRC cells,instead,it is a fragment or scattered distribution of the F-actin.The co-localization of SSH3 and LIMK1 was tested in the CRC cells using Immunofluorescent co-localization technology,and the results revealed that SSH3 and LIMK1 co-localized in CRC cells.The results of Co-IP revealed that when the protein was co-immunoprecipitated with SSH3 antibody,LIMK1 and Racl protein can be found using Westernblot.Similarly,when the protein was co-immunoprecipitated with LIMK1 antibody,SSH3 and Racl protein can be found using Westernblot.When the protein was co-immunoprecipitated with Racl antibody,LIMK1 and Racl protein can be found using Westernblot.The results of Westernblot revealed that the level of p-LIMK1 and p-Cofilin decreased when SSH3 was up-regulated,and the level increased when SSH3 was inhibited.These results suggested that SSH3 may promote the invasion and metastasis of CRC by interacting with LIMK1 and Racl,which can modulate the activity of Cofilin and the rearrangements of cytoskeleton.The public database was used to analyze the transcription factor of the SSH3 promoter.The results suggested that there were many possible c-Myc binding sites in the promoter of SSH3.The results of Luciferase reporter assay,ChIP-qPCR assay and Westernblot revealed that c-Myc can promote the transcription of SSH3.These results demonstrated that the high expression of SSH3 in CRC is due to the transcriptional activation by c-Myc.