The Title Of The Dissertation Ku80 Negatively Regulates Sternness Of HCC Via SALL4-OCT4 Pathway

The cells in the tumor are heterogenous.The unique function of cancer stem cells,as a decisive factor for tumor,plays a big role in the formation of tumor heterogeneity.SALL4 has been considered to be a novel target for HCC therapy as a newly found stem cell biomarker in liver cancers since the past two years.In the previous studys,we used mass-spectrometric technique to examine the immunoprecipitations pulled down by Anti-SALL4 antibody and then identified a cluster of potential proteins interacting with SALL4,including protein Ku80.The interaction between SALL4 and Ku80 was verified by Co-Immunoprecipitation(Co-IP)experiment.Whether Ku80 regulates the sternness of HCC cells via its interaction with SALL4 is still unknown.We also wonder if that thus affects the funtional heterogeneity of HCC cells?What is the basic principle of mechanisms with which Ku80 is involved in regulating stemness of HCC cells?Subsequently,to investigate the molecular mechanisms with which Ku80-SALL4 interaction reduced the stenmness of HCC,we not only elucidated their interaction and specific binding domains,but also evaluated the effects of Ku80 on stemness of HCC cells,and sternness-related migration and invasion.Our results showed that,? Co-IP assays confirmed Ku80 interacting with SALL4.Further analysis of binding domains indicated that Ku80 C-terminal domain extending from amino acid 449 to 732 and C2H2 motif in SALL4 N-terminal region extending from amino acid 300 to 500 were critical for their binding.? Overexpressing Ku80 in HCC cells to determine the effects of Ku80 on maglinant phenomenon such as spheroid formation,invasion and migration.The findings indicated that Ku80 significantly suppressed the number of spheroids,invading and migrating HCC cells.Furthermore,by examing the impacts of Ku80 on SALL4 expression and other sternness-related genes,our results showed that neither mRNA of these genes were altered by Ku80,nor the protein of SALL4.However,the level of OCT4 protein was obviously reduced by Ku80.In addition,treatment with NH4Cl as an inhibitor of lysosome reversed the suppression effect of Ku80 on OCT4 protein,suggesting that Ku80 inhibited the stemness of HCC cells,possibly via autophagy-lysosome pathway with which Ku80 downregulated OCT4 protein level.? Inhibition of HCC cells stemness by Ku80 depended on its interaction with SALL4.Overexpressing deletion mutants of Ku80 to examine their influence on OCT4 protein,we found that only the Ku80 mutant interacting with SALL4 can inhibit the expression of OCT4 protein.Co-IP assays revealed that competitive binding of Ku80 to SALL4 contributed to the dissociation of SALL4-OCT4 as well as the nuclear export of OCT4 and its degradation.Immunofluorescent staining and laser scanning confocal microscope observations found the translocation of OCT4 from nucleus to the cytoplasm.In summary,we for the first time found the SALL4-Ku80 interaction and verified it;we identified their specific binding domain and confirmed that Ku80 inhibits the sternness of HCC cells via competitive binding to SALL4 which can lead to the dissociation of SALL4-OCT4 and then the nuclear export of OCT4 and its degradation.