Identification And Functional Characterization Of Translocation-Related Domains On HCCS1 Protein

Purpose Hepatocellular carcinoma suppressor 1(HCCS1) has been identified as a novel tumor suppressor gene candidate by screening the high-frequency loss of heterozygosity(LOH) on chromosome 17p13.3 in HCC.And it also plays an important role in sorting of some cytoplasmic proteins,which might be involved in its tumor suppressive activity.The purpose of this study is to identify the minimum region of functional sequence in HCCS1 that relates to its subcellular localization,and to further discuss the relationship of this minimum region of HCCS1 with its protein trsportation and tumor suppressor function.Methods The expression vectors containing various lengths of HCCS1 cDNA were constructed and transfected into HeLa cells mediated by liposomes.The localizations of different HCCS1 cDNA fragments and the colocalizations of M6PR with various truncted HCCS1 cDNA were determined by laser scanning confocal microscopy, respectively.All of the constructions were transfected into HeLa cells and BEL7404 cells mediated by liposomes.The colony formation was evaluated by crystal violet staining after 2 weeks of G418 selection.All of the constructions were transfected into HeLa cells,BEL7404 cells,SMMC7721 cells and SGC7901 cells mediated by liposomes,and the cell viabilities were evaluated by MTT at different time points after G418 selection.Results Ten different vectors expressing various lengths of HCCS1 cDNA were successfully constructed.The results showed that the polar localization of HCCS1 protein and the colocalizations with M6PR disappeared when HCCS1 cDNA was truncted to 1120bp from the 3'end.The distribution of HCCS1 proteins expressed by pEGFP-C2-HCCS1-TR2(678 bp),pEGFP-C2-HCCS1-TR1(578 bp),pEGFP-C2-HCCS1△BS(478 bp) and pEGFP-C2-HCCS1△BE(339 bp) was dispersed in the cytoplasm and nucleolus.The number of colonies from cells transfected with pEGFP-C2-HCCS1(2100 bp),pEGFP-C2-HCCS1△E5(1861 bp),pEGFP-C2-HCCS1-S1(1835 bp),pEGFP-C2-HCCS1-S22(1732 bp) and pEGFP-C2-HCCS1-S39 (1571 bp) was much lower than that of the control cells transfected with vector pEGFP-C2(P＜0.05),whereas the number of colonies from cells transfected with pEGFP-C2-HCCS1△BH(1120 bp),pEGFP-C2-HCCS1△BB(884 bp) and pEGFP-C2-HCCS1-TR3(778 bp) was not obviously different with the control cells transfected with vector pEGFP-C2(P＞0.05).Furthermore,MTT assay showed that pEGFP-C2-HCCS1(2100 bp),pEGFP-C2-HCCS1△E5(1861 bp),pEGFP-C2-HCCS1-S1(1835 bp),pEGFP-C2-HCCS1-S22(1732 bp) and pEGFP-C2-HCCS1-S39 (1571 bp) could reduce the growths of HeLa cells,BEL7404 cells,SMMC7721 cells and SGC7901 cells in vitro.However,pEGFP-C2-HCCS1△BH(1120 bp),pEGFP-C2-HCCS1△BB(884 bp) and pEGFP-C2-HCCS1-TR3(778 bp) did not show the suppressive effect on the cell growth.Conclusions We identified that the minimum region of the HCCS1 cDNA related to translocation spans 451 bp at its 1571 bp 3'end.Our results showed that this minimum region of the HCCS1 cDNA was involved in its protein transportation and tumor suppressive function.And the results implied that HCCS1 transportation funcion might be related to its tumor suppressive activity.