title: The effect of NET-1 on the proliferation, migration and endocytosis             of the SMMC-7721 HCC cell line.
abstract: To explore the effect of NET-1 on the proliferation, migration and endocytosis in the hepatocellular carcinoma (HCC) cell line SMMC-7721, we constructed the pU6H1-NET-1-siRNA (NET-1siRNA) and pcDNA3.1/myc-NET-1 (myc-NET-1) vectors and transfected them into SMMC-7721 cells. The expression levels of NET-1 mRNA and protein were detected using real-time quantitative RT-QPCR and western blotting. The proliferation rates of SMMC-7721 cells were determined by CCK-8 assays, flow cytometry (FCM) and immunohistochemistry staining. The migration in two or three dimensional space of SMMC-7721 cells were determined by wound-healing assay and in vitro invasion assay. The extent of endocytosis in SMMC-7721 cells was estimated by observing the amount of transferrin (Tfn) absorbed with capture ELISA assays, and Tfn endocytosis was observed under confocal immunofluorescence microscopy. The results show that: i) after transfecting NET-1 siRNA, the expression of NET-1 mRNA and protein in SMMC-7721 cells decreased significantly, the growth of cells was suppressed, which induced cell cycle arrest, the proliferation rates were dramatically reduced and the expression of Ki67 declined, and migration and endocytosis in cells were inhibited, compared with untreated cells (every P<0.01); ii) Following transfection with myc-NET-1, the expression of NET-1 mRNA and protein in SMMC-7721 cells increased, and both the proliferation of cells and the cell cycle were promoted (P<0.01, respectively). However, the abilities of cell migration and endocytosis were not affected compared with untreated cells. These data suggest that: i) the NET-1 gene may play an important role in proliferation, migration and endocytosis of cells; ii) siRNA technology may efficiently suppress the expression and function of NET-1 in HCC, suggesting that NET-1 may be a therapeutic target for HCC.
authors.affiliation: Department of Pathological Anatomy, Nantong University, Nantong, PR China.
authors.forename: Gui-Lan
authors.initials: GL
authors.lastname: Wang
authors.forename: Li
authors.initials: L
authors.lastname: Chen
authors.forename: Ying-Ze
authors.initials: YZ
authors.lastname: Wei
authors.forename: Jia-Ming
authors.initials: JM
authors.lastname: Zhou
authors.forename: Yuan-Yuan
authors.initials: YY
authors.lastname: Wu
authors.forename: Yi-Xin
authors.initials: YX
authors.lastname: Zhang
authors.forename: Jing
authors.initials: J
authors.lastname: Qin
authors.forename: Yuan-Yuan
authors.initials: YY
authors.lastname: Zhu
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D059447:Cell Cycle Checkpoints; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D004705:Endocytosis; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D034622:RNA Interference; D034741:RNA, Small Interfering; D059470:Tetraspanins / Q000235:genetics / Q000378:metabolism*
chemical_list: D034741:RNA, Small Interfering; C508125:TSPAN1 protein, human; D059470:Tetraspanins