Effect Of HAI-1 Over-Expression On Growth And Migration/Motility Of A Human Colorectal Carcinoma Cell Line SW620 Cells

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a Kunitz-type serine proteinase inhibitor expressed on epithelial cells. HAI-1 is first produced in a membrane-integrated form with two Kunitz domains in its extracellular region, and subsequent ectodomain shedding releases two major secreted forms, one with a single Kunitz domain and one with two Kunitz domains. HAI-1 is a strong inhibitor of hepatocyte growth factor (HGF) activator and matriptase. Hepatocyte growth factor (HGF), also designated as a scatter factor (SF). is involved in the proliferation and migration of cells. HGF/SF is secreted by mesenchymal cells as an inactive single-chain precursor form (scHGF) and requires proteolytic conversion by HGF activator (HGFA) or matriptase, to initiate MET signaling, which is believed to have important roles in invasive growth of tumor cells and regeneration of injured tissue. HAI-1 is not only an inhibitor but also a specific acceptor of active HGFA, acting as a reservoir of this enzyme on the cell surface. The latter property appears to ensure the concentrated pericellular HGFA activity in certain cellular conditions, such as tissue injury and inflammation. HAI-1 may regulate the hepatocyte growth factor (HGF)/c-Met pathway by inhibiting the activity of HGF activator (HGFA) and matriptase, two serine proteases that convert pro-HGF into its biologically active form. Matriptase is a cognate proteinase of HAI-1. This proteinase activates hepatocyte growth factor (HGF) and urokinase plasminogen activator (uPA), two proteins thought to be involved in the growth and motility of cancer cells. Enhanced expression of matriptase has been reported in several types of carcinoma cells. Thus, matriptase may play an important role in the progression of carcinomas.Although the activation of hepatocyte growth factor (HGF)/scatter factor (SF) is a criticallimiting step in HGF/SF-induced signaling , we now know little of the regulation of the expression of HGF/SF in injured tissue and tumor. In an attempt to elucidate the roles HAI-1 play in tumor cell growth and migration, we subcloned full length cDNA of humanHAI-1 into mammalian expression vector pcDNA3.1(+), and transfected the construct into human colorectal cancer cell SW620. The expression of HAI-1 in transfected SW620 cells was demonstrated by PCR and Western blot.The effects of over-expression of HAI-1 on cell growth and migration/motility were evaluated. It describes as follows: 1. The effects of over-expression of HAI-1 on cell growth(1). Growth curve determined by MTT Assay was used to evaluate the effects of over-expression of HAI-1 on cell growth in vitro.Table 1. Absorbency of cells determined by MTT assayFig. 1. Growth Curve Determined by MTT Assay (2). Soft-agar colony formation assay were used to evaluate the effects of over-expressionof HAI-1 on cell growth in vitro.Table 2 Colony-Formation Assay in Soft-Agara: p=0.33, compared with SW620; b: p=0.13, compared with SW620(pcDNA3.1(+))(3). Growth curve determination made in vivo was used to evaluate the effects ofover-expression of HAI-1 on cell growth in vivo.Table 3. The volum of tumorsFig. 2. Growth Curve made in vivo(4). The weight of tumors was used to evaluate the effects of over-expression of HAI-1 on cell growth in vivo.Table 4. weight of tumorse: 0.05