Effect Of Silencing HIF-1α On The Growth,Invasion And Angiogenesis In Human Salivary Adenoid Cystic Carcinoma Cell Line SACC-83

Objectives:To investigate the expression of HIF-1α and the cellular biological behavior of human salivary adenoid cystic carcinoma SACC-83 cells in normoxia and hypoxia environment and the effects and mechanisms of silencing HIF-1α on cell growth,migration,invasion,and angiogenesis of SACC-83 cells in hypoxic environment.Methods:1.shRNA-HIF-1α and shRNA-NC were transfected SACC-83 cells.The shHIF-1α cell line was obtained by G418 selection and positive clone cell culture,and its silencing efficiency was identified by Western blot.2.Western blot and double antibody sandwich ELISA were used to detect the expression of HIF-1α and MMP-2 and the secretion of vascular endothelial growth factor(VEGF)in SACC-83 cells in normoxia(21% O2)and hypoxia(5% O2)and the effect of silencing HIF-1α on MMP-2 protein expression and VEGF secretion in SACC-83 cells in hypoxia(5% O2).3.Human umbilical vein endothelial cells(HUVEC)were used to analyze the differences of SACC-83 cell secretions on the ability of promoting angiogenesis in normoxia(21% O2)and hypoxia(5% O2)and the effect of silencing HIF-1α on angiogenesis by SACC-83 cell secretions in hypoxia(5% O2)environment.4.Cell migration and invasion ability of SACC-83 cells in normoxia(21% O2)and hypoxia(5% O2)were observed by cell scratching and transwell invasion assay and the effect of silencing HIF-1α on the migration and invasion ability of SACC-83 cells in hypoxia(5% O2).5.MTT colorimetric assay and flow cytometry were used to detect the proliferation,apoptosis and cell cycle of SACC-83 cells in normoxia(21% O2)and hypoxia(5% O2)and the effect of silencing HIF-1α on proliferation,apoptosis and cell cycle of SACC-83 cells in hypoxia(5% O2).Results:1.A stable cell line with low expression of HIF-1α was successfully constructed(SACC-83/shHIF-1α).2.The result of Western blot and ELISA showed that the expression of HIF-1α,MMP-2 and the secretion of VEGF in SACC-83 cells in a hypoxia environment was significantly increased(P <0.01),and the ability of angiogenesis was increased(P<0.05)and the ability of migration(P <0.01)and invasion(P <0.05)was stronger,but there was no significant difference in cell proliferation,apoptosis and cycle distribution(P >0.05).3.In the hypoxic environment,the content of MMP-2 and VEGF protein in the culture supernatant of SACC-83 / shHIF-1α cells were both decreased(P <0.01),the angiogenesis ability was significantly decreased(P<0.01),the ability of cell proliferation,migration and invasion was dramatically weakened(P <0.01),and the proportion of apoptotic cells increased(P <0.05),but there was no significant difference in cell cycle distribution(P >0.05).Conclusion:Hypoxia promoted the overexpression of HIF-1α,MMP-2 and the secretion of VEGF in SACC-83 cells,and enhanced the ability of angiogenesis,invasion and migration of SACC-83 cells,but not SACC-83 cell proliferation and apoptosis and cell cycle distribution.By silencing of HIF-1α in hypoxic environment,the expression of MMP-2 protein and the secretion of VEGF in SACC-83 cells can be reduced,and the ability of cell proliferation and invasion and migration can be decreased,but the cell cycle distribution can not be affected.