Guoji Yanke Zazhi (Mar 2014)
Effect of specific silencing HIF-1α on expression of Vimentin in OCM-1 cells under hypoxia
Abstract
AIM:To investigate the effect of specific silencing hypoxia inducible factor-1 alpha(HIF-1α)by small interference RNA(siRNA)on expression of vimentin in human uveal melanoma cell lines(OCM-1)under hypoxia, in order to discuss the role of HIF-1α on epithelial-mesenchymal transition(EMT)in uveal melanoma.METHODS: OCM-1 cells were cultured under normoxia and hypoxia in vitro. We added chemical hypoxia inducer cobalt chloride(CoCl2)into nutrient medium at the concentration of 100μmol/L to simulate hypoxia microenvironment inside tumor to culture cells of hypoxic group. And hypoxic group cells were divided into five groups: simple hypoxic group, interference group, positive control group, negative control group and liposome group. SiRNA(including HIF-1α-siRNA, β-actin-siRNA and negative control)were designed and synthetized in vitro. LipofectamineTM 2000 was taken to transfect siRNA into OCM-1 cells under hypoxia. RT-PCR and Western blot were used to check the expression status of HIF-1α and vimentin on mRNA and protein levels before and after hypoxia culture and cell transfection.RESULTS: Compared with normoxia group, there was no obvious change on the expression level of mRNA of HIF-1α in simple hypoxia group(P>0.05), while the expression level of its protein increased obviously(PPPPP>0.05). CONCLUSION: Hypoxia can up-regulate the expression of HIF-1α on protein level in OCM-1 cells, and activate the transcription of vimentin as the downstream gene of HIF-1α, up-regulate the expression of vimentin both on mRNA and protein levels. This hints that HIF-1α can regulate the EMT in uveal melanoma and plays an important role in the process of tumor invasion and metastasis. We successfully down-regulate the expression of HIF-1α and vimentin by transfecting OCM-1 with HIF-1α-siRNA. This suggests that suppression the expression of HIF-1α at the molecular level maybe can shut down the process of tumor invasion and metastasis and offer new directions for cancer treatment.
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