Guoji Yanke Zazhi (Jul 2015)
Effect of silencing HIF-1α by siRNA on the expression of erythropoietin in malignant melanoma of the choroid cell under hypoxia
Abstract
AIM: To silent hypoxia inducible factor-1α(HIF-1α)gene in malignant melanoma of the choroid cell by small interference RNA(siRNA)and investigate its effect on the expression of matrix metalloproteinase-2(MMP-2)in the choroid cell line human uveal melanoma cell(OCM-1)in hypoxia environment. METHODS: OCM-1 cells cultured on culture flask were divided into normal group and hypoxia group. Hypoxia group were divided into five groups: simple hypoxic group, and interference group, and negative control group, and positive control group, and liposome group. Normal group cells were cultured on DMEM culture flask with 10% FBS, 100U/mL penicillin, 100μg/mL streptomycin as well as high concentration of glucose. The cells were maintained at 37℃ in a humidified 5% CO2 incubator. Cells in good condition were selected for experiment. For hypoxia group, chemical hypoxia inducer CoCl2 was added into nutrient medium at the concentration of 100μmol/L to simulate hypoxia microenvironment. We designed and synthesised siRNA(siRNA+negative control+positive control), the target sequences of the HIF-1α to transfect hypoxic malignant melanoma of the choroid cell. SiRNA including HIF-1α siRNA, β-actin siRNA and negative control group synthesized in vitro transfected hypoxic OCM-1 cell through Lipofectamine2000. The expression of HIF-1α, MMP-2 gene and the protein were detected by RT-PCR and Western blot.RESULTS: Compared with the normal group, the expression of HIF-1α mRNA was not obviously changed(P>0.05), but the expression of HIF-1α protein and MMP-2 mRNA protein was significantly higher(PPPP>0.05).CONCLUSION: Hypoxia status may upregulate the HIF-1α in OCM-1 cells by increasing the expression of protein. Hypoxia can also inactivate MMP-2, resulting in upregulation of MMP-2 RNA and the expression of its protein. The expression of HIF-1α and MMP-2 mRNA can be down-upregulated by transfecting OCM-1 with HIF-1α siRNA.
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