目的：研究小干扰RNA （ siRNA ）特异性沉默缺氧诱导因子-1α（ HIF-1α）对缺氧状态下人眼葡萄膜黑色瘤OCM-1细胞中波形蛋白（Vimentin）表达的影响，初步探讨HIF-1α对葡萄膜黑色瘤上皮-间质转化（ EMT ）的调控作用。 方法：体外常氧培养及缺氧培养OCM-1细胞。其中，缺氧培养是通过在培养基中加入浓度为100μmol/L的氯化钴（ CoCl 2）模拟肿瘤内部缺氧微环境，并将缺氧培养细胞分为单纯缺氧组、HIF-1α干扰组、β-actin阳性对照组、无义寡核苷酸阴性对照组及空载脂质体对照组。体外设计合成siRNA（包括HIF-1α-siRNA、β-actin-siRNA及阴性对照），以Lipofectamine TM 2000为载体介导siRNA转染缺氧培养的OCM-1细胞，以RT-PCR和Western blot检测缺氧培养前后及转染前后HIF-1α、Vimentin基因和蛋白的表达。 结果：单纯缺氧组与常氧组相比，HIF-1αmRNA表达无明显差异（ P＞0．05），蛋白表达显著升高（ P＜0．01），而Vimentin mRNA和蛋白的表达均显著升高（ P＜0．01）。缺氧培养的各组之间相比，阳性对照组β-actin mRNA表达下降（ P＜0．01），证实转染成功；干扰组HIF-1α、Vimentin mRNA和蛋白表达均明显下降（ P＜0．01），阴性对照组、脂质体对照组各检测因素均无明显差别（P＞0．05）。 结论：缺氧可促使OCM-1细胞HIF-1α在蛋白水平表达升高，并可通过转录激活下游靶基因 Vimentin，使其在mRNA和蛋白水平表达均升高，提示HIF-1α可能参与调控葡萄膜黑色瘤的EMT ，进而促进肿瘤侵袭、转移；HIF-1α-siRNA转染OCM-1细胞可成功下调HIF-1α及Vimentin的表达，说明从分子水平抑制HIF-1α的表达，可能抑制肿瘤侵袭、转移，从而为肿瘤治疗提供新方向。%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 -1αon 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-1αand 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-1αin simple hypoxia group ( P>0.05 ), while the expression level of its protein increased obviously ( P0.05). CONCLUSION:Hypoxia can up-regulate the expression of HIF-1αon 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-1αcan 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-1αand vimentin by transfecting OCM-1 with HIF-1α-siRNA.This suggests that suppression the expression of HIF-1αat 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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