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(CoCl₂)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-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. Lipofectamine^TM 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(P<0.01); both mRNA and protein levels of vimentin were up-regulated(P<0.01). Compared with other hypoxic groups, the expression level of mRNA of β-actin was down-regulated in positive control group(P<0.01), which indicated that our operation of cell transfection was successful. In interference group, the expression of HIF-1α and vimentin were down-regulated obviously both on mRNA and protein levels(P<0.01). There was no significant difference in the expression of HIF-1α and vimentin in negative control group and liposome group(P>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.