摘要
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LipofectamineTM2000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＆gt;0.05 ）, while the＆amp;nbsp;expression level of its protein increased obviously （ P＆lt;0.01）; both mRNA and protein levels of vimentin were up-regulated （P＆lt;0.01）.Compared with other hypoxic groups, the expression level of mRNA of β-actin was down-regulated in positive control group （ P＆lt;0.01 ）, which indicated that our operation of cell transfection was successful.In interference group, the expression of HIF-1αand vimentin were down-regulated obviously both on mRNA and protein levels （P＆lt;0.01）.There was no significant difference in the expression of HIF-1αand vimentin in negative control group and liposome group （P＆gt;0.05）. CONCLUSION：Hypoxia can up-regulate the expression of HIF-1αon protein
出处
《国际眼科杂志》
CAS
2014年第3期411-415,共5页
International Eye Science
