摘要
目的:探讨常氧及缺氧培养条件下RNA干扰沉默人食管鳞癌细胞Eca-109缺氧诱导因子-1α(HIF-1α)对血管生成拟态相关基因表达的影响.方法:选择未转染处理的食管鳞癌Eca-109细胞和3株稳定转染HIF-1αSiRNA的Eca-109细胞,分别予以常氧和缺氧培养,缺氧培养12、24、36、48、60、72、96h,采用Western blot法和逆转录聚合酶链反应(RT-PCR)检测HIF-1α蛋白及mRNA表达,同时Western blot法检测上皮细胞激酶(EphA2)、血管上皮钙黏附素(VE-cadherin)、基质金属蛋白酶-2(MMP-2)、层粘连蛋白5γ2链(LN-5γ2)等血管生成拟态相关基因的表达.结果:缺氧条件下培养Eca-109细胞HIF-1α蛋白表达较常氧时增高,以24-48h为明显,72-96h开始减弱,而HIF-1α mRNA检测差异无统计学意义(F=0.70,P=0.67);RNA干扰后细胞在常氧下HIF-1α、EphA2、LN-5γ2蛋白几乎无表达,缺氧后亦无增强现象,VE-cadherin、MMP-2蛋白表达较未干扰细胞稍有减弱,缺氧后表达稍增强,但差异无显著性(均P>0.05).结论:RNA干扰能抑制Eca-109细胞的HIF-1α基因表达,HIF-1α基因沉默后可以不同程度减少EphA2、LN-5γ2等血管生成拟态相关基因的表达.
AIM： To investigate the effect of RNA interference-based silencing of HIF-1α gene on the expression of vasculogenic mimicryassociated genes in esophageal squamous carcinoma cells （Eca-109） cultured in either a normoxia or a hypoxia environment. METHODS： Eca-109 cells were divided into four groups, namely, untransfected Eca-109 cells and three different strains of Eca-109 cells stably transfected with HIF-1α siRNA （H2/20, H3/10 and H3/15 strains）. After the cells were cultured in a normoxia environment or in a hypoxia environment for 12, 24, 36, 48, 60, 72 and 96 h, respectively, the expression of HIF-1α protein and mRNA was detected by Western blot and semiquantitative reverse transcription-polymerase chain reaction （RT-PCR）, and the expression of EphA2, VE-cadherin, matrix metalloproteinase-2 （MMP-2） and laminin-5 gamma-2 chain （LN-5γ2） was determined by Western blot. RESULTS： After Eca-109 cells were cultured in a hypoxia environment, the expression of HIF-1α protein was upregulated, reaching its peak at 24 and 48 h and beginning to decline at 72 and 96 h. However, there was no significant difference in the expression of HIF-1α mRNA （F = 0.70, P = 0.67）. The expression of HIF-1α, EphA2 and LN-5γ2 proteins was hardly detected in HIF-1α- silenced cells, and could not be upregulated in a hypoxia environment. In contrast, the expression of VE-cadherin and MMP-2 proteins was slightly down-regulated in HIF-1α silenced cells, and could be upregulated in a hypoxia environment though the upregulation was not statistically significant （all P 〉 0.05）. CONCLUSION： RNA interference targeting the HIF-1α gene is able to efficiently silence the ,expression of HIF-1α gene in human esophageal squamous carcinoma cells （Eca-109） and thereby inhibit the expression of vasculogenic mimicryassociated genes （EphA2, VE-cadherin, MMP-2 and LN-5γ2） to a different extent.
出处
《世界华人消化杂志》
CAS
北大核心
2009年第19期1913-1918,共6页
World Chinese Journal of Digestology
基金
国家自然科学基金资助项目
No.30770991~~
