Jiang Jian, Zhang Lu, Xia Xiao-Bo
Department of Ophthalmology, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China.
Int J Ophthalmol. 2015 Oct 18;8(5):877-83. doi: 10.3980/j.issn.2222-3959.2015.05.05. eCollection 2015.
To determine whether small interfering RNA (siRNA) of PGC-1α could inhibit vascular endothelial growth factor (VEGF) expression and tube formation in human retinal vascular endothelial cells (hRVECs).
hRVECs transfected with peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) siRNA were incubated for 24h and then placed into a normoxic (20%, O2) or hypoxic (1%, O2) environment for another 16h. PGC-1α mRNA and protein levels were detected by real-time PCR and Western blot. VEGF mRNA and protein levels were detected by real-time PCR and ELISA. Cell proliferation was evaluated by BrdU incorporation assay. Forty-eight hours after siRNA transfection, hRVECs were planted into Matrigel-coated plates and cultured under normoxic (20%, O2) or hypoxic (1%, O2) conditions for another 48h. The tube formation of hRVECs was observed under an optical microscope and quantified by counting the number of branch points and calculating the total tube length.
PGC-1α mRNA and protein levels were significantly reduced by PGC-1α siRNA, and VEGF mRNA and protein levels also decreased significantly. The percentage of BrdU-labeled cells in siPGC-1α groups were significantly decreased compared with control siRNA groups under normoxia and hypoxia in cell proliferation assay. In the tube formation assay, PGC-1α siRNA treated cells formed significantly fewer tubes.
Blocking PGC-1α expression can inhibit VEGF expression in hRVECs and inhibit their ability to form tubes under both normoxic and hypoxic conditions.
确定过氧化物酶体增殖物激活受体γ共激活因子1α（PGC-1α）的小干扰RNA（siRNA）是否能抑制人视网膜血管内皮细胞（hRVECs）中血管内皮生长因子（VEGF）的表达及管腔形成。
用PGC-1α siRNA转染hRVECs，孵育24小时，然后置于常氧（20% O₂）或低氧（1% O₂）环境中再培养16小时。通过实时聚合酶链反应（PCR）和蛋白质免疫印迹法检测PGC-1α的信使核糖核酸（mRNA）和蛋白水平。通过实时PCR和酶联免疫吸附测定（ELISA）检测VEGF的mRNA和蛋白水平。采用5-溴脱氧尿嘧啶核苷（BrdU）掺入法评估细胞增殖。siRNA转染48小时后，将hRVECs接种到基质胶包被的培养板上，在常氧（20% O₂）或低氧（1% O₂）条件下再培养48小时。在光学显微镜下观察hRVECs的管腔形成情况，并通过计数分支点数量和计算总管长度进行量化。
PGC-1α siRNA显著降低了PGC-1α的mRNA和蛋白水平，VEGF的mRNA和蛋白水平也显著下降。在细胞增殖试验中，与对照siRNA组相比，常氧和低氧条件下siPGC-1α组中BrdU标记细胞的百分比显著降低。在管腔形成试验中，PGC-1α siRNA处理的细胞形成的管腔明显减少。
阻断PGC-1α表达可抑制hRVECs中VEGF的表达，并在常氧和低氧条件下均抑制其管腔形成能力。
