Kikuchi Michiharu, Zhu Cheng, Senoo Tadashi, Obara Yoshitaka, Joyce Nancy C
Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, 20 Staniford Street, Boston, MA 02114, USA.
Invest Ophthalmol Vis Sci. 2006 Nov;47(11):4803-9. doi: 10.1167/iovs.06-0521.
To determine whether small interfering (si)RNA downregulation of the cyclin-dependent kinase inhibitor p27kip1 overcomes G(1)-phase arrest and promotes cell-cycle progression in human corneal endothelial cells (HCECs) from young (<30 years old) and older (>60 years old) donors.
Transfection of siRNA was confirmed by incubating confluent cultures of HCECs with FITC-labeled nonsilencing siRNA. Confluent cultures were transfected for 48 hours with p27kip1 siRNA (2.5, 5, 25, or 100 nM) or nonsilencing siRNA, with a lipid transfection reagent. As a comparison, cultures were also transfected for 48 hours with p27kip1 antisense (AS) or missense (MS) oligonucleotides (oligo). At various times after transfection, cells were fixed for immunocytochemical localization of p27kip1 or extracted for Western blot analysis to assess relative p27kip1 protein levels. Cultures were also prepared for ZO-1 immunolocalization, to assess the effect of transfection on the morphology of the monolayer. The number of cells was counted at 0, 48, 96, 144, and 192 hours after incubation, and a cell-viability assay was performed.
A dose-dependent decrease in p27kip1 protein level was observed in Western blot analysis, and nuclear staining for p27kip1 was greatly reduced in HCECs incubated with p27kip1 siRNA. No change in p27kip1 levels or in nuclear staining was observed in the nonsilencing control. p27kip1 siRNA (25 nM) appeared to be quantitatively more efficient than antisense oligonucleotide (500 nM) in reducing p27kip1 protein levels. Viability was less affected by siRNA treatment than by AS oligo transfection. ZO-1 staining showed no effect on morphology of the monolayer. The number of HCECs from young donors (<30 years old) transfected with p27kip1 siRNA increased up to 144 hours after incubation, whereas no change in the number of cells was observed in HCECs transfected with nonsilencing siRNA. In contrast to the results from young donors, no change in the number of cells was observed at any time point tested in HCECs from older donors (>60 years old) after p27kip1 siRNA transfection.
Transfection of p27kip1 siRNA was sufficient to promote proliferation in confluent cultures of HCECs from younger, but not older donors. These results suggest that inhibition of proliferation in older donors is regulated by other mechanisms in addition to p27kip1.
确定细胞周期蛋白依赖性激酶抑制剂p27kip1的小干扰（si）RNA下调是否能克服G1期阻滞并促进来自年轻（<30岁）和年长（>60岁）供体的人角膜内皮细胞（HCECs）的细胞周期进程。
通过将HCECs的汇合培养物与异硫氰酸荧光素（FITC）标记的非沉默siRNA孵育来确认siRNA的转染。使用脂质转染试剂将汇合培养物用p27kip1 siRNA（2.5、5、25或100 nM）或非沉默siRNA转染48小时。作为对照，培养物还用p27kip1反义（AS）或错义（MS）寡核苷酸（oligo）转染48小时。在转染后的不同时间，固定细胞用于p27kip1的免疫细胞化学定位，或提取细胞用于蛋白质印迹分析以评估相对p27kip1蛋白水平。还制备培养物用于紧密连接蛋白1（ZO-1）的免疫定位，以评估转染对单层细胞形态的影响。在孵育后0、48、96、144和192小时对细胞进行计数，并进行细胞活力测定。
蛋白质印迹分析观察到p27kip1蛋白水平呈剂量依赖性下降，在用p27kip1 siRNA孵育的HCECs中，p27kip1的核染色大大减少。在非沉默对照中未观察到p27kip1水平或核染色的变化。在降低p27kip1蛋白水平方面，p27kip1 siRNA（25 nM）似乎在定量上比反义寡核苷酸（500 nM）更有效。与反义寡核苷酸转染相比，siRNA处理对细胞活力的影响较小。ZO-1染色显示对单层细胞形态没有影响。用p27kip1 siRNA转染的年轻供体（<30岁）的HCECs数量在孵育后144小时内增加，而用非沉默siRNA转染的HCECs数量没有变化。与年轻供体的结果相反，在用p27kip1 siRNA转染后，在任何测试时间点，来自年长供体（>60岁）的HCECs数量均未观察到变化。
转染p27kip1 siRNA足以促进来自年轻但非年长供体的汇合HCECs培养物的增殖。这些结果表明，除了p27kip1之外，年长供体中增殖的抑制还受其他机制调控。
