Qiu Kai, Su Yunchao, Block Edward R
Department of Medicine, University of Florida College of Medicine, Gainesville, FL 32608-1197, USA.
Mol Cell Biochem. 2006 Nov;292(1-2):69-78. doi: 10.1007/s11010-006-9219-2. Epub 2006 May 30.
In this study, we developed an adenoviral vector harboring calpain-2 siRNA expression unit in which sense and anti-sense strands composing the siRNA duplex were connected by a loop and transcribed into a siRNA in porcine pulmonary artery endothelial cells (PAEC). We screened one efficient adenoviral vector Ad/si-m187 and found that Ad/si-m187 successfully exerted a gene knockdown effect on calpain-2 mRNA transcription and protein expression levels. The protein content of calpain-2 was reduced by 30%-80% in PAEC infected with Ad/si-m187 in comparison to a control adenoviral vector Ad/si-luc. The mRNA levels of calpain-2 were measured by real-time PCR and were decreased by 60%-100% and in a dose dependent manner. In correspondence to silencing calpain-2 gene expression, calpain-2 activity was decreased significantly. We further evaluated the role of calpain-2 in endothelial cell migration and proliferation. PAEC infected with Ad/si-m187 displayed impaired migration and cell proliferation in comparison to cells infected with control adenoviral vector (Ad/si-luc). These results indicate that adenoviral vector harboring calpain-2 siRNA expression unit is a valuable tool to study the biology of calpains and that calpain-2 plays an important role in lung endothelial cell migration and proliferation.
在本研究中，我们构建了一种携带钙蛋白酶-2小干扰RNA（siRNA）表达单元的腺病毒载体，其中构成siRNA双链体的正义链和反义链通过一个环连接，并在猪肺动脉内皮细胞（PAEC）中转录为siRNA。我们筛选出一种高效的腺病毒载体Ad/si-m187，发现Ad/si-m187成功地对钙蛋白酶-2的mRNA转录水平和蛋白质表达水平发挥了基因敲低作用。与对照腺病毒载体Ad/si-luc相比，感染Ad/si-m187的PAEC中钙蛋白酶-2的蛋白质含量降低了30%-80%。通过实时定量PCR检测钙蛋白酶-2的mRNA水平，其降低了60%-100%，且呈剂量依赖性。与钙蛋白酶-2基因表达沉默相对应，钙蛋白酶-2的活性显著降低。我们进一步评估了钙蛋白酶-2在内皮细胞迁移和增殖中的作用。与感染对照腺病毒载体（Ad/si-luc）的细胞相比，感染Ad/si-m187的PAEC显示出迁移和细胞增殖受损。这些结果表明，携带钙蛋白酶-2 siRNA表达单元的腺病毒载体是研究钙蛋白酶生物学的一种有价值的工具，并且钙蛋白酶-2在肺内皮细胞迁移和增殖中起重要作用。
