Use of recombinant calpain-2 siRNA adenovirus to assess calpain-2 modulation of lung endothelial cell migration and proliferation.
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.
Qiu
Kai
K
Department of Medicine, University of Florida College of Medicine, Gainesville, FL 32608-1197, USA.
Su
Yunchao
Y
Block
Edward R
ER
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002154:Calpain / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism*; D016923:Cell Death; D002465:Cell Movement; D049109:Cell Proliferation; D004727:Endothelium / Q000166:cytology*; D015971:Gene Expression Regulation, Enzymologic; D020868:Gene Silencing; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000378:metabolism; D006801:Humans; D008168:Lung / Q000166:cytology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D002154:Calpain