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Use of recombinant calpain-2 siRNA adenovirus to assess calpain-2 modulation of lung endothelial cell migration and proliferation

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Abstract

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.

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Authors and Affiliations

  1. Department of Medicine, University of Florida College of Medicine, Gainesville, FL, 32608-1197, USA

    Kai Qiu, Yunchao Su & Edward R. Block

  2. Malcom Randall VA Medical Center, Gainesville, FL, 32608-1197, USA

    Edward R. Block

  3. Department of Medicine, University of Florida, Box 100225, MSB M452, 1600 S.W. Archer Road, Gainesville, FL, 32610, USA

    Yunchao Su

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  1. Kai Qiu
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  2. Yunchao Su
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Correspondence to Yunchao Su.

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Qiu, K., Su, Y. & Block, E.R. Use of recombinant calpain-2 siRNA adenovirus to assess calpain-2 modulation of lung endothelial cell migration and proliferation. Mol Cell Biochem 292, 69–78 (2006). https://doi.org/10.1007/s11010-006-9219-2

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  • DOI: https://doi.org/10.1007/s11010-006-9219-2

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