Small-interference RNA gene knockdown of pancreatitis-associated proteins in rat acute pancreatitis.
OBJECTIVE
Pancreatitis-associated proteins (PAPs) are induced in acute pancreatitis and antisense-mediated gene knockdown of PAP decreased PAP gene expression and worsened pancreatitis. Here, we investigated the effect of a more stable inhibition of PAP using small-interference RNA gene knockdown in vitro and in an in vivo model of experimental pancreatitis.


METHODS
Pancreatitis-associated protein-specific siRNA was administered to AR42J cell cultures or rats induced with pancreatitis. Controls included administration of scrambled siRNA or vehicle alone. After 24 hours, cells and pancreata were harvested and assessed for PAP (PAP 1, PAP 2, PAP 3) gene expression and pancreatitis severity.


RESULTS
In vitro, PAP protein, and mRNA levels were reduced (PAP 1, 76%; PAP 2, 8%; PAP 3, 24%) in cells treated with PAP siRNA. In vivo, PAP 1, and PAP 3 expressions were reduced (PAP 1, 36%; PAP 3, 66%) in siRNA-treated rats; there was no difference in PAP 2 isoform mRNA expression and serum protein levels. Serum amylase and lipase levels decreased (> or =50%) after administration of siRNA; interleukin (IL) 1beta, IL-4, and IL-6 increased, whereas C-reactive protein and tumor necrosis factor-alpha decreased when compared with vehicle control. Administration of PAP siRNA correlated with worsening histopathology.


CONCLUSIONS
siRNA-mediated gene knockdown of PAP worsens pancreatitis. Differences in gene knockdown technology may provide different approaches to study gene function.
Lin
Yin-Yao
YY
SUNY Downstate Medical Center, Brooklyn, NY 11203, USA.
Viterbo
Domenico
D
Mueller
Cathy M
CM
Stanek
Albert E
AE
Smith-Norowitz
Tamar
T
Drew
Hazel
H
Wadgaonkar
Raj
R
Zenilman
Michael E
ME
Bluth
Martin H
MH
D000208:Acute Disease; D000818:Animals; D000951:Antigens, Neoplasm / Q000235:genetics*; D014408:Biomarkers, Tumor / Q000235:genetics*; D002460:Cell Line; D005260:Female; D017353:Gene Deletion; D037181:Lectins, C-Type / Q000235:genetics*; D010195:Pancreatitis / Q000139:chemically induced / Q000235:genetics*; D000073718:Pancreatitis-Associated Proteins; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013656:Taurocholic Acid / Q000633:toxicity; D014162:Transfection
D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D037181:Lectins, C-Type; D000073718:Pancreatitis-Associated Proteins; C000612141:REG3A protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C000612146:Reg3a protein, rat; C000612144:Reg3b protein, rat; C000612145:Reg3g protein, rat; D013656:Taurocholic Acid