[Inhibition of mouse hepatocyte apoptosis by anti-caspase-12 small interfering RNA].
OBJECTIVE
To study the inhibition of primary mouse hepatocyte apoptosis by small interfering RNA (siRNAs) against caspase-12.


METHODS
The Balb/c mouse primary hepatocytes were isolated in situ with two-step liver perfusion with 0.5 g/L collagenase type IV, and apoptosis were induced with 4 micromol/L thapsigargin (TG). The three kingds of siRNAs targeting different gene sites (130, 214, 521) were synthetized chemically. The single-stranded RNAs were annealed to produce double-stranded siRNAs, then the mouse primary hepatocytes were transfected by oligofectamine package. The inhibition of caspase-12 was analyzed with RT-PCR and Western-blot. The viable hepatocytes following the induction of apoptosis were evaluated with MTT.


RESULTS
All the three kinds of siRNAs could obviously inhibit normal mouse hepatocyte caspase-12 mRNA. The siRNA (214) were more effective than the other two when the concentration was 100 nmol/L. The caspase-12 mRNA expression was inhibited by 52.08%, while that of siRNA (521) was 30.73% (t=4.30, P <0.05). However when the concentration was 200 nmol/L, the inhibitions were similar (88.07%, 86.22% and 89.41% respectively). siRNA (214) could downregulate the expression of apoptotic hepatocytes procaspase-12 by 51.43% ( t=4.30, P <0.01). Contrasted with apoptotic hepatocytes, the cell activity, which was analyzed with MTT, increased by 48.76% (t=2.23, P <0.01).


CONCLUSIONS
siRNAs could effectively downregulate the expression of caspase-12 at mRNA and protein levels and prevent mouse primary hepatocytes from apoptosis.
Liu
Hai-fang
HF
Department of Infectious Diseases, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China.
Xie
Qing
Q
Jiang
Shan
S
Li
Guang-ming
GM
Zhou
Xia-qiu
XQ
Shi
Yi
Y
Yu
Hong
H
Jin
You-xin
YX
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D053454:Caspase 12 / Q000096:biosynthesis* / Q000235:genetics; D022781:Hepatocytes / Q000166:cytology*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D053454:Caspase 12