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[Inhibition of mouse hepatocyte apoptosis by anti-caspase-12 small interfering RNA]
Hai-fang Liu1, Qing Xie, Shan Jiang
1Department of Infectious Diseases, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China.
Objectives:
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).
Conclusion:
siRNAs could effectively downregulate the expression of caspase-12 at mRNA and protein levels and prevent mouse primary hepatocytes from apoptosis.
Insights
Small interfering RNAs (siRNAs) effectively inhibit caspase-12 expression in mouse hepatocytes, preventing apoptosis. This study demonstrates siRNA efficacy in downregulating both mRNA and protein levels of caspase-12.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Context:
- Hepatocyte apoptosis plays a critical role in liver injury and disease.
- Caspase-12 is a key mediator of apoptosis in hepatocytes.
- Targeting apoptotic pathways offers therapeutic potential for liver diseases.
Purpose:
- To investigate the efficacy of small interfering RNA (siRNA) in inhibiting caspase-12 expression in primary mouse hepatocytes.
- To evaluate the role of caspase-12 in thapsigargin-induced hepatocyte apoptosis.
- To assess the potential of siRNA-mediated caspase-12 knockdown for preventing hepatocyte cell death.
Summary:
- Three distinct siRNAs targeting caspase-12 were synthesized and transfected into primary mouse hepatocytes.
- siRNAs significantly inhibited caspase-12 mRNA and protein expression in a dose-dependent manner.
- siRNA treatment, particularly siRNA (214), effectively reduced thapsigargin-induced hepatocyte apoptosis and increased cell viability.
Impact:
- Demonstrates the potent inhibitory effect of siRNAs on caspase-12, a key apoptotic mediator.
- Provides a potential RNA interference-based strategy for preventing or treating liver injury associated with hepatocyte apoptosis.
- Highlights the therapeutic promise of targeting specific apoptotic pathways in liver disease management.
